Biochimica et Biophysica Acta 1812 (2011) 333–334
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Biochimica et Biophysica Acta
j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / b b a d i s
Editorial
Preface: Zebrafish Models of Neurology☆
Forming hypotheses about the Molecular Basis of Neurological
Disease has traditionally been based on human genetics, biochemistry
and molecular biology, whereas testing these hypotheses requires us
to turn to cell culture and animal models. Zebrafish have emerged as a
tractable platform to complement and bridge these paradigms in
many diseases, allowing integration of human genetics and cell
biology within the in vivo setting. Perhaps it is in Neurology and
Neurodegeneration, however, where zebrafish have special promise
to make contributions. Four special advantages of zebrafish to
Neurology are worth highlighting here: efficient in vivo tests for
genetic interactions, conserved cellular architecture of the CNS, the
ability to create genetically mosaic animals, and tractable behaviour
and electrophysiology to assess experimental interventions.
Linkages between Neurodegenerative diseases that were once
considered distinct are being recognized and celebrated as a path
forward to understanding the molecular basis of disease spread
through the CNS. One example includes interactions between
notorious proteins in Alzheimer Disease (AD) and Prion Disease,
such that the Prion Protein may now be viewed as a receptor for Aβ
oligomers. The abundance of such interactions between Neurological
Diseases is growing, as protein interactome studies identify many
players in common, compelling the field to integrate an increasing list
of genetic interactions into their models. The ability to efficiently
knockdown and overexpress gene products in vivo is propelling
zebrafish to prominence at a rate commensurate with the complexity
of these genetic interactions.
On the other hand, various researchers have recently noted
intriguing parallels between prion diseases and the protein misfolding
in other neurological diseases. In particular, questions are being raised
as to the ability of misfolded proteins (including Tau and Aβ) to induce
the misfolding of normally folded protein, leading to a prion-like
domino effect and accumulation of protein dysfunction/aggregation
that are hallmark disease pathologies. Consistent with such findings is
the apparent spreading of disease pathology from a nucleation centre
through the CNS during clinical disease progression. Other celebrated
potential prion-like proteins include α-synuclein, polyglutamine
proteins and SOD1 misfolding in ALS.
Attention toward this prionoid nature of various neurological
diseases is substantial, in large part because it identifies a novel
therapeutic target: the mechanism of disease spreading. Unfortunately, the field knows remarkably little about disease spreading, with
concepts in high-profile reviews centering on Tunneling Nanotubes
and exosome or synaptic release. Perhaps this fundamental lack of
knowledge regarding disease spread through the CNS is the
consequence of the tools available to neurologists and neuroscience
researchers. The mechanisms underlying AD, TSEs, and ALS have
☆ This article is part of a Special Issue entitled Zebrafish Models of Neurological
Diseases.
0925-4439/$ – see front matter © 2010 Published by Elsevier B.V.
doi:10.1016/j.bbadis.2010.11.004
benefitted primarily from biochemistry/ biophysics of protein
aggregation, cell culture work and mouse models; These paradigms
are challenging to implement in a fashion that tells us how disease in
one portion of the CNS spreads to another. Zebrafish are positioned
brilliantly to fill this gap, not only because of its efficiency as a genetic
and neuroscience model, but because zebrafish have a long history as
a platform for assessing non-cell-autonomous effects of protein
perturbations in the in vivo setting. Thus it is common for zebrafish
biologists to ask if changing gene abundance or character in one group
of neurons is able to affect the fate and function of neighboring cells.
Creative and ambitious deployment of this ability will complement
established tools to promise a deep understanding of how disease
spreads between cells. A tantalizing side-benefit is the ability to
deploy the fish in high-throughput screens of protein and small
molecule effectors that represent therapeutic targets.
Finally, it is important to underline the cellular context of disease
spreading during Neurolodegeneration. As disease spreads from cell
to cell, the molecular basis of this spread is fundamentally influenced
by the cellular relationships. Importantly, The zebrafish has an
architecture of CNS cells that is conserved with that of humans.
Vasculature, blood-brain-barriers, and ventricles are all present from
a young age, permitting tissue-level spread of molecules fundamental
to disease. Perhaps more importantly, the cells of the CNS develop in
their normal environment, becoming polarized, interconnected and
ensheathed in radial glia to form ion sinks. The cover image selected is
meant to highlight that this organization exists even in young
embryos. Certainly our understanding of hallmark synaptic dysfunction during Neurodegeneration must develop from paradigms where
synapses are formed and maintained with their typical glial partners
intact. So although zebrafish is not yet as advanced in its toolkit as
Drosophila, nor as tractable as cell culture, it has a relevant cellular
architecture and genetic efficiency that make the paradigm a very
attractive in vivo complement to studies in mouse models.
This issue of BBA: Molecular Basis of Disease has invited reviews
from leading experts to consider the utility of zebrafish to Neurology.
These experts span from Neuroscientists who study fundamentals of
zebrafish Biology, through to clinical Neurologists who recognize the
zebrafish as a relevant and potent paradigm to bring their hypotheses
to bedside in as timely a manner as possible. The first contributions
have sought to critically analyze previous work regarding particular
Neurodegenerative diseases and how zebrafish have contributed to
this understanding. The subsequent contributions consider technical
opportunities and challenges of using zebrafish genetics and behavioural outputs in Neurology, and the issue ends with the promise
provided by zebrafish to understand regeneration of the CNS for
repair.
I am grateful to each of these contributors, reviewers, and the
BBA staff for their hardwork in communicating these important and
stimulating ideas.
334
Editorial
Allison is a neuroscientist with a special interest in
understanding processes of neurodegeneration and neural
regeneration. Towards this goal, Dr. Allison's research
integrates molecular biology, biochemistry, proteomics,
and electrophysiology to further develop zebrafish as a
potent animal model. Particular emphasis of his research
has been developing fish as effective models of neurodegenerative disease. He completed a Doctoral Program in
2004 with Professor Craig Hawryshyn at the University of
Victoria, Canada, where he developed a unique model of
neuronal degeneration and regeneration. This work was
funded by fellowships from the Alzheimer Society of
Canada and the Canadian Institute of Health Research
Institute of Aging. Dr. Allison went on to complete an
NSERC Post-Doctoral fellowship at the University of Michigan in the laboratory of
Collegiate Professor Pamela Raymond. Dr. Allison's work at the University of Michigan
created transgenic and mutant zebrafish has revealed genes important in the
generation, positioning, and regeneration of neurons. Dr. Allison joined the Centre
for Prions and Protein Folding Diseases as an Assistant Professor at the University of
Alberta, Canada in 2008. His appointment is through the Department of Biological
Sciences in the Faculty of Science, with a cross-appointment in the Department of
Medical Genetics in the Faculty of Medicine and Dentistry. Dr. Allison is contributing to
expanding the largest zebrafish facility in Western Canada and also maintains active
research in the field of retina development and regeneration. His current research
efforts focus on creating transgenic and mutant zebrafish to address hypotheses of
prion protein's normal function and to dissect the role of various prion protein
domains. Other efforts use similar methods to examine the amyloid hypothesis of
Alzheimer Disease. His Recent work is funded by a Recruitment Grant from PrioNet
Canada and the Alberta Prion Research Institute and by a Young Investigators Award
from the Alzheimer Society of Canada.
W. Ted Allison
University of Alberta, Centre for Prions and Protein Folding Diseases,
Departments of Biological Sciences & Medical Genetics, Edmonton,
Alberta, Canada T6G 2E9
E-mail address: [email protected].
Tel.: +1 780 492 4430; fax: +1 780 492 9234.
10 November 2010
Available online 27 November 2010
Biochimica et Biophysica Acta 1812 (2011) 335–345
Contents lists available at ScienceDirect
Biochimica et Biophysica Acta
j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / b b a d i s
Review
Zebrafish models for the functional genomics of neurogenetic disorders☆
Edor Kabashi a,b, Edna Brustein b, Nathalie Champagne a,b, Pierre Drapeau b,⁎
a
b
Center for Excellence in Neuromics, CHUM Research Center and the Department of Medicine, Université de Montréal, Montréal, QC, Canada
Department of Pathology and Cell Biology and Groupe de recherche sur le système nerveux central, Université de Montréal, Montréal, QC, Canada
a r t i c l e
i n f o
Article history:
Received 4 December 2009
Accepted 22 September 2010
Available online 29 September 2010
Keywords:
Zebrafish
Human diseases
Neurogenetic diseases
Neurodegenerative disorders
Neurodevelopmental diseases
Animal models
Genomics
Human genetics
Trangenic animals
a b s t r a c t
In this review, we consider recent work using zebrafish to validate and study the functional consequences of
mutations of human genes implicated in a broad range of degenerative and developmental disorders of the
brain and spinal cord. Also we present technical considerations for those wishing to study their own genes of
interest by taking advantage of this easily manipulated and clinically relevant model organism. Zebrafish
permit mutational analyses of genetic function (gain or loss of function) and the rapid validation of human
variants as pathological mutations. In particular, neural degeneration can be characterized at genetic, cellular,
functional, and behavioral levels. Zebrafish have been used to knock down or express mutations in zebrafish
homologs of human genes and to directly express human genes bearing mutations related to
neurodegenerative disorders such as spinal muscular atrophy, ataxia, hereditary spastic paraplegia,
amyotrophic lateral sclerosis (ALS), epilepsy, Huntington's disease, Parkinson's disease, fronto-temporal
dementia, and Alzheimer's disease. More recently, we have been using zebrafish to validate mutations of
synaptic genes discovered by large-scale genomic approaches in developmental disorders such as autism,
schizophrenia, and non-syndromic mental retardation. Advances in zebrafish genetics such as multigenic
analyses and chemical genetics now offer a unique potential for disease research. Thus, zebrafish hold much
promise for advancing the functional genomics of human diseases, the understanding of the genetics and cell
biology of degenerative and developmental disorders, and the discovery of therapeutics. This article is part of
a Special Issue entitled Zebrafish Models of Neurological Diseases.
© 2010 Elsevier B.V. All rights reserved.
1. Introduction
Zebrafish are used as a model for a wide variety of human diseases,
including cancer, cardiovascular disorders, angiogenesis, hemophilia,
osteoporosis, diseases of muscle, kidney and liver, and, last but not the
least, disorders of the central nervous system. In recent years,
zebrafish have been used to study neurodegenerative disorders.
Here we review zebrafish models of CNS diseases with an emphasis on
studies of degenerative neurological diseases. We also discuss our
recent efforts in extending these approaches to psychiatric disorders
of development. Although unlikely to yield accurate models for
complex psychiatric disorders, genetic insights from studies of
zebrafish are pertinent in helping to identify relevant molecular and
cellular mechanisms of human pathology and, at this level, promise
insight to the development of therapeutics. In particular, the in vivo
biological validation of variants identified in human genetic and
genomic studies provides an important step in defining the
pathological nature of mutations.
☆ This article is part of a Special Issue entitled Zebrafish Models of Neurological
Diseases.
⁎ Corresponding author. Department of Pathology and Cell Biology, Université de
Montréal, C.P. 6128, Succ. Centre-ville, Montréal, Québec H3C 3J7, Canada. Tel.: +1 514
343 7087; fax: +1 514 343 5755.
E-mail address: [email protected] (P. Drapeau).
0925-4439/$ – see front matter © 2010 Elsevier B.V. All rights reserved.
doi:10.1016/j.bbadis.2010.09.011
Apart from being a vertebrate with common organs and tissues
such as a brain and spinal cord with conserved organization, the
attractiveness of zebrafish as a model lies in its biology and genetics.
Zebrafish have large clutches of externally fertilized and transparent
eggs, which develop rapidly and in synchrony, with neurogenesis
starting around 10 h post-fertilization (hpf), synaptogenesis and the
first behaviors around 18 hpf and hatching around 52 hpf. Within
1 day of development, many pertinent features of the CNS appear and
can be studied in relatively simple populations of identifiable neurons.
For example, the spinal cord is divided into 30 somites each
containing fewer than a dozen cell types that form relatively simple
circuits. The rapid development of the zebrafish embryo allows for the
study of embryonic-lethal mutations as larvae can survive on their
yolk for up to a week, allowing studies of gene expression and
function throughout early developmental stages.
In addition to its advantageous biology, the second major
advantage of the zebrafish as a model is the simplicity and
effectiveness of manipulating gene expression for cell biological
observations in living embryos with relevance to human pathology.
The zebrafish genome is sequenced and, although not completely
annotated, over 80% of gene structures are available and show a high
degree of synteny (nearest neighboring genes on the chromosomes)
across vertebrate species as well as 50–80% homology with most
human sequences. Homologs for most human genes can be identified
in silico and, as described below, can be manipulated by gain- and loss-
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E. Kabashi et al. / Biochimica et Biophysica Acta 1812 (2011) 335–345
of-function approaches. However, the identification of a human
homolog in zebrafish is complicated by the fact that a large number
of genes are duplicated. This phenomenon is explained by whole
genome duplication that occurred during evolution [1]. The amino
acid identity compared to the human gene is always higher for one of
the two genes duplicated, indicating that the duplication appeared
before the species divergence. Therefore, when a gene is knocked
down, it is important to consider that the duplicated gene can
compensate for the loss of function of the targeted homolog. Also, the
function could be partitioned between the two genes or simply lost for
one of the genes. Comparative sequence analysis allows identifying
the zebrafish homolog of a human gene. Three major genome
browsers, NCBI (www.ncbi.nlm.gov), Ensembl (www.ensembl.org)
and UCSC (/genome.ucsc.edu/), can be use to search homologous gene
sequences. In many cases, the homologous gene predictions can be
found directly from either HomolGene (NCBI) or Orthologue Prediction
(Ensembl). We have found that about half of the hundred or so human
synaptic genes on chromosome X (described later) have a predicted
fish ortholog, compared to ~ 30% in Drosophila and Caenorhabditis
elegans using the same databases. If the homologous gene is not
annotated or if the transcript prediction is not complete, a BLAST
search using nucleotide or protein sequence against the whole
genome assemblies (especially scaffolds based on BAC sequencing)
can allow the identification of homologous genes. TargetP and SignalP
algorithms (www.cbs.dtu.dk/services/) can be used for signal peptide
and cleavage site prediction. An additional phylogenetic analysis and
multiple alignments of protein sequences (Clustal W) help to confirm
the orthology. The syntenic regions can also be compared using Multi
Contig View (Ensembl). For additional information, the Sanger
Institute (www.sanger.ac.uk) has excellent resources about zebrafish
sequence analysis. The sequence identity between zebrafish genes
and their human homologs is higher for ubiquitously expressed genes
than for highly specific genes such as some synaptic genes. The
divergence is mainly found in domains important for membrane
targeting as signal peptides and often in intracellular domains that
contain signaling motifs. Nonetheless, in general, the exon/intron
boundaries and regulatory sequences as well as many other types of
functional domains are evolutionarily conserved between the species.
As a simple starting point, the zebrafish homologs can be targeted for
knockdown by injection of selective anti-sense morpholino oligonucleotides (AMOs) [2]. ZFIN (zfin.org) has the principal database about
AMO gene knockdown and phenotype. The parameters to consider in
AMO design are the same as that for any oligonucleotide molecule: CG
content, stem and loop structure, and the oligo length [3]. Gene-Tools
Inc. (see www.gene-tools.com) offers an AMO design service to the
researchers purchasing their morpholinos. We recommend always
checking if the AMO can bind to off-target sequences by doing a BLAST
against NCBI or Ensembl database. AMOs can be designed to target the
ATG start site to block translation initiation or they can be used to block
splice sites to produce truncated transcripts. The latter have the
advantage of sparing maternal transcripts and permit RT-PCR amplification and quantification of the gene knockdown, which is particularly
useful when an antibody is unavailable. Further, the splice AMO can be
used to mimic splicing defects, nonsense or truncation mutations
related to disease. AMOs resist degradation by ribonucleases and are
stable for several days, and in our hands when adequately controlled,
they usually generate selective phenotypes (discussed below). However, AMOs must be injected intracellularly as they are more of a
pharmacological than a genetic tool, although they are more effective in
gene knockdown in zebrafish than interfering RNAs. Intracellular
injection is feasible in the zebrafish embryo during the first 1–2 hpf as
the blastomeres are open to the yolk prior to the 4th cell division. Freehand injection into the yolk near the blastomere border (Fig. 1) allows
for the injection of a hundred eggs or so in 1–2 h, yielding a sufficient
number of treated embryos to permit statistical analyses of phenotypes
and thus providing a genetic read-out often only 1 day later.
Replacing targeted zebrafish genes with human genes can for
many genes be done as simply as by injection of human mRNA
simultaneously with the AMO as the latter is designed to specifically
target the zebrafish and not the human sequence. We suggest using
the zebrafish homologous cDNA if your gene of interest has a highly
localized expression pattern like that of synaptic genes. Otherwise,
human, mouse, or rat cDNA can be used and easily obtained
commercially. NCBI gives the link to cDNA clones for certain genes.
The gene can be subcloned in expression vectors like pCS2 [4] or
pcGlobin [5] to synthesize the 5′-capped mRNA that will be injected in
embryos. The pcGlobin vector has the advantage to have 5′UTR and 3′
UTR from Xenopus, and we also note that pcGlobin gives a better yield
of mRNA. The cDNA can be tagged to MYC, flag, or HA epitopes to
follow the expression in fish. However, large DNA constructs such as
plasmids often yield chimeric expression patterns in limited numbers
of cells presumably as not all dividing progenitors retain the large
constructs. About half of the human genes we have tested permit
partial rescue of knockdown phenotypes, which then allows for
comparison between wild-type human mRNA and mRNA bearing
disease-related mutations. Thus, in the space of a few weeks, each
human variant can be validated in knockdown embryos once a clear
phenotype is recognized. Finally and as described below, recent
transgenic technologies permit the creation of stable, targeted (cellspecific), and inducible lines for better spatially and temporally
controlled and more detailed analyses of transgenic function.
2. Degenerative brain disorders
Degenerative disorders of the brain, including Alzheimer's, Parkinson's, and Huntington's diseases, are the first neurogenetic diseases
to have been studied using zebrafish [6,7]. Amyloid-beta plaques are
the strongest biological marker of Alzheimer's disease and the
production of amyloid-beta is regulated by the presenilins 1 and 2
(PS1, PS2). Surprisingly, wt zebrafish zPS1 was found to promote
aberrant amyloid-beta42 secretion when expressed in HEK 293 cells,
like the abnormal secretion associated with human PS1 mutations, and
mutation of one residue in zPS1 abolished this activity [8]. Truncation
of zPS1 due to loss of exons 8 and 9 upon injection of splice acceptor
AMOs had a dominant-negative effect by increasing PS1 expression [9]
and possibly the expression of other Alzheimer's-related genes [10].
Zebrafish zPS2 is expressed at later stages and is regulated by zPS1
[11,12], but its role in amyloidogenesis in zebrafish is unclear.
Zebrafish possess two homologues of amyloid precursor protein
(APP) with 63–66% homology to their human counterpart [13], and
their double knockdown causes a convergence–extension developmental defect that is rescued by human APP mRNA but not by a
Swedish mutation related to Alzheimer's disease [14]. The zebrafish
APPs possess a functional gamma-secretase complex to produce
amyloid-beta [15] and inhibition of gamma-secretase [16] or knockdown of Pen-2 [17], another member of the presinilin complex, blocks
Notch signaling to produce a severe neurogenic phenotype. Early
studies also examined the tau protein in zebrafish as tau is present in
human neurofibrillary tangles, mutations of tau are implicated in
dementia and tau may also contribute to Alzheimer's disease. The
zebrafish studies have shown that expression of human tau (driven
transiently upon injection of a construct with a neural-specific variant
of the GATA2 promoter) results by 2 days in disruption of cytoskeletal
structure, tau trafficking, and hyperphosphorylated fibrillar tau
staining [18]. More recently, stable transgenic zebrafish expressing
mutated P301L human tau and a fluorescent reporter (driven panneuronally from the HuC promoter) have permitted in vivo imaging of
defective axonal growth, tau hyperphosphorylation, and the screening
of novel therapeutic molecules [19]. These recent developments with
zebrafish tau and amyloid open the possibility of further screens for
novel therapeutics that, e.g., decrease the hyperphosphorylation of tau
E. Kabashi et al. / Biochimica et Biophysica Acta 1812 (2011) 335–345
337
Fig. 1. Rescue of knockdown phenotypes by human mRNA allows for the validation of disease-related variants.
in HuC transgenics [19] or amyloid-beta load in amyloid transgenics
[20,21].
Research into Parkinson's disease has also used zebrafish as a
model [22]. Zebrafish embryos treated with MPTP, a neurodegenerative chemical that reproduces some of the effects of idiopathic
Parkinson's disease in mammalian models, demonstrated a loss of
dopaminergic neurons, which could be rescued using the monoamine
oxidase-B inhibitor deprenyl [23–25]. Half a dozen genes have been
identified in Parkinson's disease [26], and several of these have been
studied to date in zebrafish. For example, in zebrafish, the mRNA for
the ubiquitin processing gene UCH-L1 was detected by 1 day in the
ventral region of the midbrain and hindbrain and in the ventral
diencephalon where it was co-expressed with markers of dopaminergic neurons [27]. The zebrafish DJ-1 protein has high (N80%)
homology with human and mouse DJ-1 (of unknown function) and is
expressed throughout the body [28]. Knockdown of DJ-1 in the
zebrafish did not affect the number of dopaminergic neurons, but
zebrafish embryos were more susceptible to oxidative stress and had
elevated SOD1 levels, while simultaneous knockdown of DJ-1 and p53
caused dopaminergic neuronal loss. Recently zebrafish Parkin,
another ubiquitin processing gene, was identified (62% homology
with human PARKIN) and AMO abrogation of its activity leads to a
significant decrease in the number of ascending dopaminergic
neurons in the posterior tuberculum, which is homologous to the
substantia nigra in humans [29]. Screens for compounds promoting
(like MPTP) or preventing (such as caffeine) the Parkinsonian
phenotype in zebrafish [30] should now be facilitated by the
availability of an enhancer trap line expressing GFP from the vesicular
monoamine transporter 2 promoter [31].
In zebrafish, the huntingtin (Htt) gene implicated in Huntington's
disease has a predicted 70% identity with the human protein [32] and
its knockdown disrupts a number of features [33] and causes massive
neuronal apoptosis due to reduced BDNF expression by 1 day of
development but not earlier [34]. It was recently observed that
injecting mRNA coding for the N-terminal fragment of Htt with
different length polyQ repeats led to developmental abnormalities
and apoptosis in the embryos as early as 1 day later [35]. Embryos
expressing a Q102 mRNA [35] or a Q56 plasmid [36] developed
inclusions in the cytoplasm. These Huntington's disease models are
being used to screen for novel therapeutics that could prevent
aggregate formation and clearance, such as anti-prion compounds
[35], or embryonic death, such as blockers of autophagy [37].
Another CNS disorder that has been studied recently using
zebrafish is epilepsy [38] as clonus-like seizures are induced by
convulsant agents [39–42], which has permitted forward mutagenesis
screens and the isolation of seizure-resistant mutants [43]. Zebrafish
are now being used to screen for compounds that suppress seizures
[44]. Furthermore, injection of mRNA with a mutation of the human
PRICKLE1 gene implicated in epilepsy disrupts normal function when
overexpressed in zebrafish [45], demonstrating the usefulness of
zebrafish for validating mutations of human genes causing brain
disorders.
Our group has recently collaborated on the discovery of the
MEDNIK syndrome, a rare and severe autosomal recessive neurocutaneous disorder manifested by mental retardation, enteropathy,
deafness, neuropathy, ichthyosis, and keratodermia that is often lethal
[46,47]. Affected individuals bear an A to G mutation in acceptor splice
site of exon 3 of the AP1S1 gene, which leads to a premature stop
codon (Fig. 2A [47]). The AP1S1 gene encodes the small subunit σ1A of
the first (AP-1) of four ubiquitous clathrin adaptor proteins [48–51].
Each one of the adaptor protein complexes is assembled from four
subunits, and the σ subunit, the one affected in the MEDNIK
syndrome, is part of the AP complex core and is suggested to
contribute to its stabilization. Also, together with the μ subunit, the σ
subunit is possibly involved in protein cargo selection [50,51]. To
demonstrate that the mutation in the human AP1S1 gene indeed alters
the biological function of this gene and underlies the MEDNIK
syndrome, we knocked down the function of the homologous Ap1s1
gene in zebrafish. The zebrafish Ap1s1 protein shares 91% identity
with the human protein. To inhibit zebrafish Ap1s1 mRNA translation,
two types of AMOs were designed (Fig. 2A [47]). The first AMO
targeted the N-terminal, while the second AMO targeted the acceptor
splice site of intron 2 (Fig. 2A), imitating the mutation found in
MEDNIK patients. Both AMOs caused similar, severe morphological
and behavioral deficits. The 48 hpf KD larva was smaller and had
reduced pigmentation compared to the wild-type (WT) larva. Further,
blocking Ap1s1 translation caused disorganized skin formation in
general, particularly affecting fin morphology (Fig. 2B; for further
information on the epithelial disorders, see Ref. [47]). In addition to
the morphological deficits, the 48 hpf KD larva responded abnormally
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E. Kabashi et al. / Biochimica et Biophysica Acta 1812 (2011) 335–345
to touch by coiling the tail instead of swimming away. The
compromised touch response of the KD larva could be attributed to
a massive reduction (by half) in the spinal neuron population,
specifically due to a loss in the interneuron population. The specificity
of the AMO effect was further confirmed both by Western blotting and
immunolabeling analysis (Fig. 2C and D). Most importantly, we could
rescue the knockdown phenotype by co-injecting the AMO with
human AP1S1 wild-type mRNA (Fig. 2B and D), which is not targeted
by the Ap1s1 AMO. In contrast, co-injection of AMO with mutated
human AP1S1 mRNA missing exon 3 (Fig. 2A) failed to rescue the skin
and behavioral deficits, suggesting loss of function of this truncated
form of protein (Fig. 2C) and confirming the pathogenic nature of the
mutation found in MEDNIK patients. Further, co-injection of AMO and
an additional human AP1S1 mRNA isoform found in MEDNIK patients,
containing a cryptic splice acceptor site located 9 bp downstream of
the start of the 3rd exon, rescued the phenotype (Fig. 2A). The latter
result suggests that the predicted in frame protein, lacking only 3
amino acids (Fig. 2C), is functional and may explain the viability of the
MEDNIK patients. However, the low expression level of the alternatively spliced RNA (~10%) is insufficient to sustain normal development and function, further emphasizing the importance of AP1S1 in
normal development. Our in vivo zebrafish study of Ap1s1 function
revealed its importance for appropriate neurogenesis and skin
development. Accordingly, we could speculate that MEDNIK, a novel
neurocutaneous syndrome, is caused by impaired development of
various neural networks in the spinal cord and in the brain, explaining
the multifunctional human deficits such as ataxia, peripheral
neuropathy, and mental retardation, which are concomitant with a
perturbation in epithelial cell development in the skin and in the
digestive system. We speculate that these generalized effects of AP1S1
Fig. 2. Evaluating a mutation found in human AP1S1 gene (MEDNIK syndrome) using zebrafish. (A) An illustration of the human AP1S1 gene to show the location of the mutation
found in MEDNIK patients. An A to G mutation in the acceptor splice site of exon 3 resulted in skipping of this exon and led to a premature stop codon. The use of an alternative cryptic
splice site located 9 bp downstream of the start of the 3rd exon resulted in mRNA lacking 9 bp. The location of the two different morpholinos targeting either ATG or exon 3 acceptor
splice site of the zebrafish ap1s1 gene is indicated in green. The inset on the right depicts the clathrin adaptor protein 1 (AP-1) and its 4 subunits. It is the small subunit σ1 (red),
which is mutated in MEDNIK patients. (B) Ap1s1 knockdown (KD) in zebrafish using morpholino oligo nucleotides targeting the N-terminal or the splice site (splice site, intron 2)
resulted in a similar characteristic morphological phenotype (48 h post-fertilization), which is rescued by co-injection of the wild-type human AP1S1 (rescue + WT HmRNA) or the
mRNA lacking 9 bp (rescue + −9 bp HmRNA), but not by the mutated human mRNA missing exon 3 (rescue + −exon3HmRNA). (C) Illustration of the predicated human AP1S1
proteins: normal protein containing 55AA (WT, black); truncated protein containing 19AA (red), the result of skipping exon 3; and a protein missing 3AA, a result of the alternative
splicing (blue). Western blot analysis of Ap1s1 proteins from human (left) and zebrafish (right) to show that Ap1s1 protein is hardly expressed in MEDNIK patients and in ap1s1
knockdown (KD) zebrafish larva. To normalize the Western blot analysis, proteins extracted from WT, KD, and CTRL larvae were incubated with anti-actin. CTRL = control,
WT = wild type. (D) Localization of Ap1s1 in skin cells of zebrafish wholemounts is illustrated using anti-Ap1s1 antibody immunofluorescence. Cell membrane (polygonal) and a
well-defined perinuclear ring can be nicely observed in both normal (WT) and rescued larvae (rescue + WTHmRNA), whereas only a residual and diffuse staining could be observed
in knockdown (KD) larvae (modified from Ref. [47]).
E. Kabashi et al. / Biochimica et Biophysica Acta 1812 (2011) 335–345
mutation are due to a widespread deficit in vesicular transport and
protein sorting.
3. Degenerative spinal cord disorders
Zebrafish have also been proven to be particularly effective as a
model for motor dysfunction [52]. Our group has worked extensively
on motor neuron diseases, including the most common of these
disorders, amyotrophic lateral sclerosis (ALS). The advantage of
studying motor neuron diseases in zebrafish consists in the rapid
development of the spinal cord and allowing analysis of motor neuron
branching patterns as early as 24 hpf. In addition, responses to touch
and swimming can be monitored following hatching around 48 hpf
[53]. SOD1 (ALS1) is known to cause ALS in 10–20% of familial ALS
cases mainly through an autosomal dominant toxic gain of function
and has 70% amino acid identity to the zebrafish homologue.
Overexpression of mutant SOD1 in zebrafish leads to short motor
axons with premature branching [54]. Interestingly, several other
genes implicated in motoneuron degeneration show a similar
phenotype when tested in zebrafish. Alsin (ALS2) is the gene mutated
in juvenile ALS through autosomal recessive mode of action, which
leads in most cases to protein truncation. Corresponding to this, in
zebrafish embryos, knockdown of Alsin (61% homology) by AMO
causes shortening of motor axons and also loss of neurons in the
spinal cord [55]. ELP3 was identified through association studies
performed in ALS patients and in a Drosophila screen for genes
important for neuronal survival [56]. Knockdown of this gene (ELP3)
in zebrafish caused increased branching and shortened motor axons
[56]. Recently, TDP-43 was found enriched in inclusion bodies from
spinal cord autopsy tissue obtained from ALS patients [57]. Moreover,
about 30 mutations have been identified in a considerable number of
ALS patients, suggesting that this protein plays an important role in
disease pathogenesis [58,59]. The zebrafish ztardbp gene product
(TDP-43) has 73% homology to the human protein. Knockdown of
TDP-43 (tardbp) in zebrafish embryos led to motor neuron branching
and motility deficits (Fig. 3). This AMO phenotype was rescued by coexpressing human wild-type (WT) TDP-43 (which is not targeted by
the AMO) (Fig. 3). However, three ALS-related mutations of human
TDP-43 identified in both in SALS and FALS cases (A315T, G348C,
A382T) failed to rescue these phenotypes, indicating that these
mutants are pathogenic (Fig. 3). A similar phenotype was observed
when mutant (but not wt) TDP-43 was overexpressed, with the
G348C mutation being the most penetrant (Fig. 3). These results
indicate that TDP-43 mutations cause motor defects, suggesting that
both a loss as well as a gain of function may be involved in the
molecular mechanism of pathogenesis [125]. Finally, it is interesting
to note that a number of groups generated ALS2 knockout mice yet
failed to observe motor deficits associated with motor neuron
degeneration and concluded that ALS2 was not an important gene
for ALS [55,60]. However, the first three exons of ALS2 were left intact
in these mice. We found that the motor phenotype in zebrafish upon
ALS2 KD could be partially rescued upon overexpression of the
alternative transcript of the first three exons of ALS2, indicating that
complete disruption could in fact be pathogenic in ALS [55]. This
nicely illustrates the usefulness of zebrafish knockdown models for
more complete disruption of gene function in some circumstances.
Zebrafish have been also widely used to study the functional role
of gene mutations in another motor neuron disease, hereditary spastic
paraplegia (HSP). Over 30 loci are known for this disorder, and at least
20 genes have been identified to cause spastic paraplegia [61]. Our
group identified missense variants in the KIAA0196 gene at the SPG8
locus and validated these mutations using the zebrafish model (87%
homology to the human protein) [62]. Knockdown of the zebrafish
homolog of KIAA0196 gene caused a curly-tail phenotype coupled
with shorter motor axons. This phenotype could be partially rescued
by the human WT KIAA0196 but not the two mutants identified in
339
HSP patients [62]. Recently, SLC33A1 was identified as the gene
responsible for SPG42 [63]. Knockdown of this gene in zebrafish
embryos using AMO caused a curly-tail phenotype and defective axon
outgrowth from the spinal cord [63]. Finally, knockdown of spastin
(SPG4) caused widespread defects in neuronal connectivity and
extensive CNS-specific apoptosis [64].
Spinal muscular atrophy (SMA) is an autosomally recessive motor
neuron disorder caused by mutations in the survival motor neuron
gene (SMN1). In a series of publications, Beattie's laboratory has
demonstrated that knockdown of the SMN1 in zebrafish embryos
(49% homology to human protein) causes motor axon outgrowth and
pathfinding defects in early development [52,65]. Further, in a series
of elegant rescue experiments, this group was able to show that a
conserved region in exon 7 of the SMN1 gene (QNQKE) is critical for
axonal outgrowth and that the plastin 3 (PLS3) may be important for
axonal outgrowth since overexpression of plastin 3 rescued the
phenotype caused by SMN1 knockdown [66,67]. Finally, transgenic
expression of homozygous deletion found in SMA patients that leads
to truncated SMN1 protein as well as knocking out the Smn1 gene in
zebrafish caused deficits in the neuromuscular junction formation and
death at the larval stage [68].
4. Developmental disorders
Although this developmental model is proving useful in the
study of late-onset degenerative diseases, zebrafish are just starting
to be used in the study of developmental brain diseases. Interesting
speculations have been made on the potential of zebrafish for
modeling developmental psychiatric disorders such as autism [69],
but the lag in advancing these models is perhaps because the
clinical phenotypes are so subtle and specific to humans suffering
from disorders such as autism, schizophrenia, non-syndromic
mental retardation, and others that zebrafish may appear as a
doubtful model. So far (and very recently), the only genes linked to
schizophrenia to be studied in zebrafish using AMO methods are
DISC1 and NRG1 [70]. The receptor tyrosine kinase Met has been
implicated in cerebellar development and autism, and knockdown
of either Met alone or both of its Hgf ligands (two genes in
zebrafish) together using ATG or splice junction AMOs results in
abnormal cerebellar and facial motoneuron development [71].
However, neither the human homologs of these genes nor their
disease-related mutations have been tested in zebrafish. Our new
ongoing genomics approach, based on large-scale re-sequencing of
human synaptic genes in patients suffering from some of these
disorders, is identifying mutations that can be validated, if not
exactly modeled, in zebrafish.
As a part of our “Synapse to Disease” project, we are using a
combination of AMO knockdown and gene overexpression to
functionally validate novel synaptic gene mutations identified in
large cohorts of patients with autism, schizophrenia, or nonsyndromic mental retardation. Hundreds of synaptic genes were
selected based on published studies and databases. Many genes were
chosen because they are X-linked [72] as there are many more
affected males than females in autism [73]. Other genes that were
selected are those of the glutamate receptor complex because of their
association to neurodevelopmental diseases [74]. So far, the exonic
regions of over 400 genes have been sequenced, and 15 de novo
mutations (present in the patients and not their parents) were
genetically validated (manuscript submitted). We have identified
deleterious rare de novo mutations in Shank3, IL1RAPL1, NRXN1, and
KIF17 genes, and all of these genes have orthologs in zebrafish. We
have identified a de novo mutation in the Shank3 gene in a patient
with autism [75] as Shank3 deletions or duplications were previously
identified in patients with autism [76]. Shank3 encodes a scaffolding
protein found in excitatory synapses directly opposite to the presynaptic active zone. The Shank proteins link ionotropic and
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E. Kabashi et al. / Biochimica et Biophysica Acta 1812 (2011) 335–345
Fig. 3. Motor phenotype of gain and loss of function of TDP-43 in zebrafish. (A) A schematic representation of TDP-43 protein showing the functional domains, localization of FLAG
(3 kDa) and myc tags (12 kDa), and sites where the G348C mutation found in ALS patients was introduced by site-directed mutagenesis. (B) Locomotor phenotype. Zebrafish
embryos develop a touch-evoked escape response as seen when embryos are injected with WT human TDP-43 RNA. Expression of TDP-43 G348C RNA causes a deficiency in the
touch-evoked response. A similar phenotype is observed when the zf tdp-43 expression was knocked down using a specific AMO. This phenotype was rescued with expression of WT
but not G348C RNA. (C) Motor axonal deficits. The behavioral (motor) phenotype was selectively associated with a shortening of the axon and premature branching in motor
neurons (see G348C TDP-43 and tdp-43AMO) (modified from Kabashi et al., accepted at Hum. Mol. Genet.).
metabotropic glutamate receptor complexes together and to the
cytoskeleton to regulate the structural organization of dentritic
spines. The splice mutation identified in the Shank3 gene would
result in a truncated protein lacking the Homer, Cortactin, and SAM
domains important for spine induction and dentritic targeting [77].
Zebrafish have two Shank3 homologous genes (shank3a and shank3b).
The identity to human protein is overall 65% and 62% for shank3a and
shank3b, respectively, but when we compare every individual
functional domain to the human one, the amino acids identity reach
80% and is over 90% for the PDZ domain. The phenotype of Shank3
knockdown zebrafish was not reported in the literature yet, and we
observed major motility deficits that could be rescued by the rat
mRNA but not by some of the mutations [126].
We also found de novo mutations in IL1RAPL1 in autistic patients
[78]. IL1RAPL1 and 2 are plasma membrane proteins that belong to a
class of the interleukin-1 receptor family characterized by a 150-aa Cterminal domain that interacts with Neuronal Calcium Sensor-1 (NCS1) [79,80]. A recent study from [81] used IL1RAPL1-deficient mouse to
show that IL1RAPL1 controls inhibitory network during cerebellar
development. In one patient, a de novo frameshift mutation in
IL1RAPL1 causes a premature stop of the translation (I367fs) resulting
in a truncated protein, lacking the intracellular TIR domain and the Cterminal domain interacting with NCS-1 [78]. Moreover, a large
deletion of exons 3 to 7 of IL1RAPL1 in three brothers with autism
and/or non-syndromic mental retardation was also identified in our
study. Mutations resulting in deletion of the TIR and the C-terminal
domains were previously identified in patients with non-syndromic
mental retardation [82,83], suggesting that those domains are
important to the protein function. Two genes encode IL1RAPL1 in
zebrafish (il1rapl1a and il1rapl1b) and share 69% and 75% identity,
respectively, with human protein. The divergence is mainly in the
signal peptide (il1rapl1a: 42%, b: 47%). Recently, a study using
zebrafish il1rapl1a showed that the mRNA is constantly expressed
during early embryonic development, indicating that it is maternally
provided [84]. In this study, they also showed that knockdown of the
other gene, il1rapl1b, using an ATG AMO caused a selective loss of
synaptic endings in olfactory neurons and that il1rapl1b C terminus
and TIR domains regulate the synaptic vesicles accumulation and
morphological remodeling of axon terminals during synapse formation [84]. These observations suggest that il1rapl1b may be essential
for synapse formation and indicate how a loss-of-function IL1RAPL1
mutant could affect brain neurodevelopment and its possible
association to autism and non-syndromic mental retardation. To
determine more specifically the function of IL1RAPL1 in the context of
schizophrenia, we knocked down the expression of zebrafish il1rapl1a
with an ATG AMO and observed a severe phenotype with incomplete
development of the embryo, which could not be rescued by human
mRNA. These preliminary (unpublished) results indicate that
IL1RAPL1 plays an important function during early embryonic
development. Further work, such as with splice junction AMOs that
E. Kabashi et al. / Biochimica et Biophysica Acta 1812 (2011) 335–345
spare maternal transcripts that are essential for early development, is
required in order to develop a pathogenic validation.
Neurexins are predominantly pre-synaptic cell-adhesion molecules. They can induce pre-synaptic differentiation by interacting with
neuroligins. There are three neurexin genes (NRXN1, 2, and 3), each of
which encodes two major variants (alpha and beta). Only NRXN1 gene
was shown to be disrupted using CNV analysis in autism [85,86]. More
recently, NRXN1 gene has been involved in CNV found in SCZ patients
[87,88]. We have identified an insertion of 4 bp predicted to cause a
frameshift with a premature stop affecting the two major variants
alpha and beta of NRXN1 in SCZ patient. The protein is predicted to
miss the transmembrane and intracellular domains. Two orthologs of
NRXN1 have been identified in zebrafish (Nrxn1a and 1b) with an
identity to the human protein over 70%. The greatest variability is
found in the signal peptide (Nrxn1a, 27%; 1b 32%). An expression
analysis showed that all three Nrxn genes are expressed during
zebrafish embryonic development and some specific isoforms of
Nrxn1a expressed at different stage of the development [89]. These
results indicate the potential for developing zebrafish models of
neurexin mutations. However, the fact that there exist thousands of
isoforms of Nrxn1 and the amino acids of the signal peptide are
divergent could make the functional validation in zebrafish more
challenging.
Kinesin 17 (KIF17) is a member of the kinesin superfamily
containing a motor domain for ATP hydrolysis. KIF17 binds to the
scafolding Mint1 to transport the NMDA receptor subunit 2B (NR2B)
to the dendrites along microtubules [90,91]. In addition, the K+
channel Kv4.2, a major regulator of dendritic excitability, is also
transported to the dentrites by KIF17 [92]. In mice, overexpression of
Kif17 enhances spatial and working memory [93]. The expression of
KIF17 was shown to be altered in a mouse models for Down syndrome
(trisomy 21) leading to learning deficit [94]. We have identified a
nonsense mutation resulting in a protein that lacks the tail domain in
a SCZ patient. This domain was shown to interact with Mint1
following its phosphorylation by CaMKII [95]. One orthologous
sequence was identified in zebrafish with an identity of 61% compare
to the human amino acids. The divergence was mainly observed in the
motifs important for cargo binding. The zebrafish KIF17 is widely
expressed in the nervous system and retina [96]. By using low doses
splicing AMO, this group demonstrates that KIF17 is essential for
vertebrate photoreceptor development and seems to have little effect
on the global zebrafish development. At higher doses of either an ATG
or a splice junction AMO that micked the effect of the de novo
nonsense truncation mutation in schizophrenia, we observed a
characteristic dose-dependent morphological trait with stunted,
curly embryos [127]. This validates that the nonsense mutation is a
loss-of-function pathogenic effect.
Together, these results with several zebrafish developmental
genes indicate the potential usefulness of this model for validating
brain disease mutations, but clearly each gene requires its own set of
carefully designed experiments. Whereas some human genes can be
more difficult to study in zebrafish, with others, one can observe
faithful disease phenotypes that can be characterized in detail such as
for AP1S1 in MEDNIK and ALS2 in ALS and thus provide novel insights
to these disorders. We have observed some knockdown phenotypes in
zebrafish that are not as obvious in knockout mice, which may reflect
a lower degree of compensation in zebrafish, perhaps because their
reproductive biology is based more on the quantity of offspring rather
than necessarily on the quality of developmental compensation.
5. Future directions
Several approaches have the potential of further advancing
zebrafish models of human CNS disorders and making them uniquely
useful, in particular targeted expression, multigenic analysis and
chemical genetics.
341
5.1. Targeted expression
Most of the approaches taken to date to express foreign genes in
zebrafish are based on injection of mRNA for transient expression
throughout the embryo. This simple approach is valid for ubiquitously and early expressed genes such as AP1S1, SOD1, TARDBP and
members of the ubiquination and beta-secretase complexes discussed above. However, mRNA injection risks expressing genes
ectopically, such as in the case of synaptic genes, and is usually only
efficient at embryonic stages, prior to mRNA degradation that occurs
within a few days. The latter thus limits the time course for
phenotypic analysis, which is particularly problematic for studies of
neurogeneration in (often) adult-onset human diseases. More
specific expression patterns can be achieved by stable transgenesis
using the efficient Tol2 transposon system [97], but this is best suited
for generating long-term models (rather than for preliminary or
large-scale screens) as it is a slower process that requires the raising
of founder fish and their out-crossing to verify stable and specific
transgenic lines. A number of promoters are available for neuralspecific transgenic expression. For example, the α-tubulin promoter
[98] was the first used to drive pan-neuronal expression and, as
described above, the HuC promoter expressed in post-mitotic
neurons [19] and a neural-specific variant of the GATA2 promoter
[18] are also effective at early stages. More selective subsets of
neurons can be targeted using more restrictive promoters such as the
dopaminergic-specific vesicular monoamine transporter 2 promoter
[31]. In the spinal cord, which is an important region for studies of
motor dysfunction and synaptic transmission, the developmentally
conserved transcriptional code [99] has permitted the generation of
transgenic lines for selective types of neurons. These include
motoneurons: HB9 [100], Isl-1 [101], commissural neurons: Evx1
[102], mostly glycinergic neurons: Pax2.1 [103]; vGlyT2 [104], mostly
glutamatergic neurons: Alx/Vsx2 [105], as well as sensory neurons:
Ngn1 [106] and oligodendrocytes: Nkx2.2a [107], olig2 [108] and
sox10 [109].
However, these stable transgenic models also have their limitations, and here, we consider three major ones that can be at least
partially avoided depending on necessity: ectopic expression, toxic
expression, and genetic background. Although promoter-based stable
transgenic lines confer more selective expression patterns, rarely do
they perfectly recapitulate the natural expression patterns as often
only subsets of enhancer elements are used in generating the
transgenic constructs. Indeed, this is a limitation of the lack of a
knock-in technology in zebrafish, requiring the integration of novel
constructs in a wild-type background (considered below). However,
perfectly faithful expression patterns are not always essential as
expression in incomplete or expanded subsets of neurons can be
useful to study. An approach that provides a more accurate expression
pattern is the use of bacterial artificial chromosomes (BACs), such as
with the amyloid line mentioned above [21]. In principle (although
we are unaware of its practice with zebrafish), humanized models
could be generated based on transgenesis with human BACs, as is
commonly done with mice. However, vertebrate genes can be
hundreds of kilobases in size and are not always completely contained
within single BACs. As an alternative, pufferfish (Takifugu rubripes)
BACs can be used as the intergenic regions are much smaller, an
approach we have taken in generating an Evx1 transgenic line [102],
although even in this case less than 80% of the neurons labeled by a
selective antibody to evx1 also expressed GFP, indicating that not all
evx1 cells transgenically express GFP.
A second issue in considering stable transgenic lines is toxicity, as
many of the transgenes bearing disease-related mutations can be
embryonic-lethal. A simple approach is to drive expression from a
heat shock promoter [110], although this may lose in generalized
spatial expression what it gains in restricted temporal activation. A
powerful alternative is the use of combinatorial expression systems,
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such as the cre-loxP system developed in mice and the yeast Gal4-UAS
binary system popular for invertebrate genetics, both of which have
found applications in zebrafish [111,112]. Examples are the Evx1-Gal4
[102] and HuC-Gal4 [19] lines described earlier. The latter drives
transcription in both directions from the UAS in order to express two
constructs simultaneoulsy, such as a gene of interest (in this case
human P301L tau) and a reporter (dsRed) in the same cells. Another
technique for expressing more than one gene from the same construct
is to use the self-cleaving viral 2A peptide as a linker between the
genes of interest [113].
A final concern is the creation of transgenic lines in a genetic
background containing the endogenous gene as (in the absence of
knock-in technology) this results in the addition of another gene. One
possibility that is rarely used is to create a transgenic in a knockout
background. In principle, this can be done if mutants have been
isolated in screens for related phenotypes or by TILLING (Targeted
Induced Local Lesions in Genomes) [114] available through a new
consortium (see https://webapps.fhcrc.org/science/tilling/). A new
technology is the targeted lesioning of genes by zinc finger nucleases
(ZFN) [115], although this is a complex and expensive technology that
at the moment is beyond the reach of small laboratories. Preliminary
success in targeting genes in zebrafish is encouraging, and the
possibility to use homologous recombination, rather than nonhomologous end joining, to repair ZFN-induced double-strand breaks
may permit the development of a knock-in approach for zebrafish
[116]. Much works remain to be done before this enticing possibility
becomes practical, but nonetheless, a number of transgenic
approaches are now available for refined studies of gene expression
and function in zebrafish, including human genes bearing diseaserelated mutations.
5.2. Multigenic analysis
Although highly penetrant mutations of single genes are often
implicated in CNS disorders, such as the majority considered in this
review, most diseases are multigenic in nature, requiring multiple
weakly penetrating mutations in predisposing genetic backgrounds in
order to produce the disease. Existing genetic models most easily
address single gene mutations (or rather have focused on these
simple cases), but clearly, the future of these models lies in their
ability to provide insights to the nature of genetic interactions in
disease. The zebrafish may prove to be particularly amenable to
multigenic analysis by a combination of the approaches described
above. The use of two or even three AMOs is a simple solution once
each AMO is validated. For example, in the context of Parkinson's
disease, simultaneous knockdown of DJ-1 and p53 was found to cause
dopaminergic neuronal loss [28] and knockdown of both Hgf ligands
was necessary to induce Met-related cerebellar defects [71]. Also,
photoactivatable caged AMOs can be used to release these at specific
time points [117]. This approach seems promising, but unfortunately,
caged AMOs are not available commercially yet. Another major
limitation of the AMO is the necessity to inject in embryo at 1 or 2 cells
stage. An interesting alternative would be to use the vivo-AMOs that
allow making gene knockdown and splice modification in adult
animals. The vivo-AMOs are modified AMO able to enter cells by
endocytosis. They could be conjugated with a dendrimeric octaguanidine (Gene Tools) or a cell-penetrating peptide linked to phosphorodiamidate (PPMO) (AVI BioPharma Inc.). Vivo-AMOs were shown to
be effective in mice [118,119], and they are currently tested in adult
zebrafish [3].
By performing AMO injections in mutants or transgenics, either
stable or inducible, it should in principle be possible to readily test
several genes simultaneously or in different combinations, bestowing
zebrafish with a polyvalence that is difficult to envisage for other
models such as mice. In the case of mutant and transgenic backgrounds, comparative microarray screening [120] becomes feasible,
particularly as multiple conditions (e.g., different mutant alleles)
permit internal verification and comparison of the array results. A
pertinent example is the recent microarray analysis [121] of three
alleles of the neurogenic mindbomb mutant affecting Notch signaling
for which each allele affected the expression of hundreds of genes but
less than 100 were commonly affected. Thus, zebrafish have a unique
potential for rapidly analyzing multiple genes and dissecting complex
genetic pathways.
5.3. Chemical genetics
While zebrafish retain advantages for studying the genetics,
biology, and pathobiology of disease genes of interest, they also
have the major advantage of being the only vertebrate model
amenable to large chemical genetic screens [122]. Small chemical
libraries of over 10,000 molecules, including approved drugs and
randomly synthesized organic molecules of unknown function, have
been screened in zebrafish models of cancer [123] and cardiovascular
disorders [124], and some of these are in clinical testing. With the
numerous models of brain diseases being developed in zebrafish, this
approach holds much potential for drug discovery. This is particularly
attractive in zebrafish as functional chemical genetic screens in vivo
with living embryos offer the potential of discovering therapeutics
without prior knowledge or need for rational screening designs. Thus,
zebrafish are a unique model ranging from molecular genetic
validation to drug discovery with important applications on the
horizon for brain diseases, including many complex and untreatable
disorders.
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Biochimica et Biophysica Acta 1812 (2011) 346–352
Contents lists available at ScienceDirect
Biochimica et Biophysica Acta
j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / b b a d i s
Review
Zebrafish as a tool in Alzheimer's disease research☆
Morgan Newman a,1, Giuseppe Verdile b,1, Ralph N. Martins b, Michael Lardelli a,⁎
a
b
Discipline of Genetics, School of Molecular and Biomedical Science, The University of Adelaide, SA 5005, Australia
Centre of Excellence for Alzheimer's Disease Research and Care, School of Exercise, Biomedical and Health Sciences, Edith Cowan University, Joondalup, WA 6027, Australia
a r t i c l e
i n f o
Article history:
Received 27 November 2009
Accepted 21 September 2010
Available online 12 October 2010
Keywords:
Zebrafish
Alzheimer's disease
Presenilin
a b s t r a c t
Alzheimer's disease is the most prevalent form of neurodegenerative disease. Despite many years of intensive
research our understanding of the molecular events leading to this pathology is far from complete. No effective
treatments have been defined and questions surround the validity and utility of existing animal models. The
zebrafish (and, in particular, its embryos) is a malleable and accessible model possessing a vertebrate neural
structure and genome. Zebrafish genes orthologous to those mutated in human familial Alzheimer's disease have
been defined. Work in zebrafish has permitted discovery of unique characteristics of these genes that would have
been difficult to observe with other models. In this brief review we give an overview of Alzheimer's disease and
transgenic animal models before examining the current contribution of zebrafish to this research area. This article
is part of a Special Issue entitled Zebrafish Models of Neurological Diseases.
© 2010 Elsevier B.V. All rights reserved.
1. Alzheimer's disease
Alzheimer's disease (AD) is the most common form of neurodegenerative disease. Globally, the incidence of dementia was estimated in 2001
to be 24.3 million with an estimated increase to 81.1 million in 2040 [1].
Clinically, AD is characterised by progressive memory loss and can include
impairment of speech and motor ability, depression, delusions, hallucinations, aggressive behaviour and, ultimately, increasing dependence upon
others before death (recently reviewed by Voisin and Vellas [2]). The
major neuropathological hallmarks of the disease were first described by
Alois Alzheimer as “fibers” and “small military” foci (English translation,
[3]) Today these are commonly referred to as neurofibrillary tangles
(NFTs) and amyloid plaques. Neurofibrillary tangles are hyperphosphorylated forms of the tau protein, while the major protein component
of amyloid plaques is a small peptide referred to as amyloid beta (Aβ).
Other forms of lesion have also been found in AD brains (reviewed by
Duyckaerts et al. [4]).
Alzheimer's disease can be classified broadly into two groups, late
onset AD (LOAD, occurring N65 years) and early onset AD (EOAD,
occurring b65 years). LOAD accounts for the vast majority of AD cases
(95%) and is associated with many risk factors. It is well established that
age and possession of the ε4 allele of the APOLIPOPROTEIN gene (APOE ε4)
are the major risk factors for most of the population (reviewed by Martins
et al. [5]). However, other risk factors such as hormonal changes
associated with ageing, diet and lifestyle factors play very important
roles. Cardiovascular risk factors such as hypercholesterolemia, increased
☆ This article is part of a Special Issue entitled Zebrafish Models of Neurological
Diseases.
⁎ Corresponding author. Tel.: +61 8 83033212; fax: +61 8 83034362.
E-mail address: [email protected] (M. Lardelli).
1
Made equal contributions to this paper.
0925-4439/$ – see front matter © 2010 Elsevier B.V. All rights reserved.
doi:10.1016/j.bbadis.2010.09.012
LDL and reduced HDL levels, hypertension, obesity and type II diabetes
make important contributions to the risk of developing AD (reviewed by
Martins et al. [5]). Genome-wide association studies in humans have
identified a number of possible additional loci associated with risk for
LOAD (reviewed by Rademakers and Rovelet-Lecrux [6]. In particular, two
recent studies identified overlapping sets of candidates (see [7,8] and
Table1). Individuals carrying the APOE ε4 allele also bear some risk of
developing the early onset form of AD. Overall, EOAD accounts for only a
small percentage of AD cases but is the most severe form of the disease.
The majority of EOAD cases are familial forms of AD showing dominant
inheritance.
Familial AD (FAD) is typically associated with an early age of onset
as young as 25 years (reviewed by Verdile and Martins [20]). Most of
our knowledge of the molecular events underlying the development
of Alzheimer's disease comes from FAD since we can use genetic
analysis to identify the genes and proteins involved. Mutations at only
three loci are known to cause FAD although other loci may yet be
found (see review by Rademakers and Rovelet-Lecrux [6]). One locus
hosts the Amyloid Precursor Protein (APP) from which Aβ is cleaved
by the action of two “secretases,” β-secretase and γ-secretase (see
Fig. 1). Mutations in APP alter these cleavages so that Aβ production is
increased or more aggregation-prone forms of Aβ are formed
(reviewed by Verdile et al., [20]). The remaining two loci host the
presenilin genes PSEN1 and PSEN2 with mutations in these genes
accounting for 50–70% of EOFAD cases [21]. The presenilin proteins
form the catalytic core of protein complexes with γ-secretase activity.
Mutations in the presenilins also appear to alter the rate or form of Aβ
production and many LOAD associated risk factors also regulate Aβ
levels in the brain. Therefore, it is not surprising that the currently
dominant hypothesis for the cause of FAD (and LOAD) is the “amyloid
hypothesis” that postulates that toxic oligomerisation of Aβ is the
initiating factor beginning a cascade of consequences such as synaptic
M. Newman et al. / Biochimica et Biophysica Acta 1812 (2011) 346–352
347
directed at neutralising Aβ toxicity (i.e. PBT2) are addressing such issues
(reviewed by Wisniewski, 2009 [29]). There are also therapies not
directed at Aβ that show promise including the anti-histamine Latrepirdine (formerly known as Dimebon™) which has completed phase IIIa
trials and is currently in IIIb trials [30]. The mechanism(s) underlying the
benefits of Latrepirdine is unclear but may include improving mitochondrial activity. Another agent that has shown some promise is Rember™
(methylthionium chloride) which reduces tau aggregation (see http://
www.taurx.com/) although this drug was not seen to affect the
histological or behavioural phenotypes of transgenic zebrafish expressing
mutant human MAPT (see 2009 ICAD conference report at www.
alzforum.org/new/detail.asp?id=2203). Methylthionium chloride is better known to those working with zebrafish as methylene blue which is
used to inhibit fungal growth and to stain the yellow pteridine-containing
pigment cells of fish, xanthophores [31]. Recently, the combination of
Dimebon (Latrepirdine) and methylene blue has also been shown to
inhibit aggregation of TDP-43 [32], a protein component of ubiquitinated
inclusions found in the neurodegenerative diseases amyotrophic lateral
sclerosis (ALS) and frontotemporal lobar degeneration (FTLD) (reviewed
by Mackenzie [33]). The beneficial outcomes of these non-Aβ directed
agents may provide further evidence for alternatives to the amyloid
hypothesis, However, it is conceivable that these agents may be acting to
ameliorate the consequences of Aβ toxicity e.g. by protecting neurons
from insults such as aggregation of Aβ or other proteins.
Fig. 1. The γ-secretase complex consists of four protein components including PSEN1 or
PSEN2 that reside within lipid bilayers and cleave type 1 transmembrane domain proteins
at a catalytic site dependent upon two aspartate residues (stars). APP is initially cleaved
either by α- or β-secretase to produce APPα or APPβ. Only β-secretase cleavage permits
Aβ peptide production after secondary cleavage by γ-secretase that also releases APP's
intracellular domain that can enter the nuclear to contribute to gene regulation.
dysfunction, inflammation and oxidative damage causing neuronal
dysfunction, degeneration and death (see review in [22]).
The amyloid hypothesis is an attractively parsimonious explanation
for AD pathogenesis and finds support from numerous phenomena
observed in EOFAD and LOAD brains. However, it fails to explain some
observations in animal models and the outcomes of a number of human
clinical trials of drugs intended to ameliorate Aβ toxicity. For example,
the majority of transgenic mice harbouring FAD-causing mutations
leading to accumulation of human Aβ in their brains do not show
dramatic neurodegeneration [23] (see the discussion in the 2008 paper
by Chen et al. [24]). However, these transgenic mice do show cognitive
deficits in behavioural tests and show reduction in long-term
potentiation, most likely indicating loss in synaptic activity [25]. To
obtain significant neurodegeneration or neuronal loss in transgenic
mice requires expression of three or more mutations in AD-associated
genes. Examples of mouse AD models used currently include “3xTg-AD”
mice bearing mutant alleles of PS1, APP and MAPT [26] and “5xFAD” mice
bearing three APP and two PS1 mutant alleles [27]. However, the coexistence of three or more mutations in AD-associated genes is not a
situation that occurs in humans. A more physiologically relevant animal
model is required that more closely mirrors human AD neuropathology
including the observed neurodegeneration and neuronal loss (see the
critical review by Buxbaum [28]).
A number of therapeutic agents aimed at reducing Aβ production or
accumulation/aggregation have failed human clinical trials. These include
peptide mimetics that inhibit Aβ aggregation (i.e., Alzhemed™,) or agents
that either inhibit γ-secretase activity (e.g. LY-411,575 and LY-450,139 )
or target the production of the more neurotoxic Aβ42 (e.g. the NSAID,
Flurizan™ and refer to www.alzforum.org regarding information on AD
drugs in clinical trials). This has raised questions over the validity of the
amyloid hypothesis. However, issues surrounding penetration through
the blood–brain barrier and the advanced state of neurodegeneration
when these drugs were given may explain their failure. Apparently
successful therapeutics currently in phase II/III trials such as the
immunotherapeutics Bapinezuimab™ and Solabezumab™ or agents
2. Presenilin activity is involved in many aspects of AD pathology
The presenilins proteins are known to interact with both the APP and
tau proteins [34–38] providing one possible link between Aβ production
and changes in tau phosphorylation. Mice lacking expression of both
Psen1 and Psen2 in forebrain tissue show a number of phenotypic effects
resembling Alzheimer's disease, i.e. neurodegeneration such as hyperphosphorylation of tau, formation of intracellular tau inclusions, gliosis,
expansion of brain ventricles and neuronal death all in the absence of
any Aβ production [24]. Some characteristics of Alzheimer's disease,
such as increased formation of glia, may result from changes in
presenilin activity since γ-secretase complexes cleave not only APP
but also a large collection of other transmembrane proteins such as
NOTCH, E-cadherin, neuregulin-1 and syndecan (reviewed in Ref. [39]).
Presenilins also function in a number of cellular processes, at least some
of which appear independent of γ-secretase activity. For example, Kang
et al. [40] showed that mouse Psen1 represents a second pathway for
phosphorylation of β-catenin independent of Axin. Presenilins also
appear to act as Ca2+ leak channels in the endoplasmic reticulum [41].
Presenilins are also found in interphase kinetochores [42] (although
this observation has not been replicated) and changes in presenilin
activity can cause aneuploidy ([43,44]) and dysregulation of cell cycle
proteins [12,45]. Indeed, the activity in γ-secretase of at least some of
the proteins comprising these complexes does not appear to be their
original function. Homologues of presenilins exist in plants and this
kingdom does not display γ-secretase activity [46].
The great majority of the mutations causing FAD are found in PSEN1.
Currently over 180 different missense mutations are known (data on
mutations are collated at Alzheimer Disease and Frontotemporal
Dementia Mutation Database, http://www.molgen.ua.ac.be/ADMutations/). There is also evidence that oligomeric forms of Aβ can feed back
onto the activity of γ-secretase ([47]). Thus it is possible that mutations
that cause production of more Aβ, or more aggregation-prone forms of
Aβ, may affect presenilin function and that changes in presenilin
function may then affect tau phosphorylation, cell differentiation (e.g.
via Notch signalling), calcium homeostasis, etc.
3. Using zebrafish to study genes mutated in FAD
Mice are the dominant model used for modelling of Alzheimer's disease
pathology (reviewed in Ref. [48]) but non-mammalian organisms have also
348
M. Newman et al. / Biochimica et Biophysica Acta 1812 (2011) 346–352
enlightened us. Mutation screening in the nematode, Caenorhabditis elegans
first showed the connection between the presenilins and Notch signalling
[49,50]. Expression of human Aβ in C. elegans [51] and Drosophila [52] has
been seen to cause amyloid deposits in muscle and neurodegeneration
respectively. Aβ toxicity can also be studied using yeast [53]. Use of these
organisms has opened up possibilities for screening drug libraries to find
compounds blocking Aβ toxicity.
As a model organism, zebrafish presents many advantages for the
study of human disease. The vertebrate zebrafish genome and anatomy
show only ~420 million years (Myr) of divergence from the human
lineage [54] rather than the ~600 Myr of the ecdysozoans (Drosophila
and C. elegans, [55]). Therefore, most human genes have clearly
identifiable orthologues in zebrafish (however, a genome duplication
early in the teleost (bony fish) lineage means that, in many cases, two
zebrafish genes together perform the function of an orthologue). The
numerous and externally fertilized embryos of zebrafish are easily
amenable to methods for manipulating gene and protein activity such as
injection of antisense oligonucleotides, mRNAs or transgenes and they
can be arrayed in microtitre plates for screening of drug libraries. Cell
labelling and transplantation for tracking the behaviour and interactions
of living cells is also routine. A good indication of the potential utility of
zebrafish for Alzheimer's disease research is the attempts to obtain
patent coverage of wide areas of the field (e.g. WO/2006/081539).
Zebrafish possess genes orthologous to all the genes known to be
involved in Alzheimer's disease (Table 1) The genes appa and appb [9]
are duplicates of an ancestral teleost orthologue of human APP. The
genes psen1 [11] and psen2 [16] are orthologues of human PSEN1 and
PSEN2, respectively. Our laboratory has also identified duplicates of an
ancestral teleost orthologue of the MAPT gene encoding tau protein,
mapta and maptb [56] (discussion of tau activities in zebrafish
including the MAPT-based model of neurodegeneration of Paquet et
al. 2009 [57] is to be found in an accompanying paper by Ed Burton).
The body of research literature examining the endogenous zebrafish
appa, appb, psen1 and psen2 genes is still quite limited. Musa et al. [9]
detected widespread and overlapping transcription of both appa and
appb by whole mount in situ transcript hybridisation (WISH) from midgastrulation. Later in development these two genes show differing
patterns of transcription but, notably, at 24 hpf both are expressed in the
developing forebrain (telencephalon) and ventral diencephalon. In
addition, appb is expressed in the ventral mesencephalon, a series of
nuclei in the hindbrain and in the spinal cord. Joshi et al. [10] recently
examined the effects of reduction of appb function through use of
antisense morpholino oligonucleotides blocking translation (while they
also attempted to reduce appa mRNA translation it is not clear that they
were able to do this. Also, their appb analysis utilized only one
morpholino so non-specific effects are not excluded). They observed
defective convergent extension movements and reduced body length.
Interestingly, injection into zebrafish embryos of mRNA encoding
normal human APP was more effective at ameliorating the loss of
appb translation than an Alzheimer's mutant form (“APPswe” containing
the “Swedish” double mutation K595N with M596L [58]). This
demonstrates that zebrafish embryos can be exploited for analysis of
differences in the activity of different mutant forms of APP [10].
Two papers examining transcriptional control of appb have been
published [59,60]. Both emphasise the importance for expression of a
regulatory element in the first intron of the gene. Approximately 14
kb of DNA from the promoter region and first intron of the gene can
largely recapitulate the embryonic transcription pattern of appb [59].
Attempts to model Aβ toxicity have, so far, proven difficult and no
studies have yet been published. The existence of transgenic fish
expressing human APPswe in neurons in zebrafish the under the control
of the promoter of the elavl3 gene (previously known as HuC) was
reported at the 6th European Zebrafish Development and Genetics
Meeting in Rome in July 2009 (Hruscha, Teucke, Paquet, Haass and
Schmid) but there is no data yet on any toxic effects. In our laboratories
we have followed a number of approaches to analysing Aβ toxicity.
Simple incubation of zebrafish embryos in media containing Aβ peptide
appears to produce effects on neural development as monitored by
changes in the patterns of neurons observed in non-transgenic
zebrafish. It can also induce cell and embryo death (Frederich-Sleptsova
et al. manuscript in preparation). We also attempted to generate a
transgenic zebrafish model to facilitate screening for drugs suppressing
Aβ toxicity by expressing the 42 amino acid residue form of human Aβ
(Aβ42) in the melanophores (melanocytes) that constitute the zebrafish's dark surface stripes. The hope was to create a highly visible but
nevertheless viable phenotype in zebrafish larvae that could then be
arrayed in microtitre dishes for exposure to various chemical compounds. For this we used a DNA fragment from the promoter of the
zebrafish gene mitfa (previously nacre) that can specifically drive
transcription in zebrafish melanophores [61]. This was coupled to DNA
coding for a secretory signal fused to Aβ42. Unfortunately, fish bearing
this transgene only showed a disturbed pigmentation pattern at 16
months of age—too late for use in drug screening [81].
Analysis of presenilin function in zebrafish has tended to focus on
psen1 due to the more frequent role played by mutations of its human
orthologue, PSEN1 in AD. Zebrafish psen1 sequence and activity were
first analysed by Leimer et al. in 1999 [11] who noted extensive
conservation of protein primary structure. Gene transcripts are
Table 1
Zebrafish genes orthologous to those involved in Alzheimer's disease.
Zebrafish orthologue
Accession number
Zebrafish references
appa
appb
psen1
psen2
NM_131564
NM_152886
NM_131024
NM_131514
[9,10]
Clusterin (CLU)
Phosphatidylinositol Binding Clathrin Assembly Protein (PICALM)
Complement Component (3b/4b) Receptor 1 (CR1)
apoeb
mapta
maptb
Clu
picalm
Unknown
NM_131098
N/A
N/A
NM_200802
NM_200927
Loci for γ-secretase complex members
Nicastrin (NCT)
Anterior Pharynx Defective 1 Homolog A (APH1a)
Anterior Pharynx Defective 1 Homolog B (APH1b)
Presenilin Enhancer 2 (PSENEN)
ncstn
unknown
aph1b
psenen
NM_001009556
[17]
NM_200115
NM_205576
[18]
[18,19]
Loci for dominant mutations in FAD
Amyloid Precursor Protein (APP)
Presenilin 1 (PSEN1)
Presenilin 2 (PSEN2)
Loci associated with risk for ADa
Apolipoprotein E (APOE)
Microtubule-Associated Protein Tau (MAPT)
a
A large number of additional associated genes have been reported and are reviewed in Rademakers and Rovelet-Lecrux, 2009.
[11–15]
[12,15,16]
M. Newman et al. / Biochimica et Biophysica Acta 1812 (2011) 346–352
apparently present in all cells at all developmental times examined.
When zebrafish Psen1 protein expression was driven at high levels in
cultured human HEK293 cells the zebrafish protein (as expected)
displaced human PSEN1 from γ-secretase complexes indicating
sufficient structural conservation to interact with other complex
components. However, the C-terminal fragment (CTF) of zebrafish
Psen1 generated by the endoproteolysis of the holoprotein (required to
activate γ-secretase activity [62]) is sufficiently different in size that it
can be distinguished from the CTF of human PSEN1 on western blots.
Also, zebrafish Psen1 possesses sufficient primary structural differences
from human PSEN1 that its γ-secretase cleavage of APP was abnormal
leading to increased production of Aβ42 relative to production of the less
aggregation-prone 40 amino acid residue form of Aβ (Aβ40). These
authors also demonstrated that mutation of one of the two critical
catalytic aspartate residues in zebrafish Psen1 could abolish its γsecretase activity.
As expected from the expression of the psen1 transcript, we showed
that Psen1 protein can be observed from the earliest stages of
development [14]. Interestingly, this did not appear to be the case for
zebrafish psen2 that we identified in 2002 [16]. We detected the
ubiquitous presence of psen2 transcripts at all stages but an antibody
raised against an oligopeptide corresponding to N-terminal fragment
(NTF) sequence from Psen2 protein only revealed higher expression of
Psen2 after 6 hpf [16]. However, there is evidence from humans [63,64]
and from our own unpublished work of alternative splicing and
phosphorylation of PSEN2 orthologous proteins so we presume that
species of zebrafish Psen2 protein do, in fact, exist at the earliest
developmental stages in this organism.
The essential role of presenilin activity in Notch signalling during
development and the resorption of dead embryos that occurs in mice
has hindered the analysis of the roles of presenilin during vertebrate
development. Resorption is not a problem in zebrafish so we have been
keen to exploit morpholino antisense technology to block the activity of
the zebrafish psen1 and psen2 genes. In mice, loss of Psen1 activity is
lethal during development and Psen1−/− embryos show a similar
phenotype to those lacking mouse Notch1 activity [65,66]. In contrast,
mouse Psen2−/− embryos are viable with no obvious developmental
defects [67,68] (although adults do display haemorrhaging and
histological changes in lung tissue [69]. However, mice lacking both
Psen1 and Psen2 activity have a more severe phenotype and loss of γsecretase activity than when only Psen1 activity is absent [67]. When we
blocked psen1 translation in zebrafish we observed similarly irregular
delineation of somites to that seen in Psen1−/− mouse embryos and the
cyclic expression of the her1 gene that controls somite formation was
disturbed in the presomitic mesoderm [13]. Interestingly, blockage of
translation of zebrafish psen2 transcripts also produces significant
changes in embryo development including expansion of brain ventricles
at 48 hpf and decreased trunk neural crest formation with increased
numbers of neurons at 24 hpf. The latter two defects at least can be
attributed to decreased Notch signalling indicating that Psen2 in
zebrafish plays a more prominent role in Notch signalling than its
mammalian orthologue [15]. Indeed, in unpublished work, van Tijn et al.
[70] have shown that zebrafish homozygous for null mutations in psen1
are, nevertheless, viable and fertile indicating that psen2 activity can
satisfy all essential psen1 functions, at least in a laboratory setting.
The viable homozygous null psen1 mutant zebrafish of van Tijn et
al. [70] show decreased cell proliferation and de novo neurogenesis.
Such fish also present an opportunity for in vivo analysis of the effects
on psen1 function of mutations causing Alzheimer's disease [70,71].
Since presenilin proteins are ubiquitously expressed, simple ubiquitous transgenic expression of mutated psen1 genes in fish lacking
endogenous psen1 activity can allow analysis of their function in vivo.
The ability to analyse the function of genes involved in FAD in vivo is
important since gene regulatory and functional data from cultured cells
and in vitro assays is sometimes not consistent with what occurs in the
whole organism. This may be due to genetic and epigenetic changes that
349
occur in cultured cells during successive passages and/or immortalisation. Also, cultured cells exist in a highly abnormal environment without
the normal three-dimensional context of cellular contact and signalling.
This may explain, to some extent, differences that we have observed
while analysing the function of presenilins in zebrafish embryos
compared to what has been observed from mammalian cell culture.
For example, when we overexpressed Psen2 protein in zebrafish
embryos at relatively high levels by mRNA injection we did not see a
decrease in Psen1 levels [13] whereas transfection of constructs
expressing presenilin proteins into cultured mammalian cells is
known to reduce the levels of endogenous presenilin proteins [72].
A fertilized zebrafish egg represents a macroscopic single cell in
which the activity of one or multiple endogenous genes/proteins can be
subtly manipulated to obtain a phenotypic or biochemical readout. We
attempted to model the molecular effects of mutations that alter human
PSEN1 splicing to cause FAD by altering the splicing of zebrafish psen1
using injected morpholino antisense oligonucleotides. Mutations
causing increased levels of a PSEN1 transcript isoform lacking exon
8 or that cause loss of exon 9 can cause a form of FAD with a peculiar
histology of plaques lacking neuritic cores and showing a “cotton wool”
morphology [73,74]. Unfortunately, when morpholinos binding over
the splice acceptor sites of zebrafish exons cognate to human exon 8 or
exon 9 were injected we did not observe the desired exon loss from
transcripts. Instead we saw inclusion of cognate introns 7 or 8,
respectively, in a proportion of transcripts causing truncation of the
open reading frames after exons 7 or 8. Interestingly this produced very
strong dominant negative effects in the case of truncation after exon 7
sequence but not for truncation after exon 8 (which is very similar to a
normal Psen1 NTF). The dominant negative effects included crossinhibition of psen2 activity [14]. Injection of mRNAs encoding Psen1
proteins truncated after exon 7 or exon 8 sequences produced similar
results ([14] and unpublished data). Tests with further morpholinos and
mRNAs have shown that transcripts producing dominant negative
truncated proteins are those that code for part or all of sequence cognate
to human PSEN1 exons 6 and 7 but not exon 8. (Curiously one of two
nonsense mutations in zebrafish psen1 currently under analysis in the
laboratory of Dana Jongejan-Zivkovic falls in this region but there
appears to be no dominant negative effect probably due to nonsense
mediated decay of the transcript before it can be translated into a
truncated protein. (Jongejan-Zivkovic personal communication). The
discovery of potently dominant negative forms of truncated presenilin
may be important since it has been suggested that a unifying feature of
all the 180+ mutations in human PSEN1 may be that they are
hypomorphic with respect to γ-secretase activity [75]. It is possible
that the truncated forms of numerous proteins that are seen to
accumulate in the brains of LOAD individuals may represent a failure
of RNA quality surveillance mechanisms such as nonsense mediated
decay [76]. This, combined with increased rates of aberrant splicing, etc.
seen in aged cells [77] may allow accumulation of dominant negative
truncated forms of presenilin in ageing brains that could suppress γsecretase activity and thus contribute to LOAD.
A zebrafish model of FAD or LOAD would be very useful for screening
for drugs to prevent or ameliorate these diseases. However, even
embryo development in non-transgenic zebrafish can provide assays
for the actions of drugs relevant to Alzheimer's disease. The drug N-[N(3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl ester (DAPT,
[78]) is now commonly used to inhibit γ-secretase activity in zebrafish
[79]. Unfortunately, it is commonly and incorrectly assumed to be
blocking only Notch signalling which, while very important, is not
the only influential signalling pathway facilitated by γ-secretase. For
example, as noted above, Joshi et al. [10] showed that inhibition of appb
activity causes aberrant convergence-extension movements although it
has not been shown that γ-secretase activity is required for this
and treatment with DAPT [79,15] or simultaneous inhibition of
translation of both Psen1 and Psen2 by morpholino injection [15]
does not produce this effect. We have previously shown that numbers of
350
M. Newman et al. / Biochimica et Biophysica Acta 1812 (2011) 346–352
a particular spinal cord cell type, the Dorsal Longitudinal Ascending
(DoLA) interneuron, appear dependent upon Psen2 but not Psen1
activity [14,15]. We used this as an assay for Psen2 activity to show that
truncated Psen1 could suppress Psen2 activity [14]. Finally, in a
publication from the Xia laboratory, Yang et al. [80] used zebrafish
embryos to observe the differential effects on development of drugs
inhibiting primarily the APP or NOTCH cleavage activities of γ-secretase.
Thus, zebrafish embryos can provide a valuable tool for discovery of
additional compounds that show differential inhibitory effects on γsecretase. The Xia laboratory has also used injection of different
combinations of morpholino antisense oligonucleotides to analyse
the effects of inhibiting translation of additional protein components of
γ-secretase complexes such as aph1b (orthologous to human APH1B)
and psenen (orthologous to human PSENEN and previously named
PEN2). In this way they demonstrated that zebrafish Psenen interacts with NF-kappaB [19] and the p53-dependent apoptosis pathway
[18].
4. Concluding remarks
Despite many years of intensive research uncertainty still surrounds
the fundamental mechanisms underlying AD pathology. There is
considerable doubt about the relevance of existing transgenic mouse
models of the disease. Our lack of understanding has slowed the
development of therapies to treat or prevent AD. The vertebrate genome
and neurobiology of zebrafish and our rapidly expanding ability to
manipulate gene activity in this organism offer alternative pathways in
which to probe the mysteries of AD pathology. In particular, zebrafish
are well suited to biochemically-based approaches whereby gene
activities are manipulated transiently and then the consequences
examined in an otherwise normal cellular environment. As genomewide association studies reveal additional genes involved with
development of AD pathology these biochemically-based approaches
can rapidly contribute to our understanding of the relevance of these
genes while reducing the chance of being misled by experimental
results from tissue culture systems that may not reflect in vivo reality.
Acknowledgments
This work was supported by a project grant from the National
Health and Medical Research Council of Australia (453622), the
Cancer Council of South Australia (S 24/04) and the School of
Molecular and Biomedical Sciences of The University of Adelaide.
Professor Martins is supported by grants from the McCusker
Foundation for Alzheimer's Disease Research, Department of Veterans
Affairs, National Health and Medical Research Council (NHMRC),
Centre of Excellence for Alzheimer's disease Research and Care
(RNM). Dr Verdile is generously supported by grants from Mr.
Warren Milner (Milner English College, Perth, Western Australia) and
Ms Helen Sewell. The sources of funding listed above played no role in
study design; in the collection, analysis and interpretation of data; in
the writing of the report; or in the decision to submit the paper for
publication. We are grateful to Prof. Christian Haass and others for
valuable comments on the manuscript. The authors declare that they
have no conflict of interest in this work.
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Contents lists available at ScienceDirect
Biochimica et Biophysica Acta
j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / b b a d i s
Review
Zebrafish models of Tauopathy☆
Qing Bai a,b, Edward A. Burton a,b,c,d,e,f,⁎
a
Pittsburgh Institute for Neurodegenerative Diseases, University of Pittsburgh School of Medicine, USA
Department of Neurology, University of Pittsburgh School of Medicine, USA
c
Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, USA
d
Department of Neurology, VA Pittsburgh Healthcare System, USA
e
Geriatric Research, Education and Clinical Center, VA Pittsburgh Healthcare System, USA
f
Division of Movement Disorders, University of Pittsburgh Medical Center, USA
b
a r t i c l e
i n f o
Article history:
Received 24 January 2010
Accepted 8 September 2010
Available online 16 September 2010
Keywords:
Tau
Tauopathy
Progressive supranuclear palsy
Zebrafish
Alzheimer's disease
Transgenic
Neurodegeneration
a b s t r a c t
Tauopathies are a group of incurable neurodegenerative diseases, in which loss of neurons is accompanied by
intracellular deposition of fibrillar material composed of hyperphosphorylated forms of the microtubuleassociated protein Tau. A zebrafish model of Tauopathy could complement existing murine models by providing a
platform for genetic and chemical screens, in order to identify novel therapeutic targets and compounds with
disease-modifying potential. In addition, Tauopathy zebrafish would be useful for hypothesis-driven
experiments, especially those exploiting the potential to deploy in vivo imaging modalities. Several
considerations, including conservation of specialized neuronal and other cellular populations, and biochemical
pathways implicated in disease pathogenesis, suggest that the zebrafish brain is an appropriate setting in which
to model these complex disorders. Novel transgenic zebrafish lines expressing wild-type and mutant forms of
human Tau in CNS neurons have recently been reported. These studies show evidence that human Tau undergoes
disease-relevant changes in zebrafish neurons, including somato-dendritic relocalization, hyperphosphorylation
and aggregation. In addition, preliminary evidence suggests that Tau transgene expression can precipitate
neuronal dysfunction and death. These initial studies are encouraging that the zebrafish holds considerable
promise as a model in which to study Tauopathies. Further studies are necessary to clarify the phenotypes of
transgenic lines and to develop assays and models suitable for unbiased high-throughput screening approaches.
This article is part of a Special Issue entitled Zebrafish Models of Neurological Diseases.
Published by Elsevier B.V.
1. Introduction
The microtubule-associated protein Tau (MAP-τ, ‘Tau’) undergoes
biochemical alterations, cellular redistribution, and deposition as
insoluble intraneuronal fibrils (Fig. 1), in a variety of neurodegenerative conditions that are collectively termed ‘Tauopathies’. Together,
these diseases, which include Alzheimer's disease, progressive supranuclear palsy and other conditions (Table 1), are an important cause
of morbidity and mortality, with diverse clinical manifestations. No
currently available treatments improve the prognosis of any of these
relentlessly progressive diseases. Consequently, investigations aimed
at determining the underlying pathophysiology of Tauopathies, and
isolating novel therapeutic agents that prevent disease progression,
are of great importance. In this review, we consider recent developments concerning the possibility that a zebrafish Tauopathy model
might be useful for therapeutic target and drug discovery in vivo. After
☆ This article is part of a Special Issue entitled Zebrafish Models of Neurological
Diseases.
⁎ Corresponding author. 7015 BST-3 3501 Fifth Avenue, Pittsburgh, PA 15217, USA.
Tel.: +1 412 640 8480; fax: +1 412 648 8537.
E-mail address: [email protected] (E.A. Burton).
0925-4439/$ – see front matter. Published by Elsevier B.V.
doi:10.1016/j.bbadis.2010.09.004
briefly reviewing current knowledge and murine models of Tauopathy, we discuss the possible advantages of a zebrafish model and
whether a truly representative model encompassing key biochemical
events underlying Tauopathy can be recapitulated in the zebrafish
central nervous system. Finally, we review recent publications
demonstrating initial proof of concept that Tauopathy zebrafish
models recapitulate core features of the human disorders.
2. The microtubule-associated protein Tau
Neurons rely on fast axonal transport to shuttle organelles and
macromolecules over long distances, allowing their physiological
distribution and turnover within axons and dendrites. Microtubules,
which provide the tracks along which molecular motors rapidly
transport these diverse cargos, are composed of polymerized tubulin
monomers. Assembly of tubulin into microtubules is promoted by
microtubule-associated proteins, the first of which to be identified
was termed ‘Tau’ (τ was used to denote a factor essential for tubule
formation) [1,2]. Tau is expressed widely in neurons, where it is
enriched in the axonal compartment [3]. The microtubule-binding
domain of Tau localizes to the C-terminal half of the protein [4,5]
(Fig. 2). The N-terminal, or projection domain, contains a proline-rich
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isoforms. In adult human brain, six isoforms are expressed, produced
by alternative splicing of exons 2, 3 and 10 (exons 4A, 6 and 8 are not
expressed in the CNS). Tau isoforms in the CNS contain either three or
four copies of a tandem repeat containing tubulin-binding sequences,
referred to as 3R- and 4R-Tau respectively (Fig. 2). Exon 10 encodes
the second microtubule-binding repeat, such that inclusion of exon 10
in the mRNA results in translation of 4R-Tau, whereas exclusion of
exon 10 results in a transcript encoding 3R-Tau [6,7]. Optional
inclusion of exon 2, or exons 2 and 3, gives rise to N-terminal
insertions of 29 (‘1N’) or 58 (‘2N’) amino acids respectively [8]. The six
resulting CNS isoforms are shown in Fig. 2. Additional isoforms
generated by incorporation of exon 4A (‘large Tau’) are expressed in
the peripheral nervous system [9].
2.2. Functions of Tau
Fig. 1. Neurofibrillary tangles in Alzheimer's disease. A: Neurofibrillary tangles in
Alzheimer's disease prefrontal cortex are demonstrated using the Gallyas silver method
[127]. NFTs are seen as numerous argyrophilic (black) fibrillar intraneuronal inclusions
(arrows). Neuropil threads, axonal abnormalities also caused by accumulations of
fibrillar Tau, are also seen (arrowheads). B: Neurofibrillary tangles in Alzheimer's
disease hippocampus are demonstrated by immunohistochemistry, using an antibody
(AT8) that detects phospho-S202/T205-Tau. NFTs are seen as abundant immunoreactive (red) intraneuronal inclusions (arrows). Accumulations of phospho-Tau are also
apparent in abnormal axonal terminals surrounding amyloid plaques (arrowheads).
region and multiple potential serine–threonine phosphorylation sites,
and is thought to be involved in interactions with other cellular
components.
Association of Tau with tubulin promotes microtubule formation
[1,2], and association of Tau with microtubules is thought to enhance
their stability [10]. 4R-Tau binds to microtubules with greater avidity
than 3R-Tau isoforms [4,11,12] (this is attributable to sequences in the
first inter-repeat segment that are unique to 4R proteins, rather than
to the additional copy of the microtubule-binding tandem repeat
[13]), suggesting that expression of 4R-Tau may favor microtubule
formation and stability. In healthy adult brain tissue, the 4R-/3R-Tau
ratio is approximately 1, and disruption of this balance in the human
brain may have serious consequences (see below) [14]. However,
there are situations in which the 4R-/3R-Tau ratio may be altered
physiologically. For example, during embryogenesis, Tau is exclusively expressed in the brain as the 3R/0N isoform [8]. It has been argued
that this might promote plasticity of neuronal processes during
development by reducing microtubule stability. Recent work has
suggested that Tau interacts with motor proteins involved in axonal
transport, provoking different functional consequences for distinct
types of motor molecules [15]. Kinesin, which transports cargo
towards the distal end of the axon, tended to detach from the
microtubule on encountering Tau, whereas the proximally-directed
motor Dynein showed more directional reversal than detachment.
These effects were apparent in the presence of 3R/0N Tau, and suggest
that association of Tau with microtubules may modulate axonal
transport in addition to promoting microtubule stability.
2.3. Tau phosphorylation
2.1. The MAPT gene
Tau is encoded by the MAPT gene, which is located on chromosome
17 and contains 16 exons. Alternative splicing of the primary
transcript leads to a family of mRNAs, encoding different protein
Tau undergoes post-translational changes, including physiological
serine–threonine phosphorylation [16] and O-glycosylation [17], in
addition to tyrosine phosphorylation [18], SUMOylation [19] and
pathological nitration [20]. Of these changes, phosphorylation has
Table 1
Neurodegenerative diseases associated with prominent Tau pathology.
Disease
Typical clinical presentations
Typical Tau pathology
Tau filaments
Tau species
Etiology
Alzheimer's disease (AD)
Memory disorder, dysphasia, frontal
lobe cognitive–behavioral disorder,
dementia
Falls, rigidity, oculomotor disorder,
cognitive–executive disorder
Flame-shaped
neurofibrillary tangles
Paired helical
filaments, straight
filaments [123]
Straight filaments
[124]
4R- and 3R-Tau
Predominantly
4R-Tau
Mostly unkown, few cases
caused by mutations in APP, PS1
or PS2 genes
Unknown, linked to H1
haplotype at MAPT locus
Twisted ribbon
filaments [125]
Predominantly
4R-Tau
Unknown, linked to H1
haplotype at MAPT locus
Helical filaments;
straight filaments
[126]
Variable
Predominantly
3R-Tau
Unknown
Variable
(see text)
Mutations in MAPT gene
encoding Tau
Progressive supranuclear palsy (PSP)
Corticobasal degeneration (CBD)
Dystonia, myoclonus, apraxia,
cortical sensory loss, dementia
Pick's disease (PiD)
Frontal lobe cognitive–behavioral
disorder, dementia
Fronto-temporal dementia and
parkinsonism linked to
chromosome 17 (FTDP17)
Variable Parkinsonian motor
disorder and/or frontal lobe
dementia
Globose neurofibrillary
tangles, tufted
astrocytes
Ballooned neurons,
pre-tangles, astrocytic
plaques
Pick bodies
Variable
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355
Fig. 2. Isoforms of the microtubule-associated protein Tau. The schematic depicts the six Tau isoforms expressed in the adult human brain, labeled to the left of each protein. Positions
of major protein domains are shown above the longest isoform. The N-terminal insertions encoded by exons 2 and 3, and the four tandem repeats (R1–4) in the microtubule-binding
domains are indicated. Sites of known phosphorylation are shown above the longest isoform. The table to the right of the figure shows the number of amino acids, predicted
molecular mass and electrophoretic mobility of each protein isoform.
been most extensively studied, since pathological hyperphosphorylation
is associated with Tauopathy [21]. Approximately 45 phosphorylation
sites have been identified, which cluster in the proline rich C-terminal
domains of the protein [22]. Phosphorylation of Tau, in particular at S262
and S356 within the microtubule-binding domain, promotes its
detachment from microtubules, suggesting a potential mechanism in
regulating microtubule stability and axonal transport [23,24]. In addition
to microtubule-binding affinity, phosphorylation may have other effects,
such as modulating binding to the chaperone Pin1 [25] and possibly in
regulating putative signal transduction functions of Tau through
phosphorylation of SH3 domains [26].
Hyperphosphorylation of Tau is a characteristic feature in Tauopathy
specimens. No single phosphorylation site is specific for Tauopathy.
Although some phospho-epitopes may be more readily detected in
pathological specimens compared with non-Tauopathy material, most of
the known phosphorylation sites have been demonstrated in normal
biopsy material [16]. Hyperphosphorylation is a quantitative rather than
qualitative phenomenon, defined by the degree to which Tau is
phosphorylated at multiple sites. Phosphorylation is a dynamic process
and Tau is rapidly de-phosphorylated post mortem, accounting for the lack
of detectable phospho-epitopes in control autopsy specimens. It is unclear
whether a functional abnormality of phosphatase activity is present in
Tauopathy samples, or whether the failure of de-phosphorylation in
Tauopathy autopsy material is attributable to changes in the properties of
the hyperphosphorylated molecule as a phosphatase substrate. During
normal development, Tau is present in a highly phosphorylated state
[27,28], suggesting that the balance of Tau phosphorylation and dephosphorylation can change physiologically, without necessarily provoking neurodegeneration.
phosphorylates all of the residues found in Tauopathy specimens.
Furthermore, there are examples in vitro of cooperation between
kinases; for example, cdk5-mediated phosphorylation of Tau promotes further phosphorylation by GSK3β [37,38]. Consequently, it is
thought that the transition from normal to hyperphosphorylated Tau
may be a hierarchical or sequential process dependent on phosphorylation by multiple different kinases. Several different protein
phosphatases dephosphorylate Tau in vitro, including PP1, PP2A,
PP2B and PP2C. Their specificities are overlapping and their roles in
vivo are incompletely understood. PP2A has emerged as a strong
candidate, since it appears to be the major brain-derived phosphatase
activity directed towards Tau in the rat brain [39] and is localized to
microtubules (both through an association with Tau and also directly,
which may regulate its activity in vitro [40]).
2.5. Tau deposits in sporadic neurodegenerative diseases
Tauopathies are characterized pathologically by the presence of
fibrillar deposits of hyperphosphorylated Tau. The histological
appearance and distribution of Tau deposits differs between diseases,
as does the ultrastructural appearance of Tau filaments that compose
neurofibrillary tangles, Pick bodies and other inclusions (Table 1).
Formation of neurofibrillary tangles (NFTs) likely occurs through
oligomeric intermediates, and there is some evidence that these are
the primary pathogenic species, rather than mature NFTs (see
below).
The predominant Tau isoforms deposited in neurons differ between
diseases. In Alzheimer's disease, SDS-PAGE analysis showed three major
2.4. Tau kinases
Several cellular kinases are able to phosphorylate Tau in vitro or in
cell culture (Table 2; reviewed in [22]). Many of these phosphorylate
multiple residues of Tau, and individual residues can be phosphorylated by multiple different kinases, suggesting that Tau is a
promiscuous kinase substrate. Consequently, it is difficult to be
certain which kinase(s) are responsible for physiological or pathological phosphorylation of Tau in vivo. Kinases that have emerged as
strong in vivo candidates include glycogen synthase kinase-3β
(GSK3β) [29–33] and cyclin-dependent protein kinase-5 (cdk5)
[34,35]; both are expressed abundantly in neurons and associate
with microtubules (cdk5 is anchored to microtubules by Tau [36]).
However, it is likely that more than one kinase participates in the
events leading to hyperphosphorylation, since no single kinase
Table 2
Phylogenetic conservation of kinases implicated in tau physophorylation.
Tau Kinase
Protein accession numbers
%
identity
%
consensus
Human
Zebrafish
Glycogen synthase kinase
3β (GSK3β)
Cyclin-dependent kinase
5 (cdk5)
Mitogen activated protein
kinase 1 (MAPK1, ERK1)
Microtubule Affinity
Regulating Kinase 1
(MARK1)
Casein kinase 1α (CK1α)
NP_001139628
NP_571456
94.5
97.4
AAP35326
AAH85381
96.2
98.6
NP_620407
BAB11813
90.8
93.5
CAH72462
AAI55560
79.3
85.5
NP_001883
NP_694483
99.3
99.3
356
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Tau immunoreactive bands of 68, 64 and 60 kDa present in detergentextracted material containing paired helical filaments [41]. After dephosphorylation of the same material, all six Tau isoforms were resolved
by electrophoresis, showing that hyperphosphorylation of Tau was
responsible for the alteration in its electrophoretic mobility, and that AD
filaments are composed of all six Tau isoforms. Similar analyses of
specimens derived from other types of Tauopathy have revealed
different results. In progressive supranuclear palsy and cortico-basal
degeneration, two major Tau bands of 68- and 64 kDa are derived from
hyperphosphorylation of 4R-Tau species [42,43]. Conversely, material
derived from Pick's disease brain shows two bands of 64- and 60-kDa,
dephosphorylation of which reveals the presence of 3R-Tau species
[43,44]. Interestingly, genetic data shows association between an
extended haplotype of markers across the MAPT locus, and PSP/CBD
[45]; recent data shows that this haplotype favors the splicing of exon
10+ transcripts encoding 4R-Tau [46], suggesting that geneticallydetermined relative over-production of 4R-Tau might be one factor
in the pathogenesis of these sporadic diseases.
2.6. Hereditary fronto-temporal dementia and Parkinsonism linked to
chromosome 17
Definitive evidence that the development of Tau pathology may be
mechanistically important in common sporadic Tauopathies comes
from the study of rare families, in which mutations within the MAPT
gene are sufficient to cause a neurodegenerative disease with
prominent Tauopathy [14,47]. FTDP17 is inherited as an autosomal
dominant trait and can manifest as a Parkinsonian movement
disorder or as a cognitive–behavioral disorder, resembling other
forms of fronto-temporal dementia (reviewed in [48]). To date, 39
different pathogenic MAPT mutations have been identified in FTDP17
families. Mutations clustered around the 5′ boundary of intron 10
alter the regulation of splicing, most frequently resulting in relative
over-production of exon 10+ transcripts and deposition of hyperphosphorylated 4R-Tau isoforms. Missense mutations within exon 10 also
provoke a 4R-Tauopathy, by changing the biophysical properties of
4R-Tau without altering the ratio of splice isoforms; these mutations
also result in 4R-Tau deposition. Finally, mutations outside exon 10
are expressed in both 3R- and 4R-Tau, but may provoke selective
deposition of 3R- or 4R-, or both isoforms of Tau. A variety of
mechanisms has been proposed to account for the pathogenicity of
MAPT missense mutants, including promotion of Tau fibril formation,
impairment of microtubule-binding and introduction or removal of
key phosphorylation sites [49]. Many of the mutants show multiple
functional abnormalities, such as alterations in 4R-/3R-splice ratio and
reduced propensity to promote the stabilization of microtubules.
Although FTDP17 is an uncommon disease, the discovery of
pathogenic MAPT mutations has provided important confirmation
that primary abnormalities of Tau, including alterations in the relative
abundance of 3R- and 4R-isoforms, can result in neurodegeneration
and Tauopathy. Given the close similarity between FTDP17 and
sporadic Tauopathies in some cases, it is reasonable to conclude that
abnormalities of cellular Tau metabolism may be central to neurodegeneration in common sporadic Tauopathies. Consequently, study of
model systems in which Tauopathy is induced by molecular
manipulations that mimic the genetic mechanisms of FTDP17 might
yield important insights into pathophysiology and identify potential
treatment targets for common sporadic Tauopathies.
3. Transgenic mouse models of Tauopathy
A variety of different transgenic lines, over-expressing wild-type
or FTDP17 mutant human Tau, have been described (reviewed in
[50,51]). Several important themes that have emerged from studies of
these animals are briefly considered here.
First, dissociation between NFT formation and neuronal dysfunction or death was demonstrated in some of these models, suggesting
that Tau hyper-phosphorylation, relocalization or oligomerization
may be more critical for the emergence of phenotypic abnormalities in
these models than formation of NFTs. Abnormalities of hippocampal
synaptic protein expression and physiology preceded the formation of
NFTs in mice expressing P301S mutant Tau [52]. Studies using a
different model in which P301L Tau was expressed under an inducible
promoter showed that suppression of transgene expression after the
onset of phenotypic abnormalities prevented neuronal loss and
behavioral abnormalities but did not affect neurofibrillary tangle
formation [53], and there was regional dissociation between NFT
formation and cell loss [54]. In addition, immunization of Tau/Aβ
transgenic mice reduced soluble Tau in the brain but did not affect NFT
formation — the animals showed phenotypic improvement, suggesting that neurobehavioral abnormalities were attributable to a soluble
Tau species rather than NFT [55].
Second, Tau transgenic mice have provided a powerful means to
test the interactions between different biochemical pathways implicated in neurodegeneration. For example, evidence of an in vivo
interaction between Tau and Aβ in the pathogenesis of Alzheimer's
disease was demonstrated by intracerebral inoculation of the
Alzheimer's disease-associated amyloid peptide Aβ1–42, which
enhanced the formation of NFTs in a mouse P301L Tau-expressing
model [56]. Furthermore, P301L Tau/APP double transgenic animals
showed exacerbation of NFT pathology in the presence of the APP
transgene, whereas Aβ plaque formation was unaffected by the overexpression of mutant Tau, suggesting that β-amyloid formation or
another function of APP might be upstream of Tauopathy in this
model [57].
Finally, murine models have provided powerful means to test
hypotheses concerning experimental treatment approaches, and
biochemical interventions aimed at understanding disease pathogenesis in vivo (reviewed in [58]). For example, administration of Li+, an
inhibitor of GSK3, reduced hyperphosphorylation of Tau in vivo and
ameliorated some phenotypic abnormalities [31], and another kinase
inhibitor targeting cdk5, GSK3 and MAPK1 delayed the onset of a
motor deficit in Tau transgenic animals [59]. Other studies have
deployed pharmacological interventions in order to clarify the
potential therapeutic utility of interventions directed at restoring
loss of Tau microtubule-stabilizing function [60], enhancing clearance
of pathological Tau species from neurons by inhibiting refolding
functions of the HSP90 complex to promote Tau degradation [61], and
curtailing a potentially pathogenic neuroinflammatory response by
targeting microglial activation using immunosuppressive drugs [52].
Together, these observations show that experimental genetic
manipulations, which mimic the mechanisms underlying FTDP17 in
humans, provoke phenotypic changes relevant to human Tauopathy
in transgenic animals. These models have been valuable for
hypothesis-driven experiments aiming to elucidate the pathogenesis
of Tauopathy and for testing putative therapeutic approaches.
4. Why would a zebrafish Tauopathy model be useful?
The zebrafish is a small freshwater fish that has been used
extensively in laboratory studies of vertebrate development. Fish
embryos develop externally allowing direct observation of embryogenesis. Many morphological and molecular parallels are shared
between fish and other vertebrates allowing insights gained in the
zebrafish model to be applied to other systems. Stemming from the
use of zebrafish in developmental studies, an extensive array of
experimental methodologies has been developed. Straightforward
techniques are available for over-expression [62,63] or targeted
knockdown of genes of interest [64], allowing the consequences of
altered gene function to be determined readily in vivo. The
deployment of fluorescent reporters [65] allows direct observation
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357
of morphology or physiology of individual cells in vivo [66–68]. The
ability of zebrafish to breed regularly and produce sizeable clutches of
embryos, and the practicable housing of significant numbers of
animals, has allowed development of techniques for large-scale
genetic [69–72] and chemical modifier [73–75] screens. These have
provided numerous novel molecular insights into development,
through unbiased phenotype-driven approaches. More recently, it
has been demonstrated that intact zebrafish larvae provide an in vivo
neurobehavioral platform suitable for screens to find chemical
modifiers of vertebrate behavior [76].
It is possible that applying these types of analysis to zebrafish
models of neurodegenerative diseases would provide novel molecular
insights into disease pathogenesis, and allow identification of
potentially therapeutic compounds. In particular, screening studies
carried out in the intact vertebrate CNS in vivo might uncover drug
targets that are not present in cell culture models commonly used for
discovery-driven approaches, such as pathogenic mechanisms that
are not cell-autonomous, or which are only expressed in enddifferentiated neurons. Consequently, there has been significant
recent interest in the development of zebrafish models of Tauopathy.
to functional sub-specialization, suggesting that the zebrafish may be
a powerful tool in which to dissect multiple cellular roles of Tau. The
two proteins encoded by the zebrafish genes have not yet been
detected, but both mapta and maptb mRNAs were expressed in the
developing CNS [97]. A complex pattern of alternative splicing of the
mapta and maptb transcripts suggests that, like human Tau, zebrafish
Tau isoforms with different numbers of microtubule-binding repeats
are expressed in the CNS, and larger forms of Tau are expressed in the
peripheral nervous system. Interestingly, mapta gives rise to transcripts encoding 4–6 microtubule-binding repeats, whereas maptb is
predominantly expressed as a 3-repeat isoform, raising the fascinating possibility that conserved functions of mammalian 3R- and 4R-Tau
are distributed between the two zebrafish genes [97].
5. Is the zebrafish a suitable organism in which to study Tauopathy?
6.1. Techniques for establishment of transgenic lines
The proposed use of a small aquatic creature to study complex
human neurobehavioral diseases naturally raises questions about
relevance — will it be possible to generate a truly representative
model, with potential to yield important insights into the human
disorder? A variety of considerations suggest that the zebrafish may
be a good model system in which to study human neurological
diseases. With respect to the human CNS, the zebrafish shows
conservation of basic brain organization [77] and many key
neuroanatomical [78,79] and neurochemical [80,81] pathways of
relevance to human disease; the zebrafish CNS contains microglia
[68], cells with astrocytic properties [82,83], oligodendrocytes and
myelin [84–87], and a blood–brain barrier [88], all of which have been
implicated in the pathogenesis of neurological disease. These
observations imply that the tissue environment in the zebrafish CNS
may present appropriate conditions in which to recapitulate pathological changes representative of the human disorders. In addition,
there is a striking degree of phylogenetic conservation of genes
implicated in the pathogenesis of neurodegenerative diseases, and a
variety of other CNS functions [89–96]. This suggests that it will be
possible to provoke neurodegeneration in zebrafish through conserved biochemical pathways that are sufficiently close to those
underlying human disorders that findings in the zebrafish model will
yield relevant insights into diseases mechanisms. It is noteworthy that
the zebrafish genome contains highly conserved orthologues of each
of the kinases implicated in Tau phosphorylation (Table 2 and Fig. 3),
which may be of particular relevance to Tauopathy in view of the
proposed central role of hyperphosphorylation in pathogenesis.
Similar considerations apply to other conserved pathways, and it
seems likely that findings in zebrafish models will be representative of
the mechanisms underlying the pathogenesis of human Tauopathies
and will therefore be relevant to their understanding.
Transgenic zebrafish can be generated by micro-injecting linearized
plasmid DNA into the cytoplasm of one-cell stage embryos [100], but
this gives rise to inefficient genomic integration and significant
mosaicism. Two technical advances, meganucleases and transposons,
have substantially improved the efficiency by which foreign DNA can be
introduced into the zebrafish genome. (i) The meganuclease I-sce1 is an
intron-encoded endonuclease from Saccharomyces cerevisiae [101],
which cleaves DNA at an 18 bp sequence-specific recognition site not
found within the zebrafish genome. In meganuclease-mediated
transgenesis, the transgene plasmid is designed so that the expression
cassette is flanked by I-sce1 sites, and is injected into zebrafish embryos
with I-sce1 enzyme. Integration of a low transgene copy number usually
occurs at a single site, with substantially enhanced efficiency, and
reduced mosaicism, compared with naked DNA injection. This improves
the rate of transmission of the transgene from the F0 to the F1
generation [102] and results in simple Mendelian transmission of
transgenes in subsequent crosses [103]. (ii) Type 2 transposons are
mobile DNA elements encoding a transposase, which mediates excision
of the transposon from the genome and its re-insertion at another
location. The Tol2 transposon, found in the genome of medaka [104], has
been engineered by deletion of the transposase gene to form a nonautonomous mobile element, which can insert into the genome in the
presence of transposase supplied in trans [105]. In this approach to
transgenesis, the transgene expression cassette is flanked by transposon
elements, and injected into zebrafish embryos with mRNA encoding
transposase, resulting in highly efficient, usually multiple, single-copy
integration events. The technique has been used increasingly since its
introduction and subsequent refinement [62], because it is necessary to
inject and screen a relatively small number of embryos in order to
identify stable transgenic lines.
6. Tools for the generation and analysis of zebrafish Tau models
Generation of transgenic Tau zebrafish requires availability of
appropriate methods for introducing exogenous DNA into the germline, and resources to direct MAPT transgene expression in the desired
temporal and spatial pattern.
6.2. Promoter resources for generation of neurodegeneration models
5.1. Zebrafish paralogues of MAPT
Recent work has identified two paralogues of MAPT in zebrafish,
annotated mapta and maptb [97]. Analysis of homology between
zebrafish and mammalian MAP-encoding genes, and examination of
synteny between zebrafish mapt and mammalian MAPT loci, strongly
suggest that the two zebrafish genes arose from duplication of an
ancestral mapt locus. A genome duplication event is thought to have
taken place during the evolution of teleosts [98]; consequently, there
are dual zebrafish paralogues of many mammalian genes [92,99]. It is
possible that divergent evolution of the two mapt genes has given rise
Most transgenic zebrafish lines have been constructed by expressing
a cDNA under control of a characterized heterologous promoter
fragment; cis-acting regulatory regions derived from zebrafish genes
are usually employed for this purpose, because they may be associated
with more reliable transgene expression [106,107]. For the generation of
a Tauopathy model, desirable attributes for the promoter would include
pan-neuronal activity and sufficient expression to provoke pathology.
Three zebrafish promoter elements that drive transgene expression
widely in CNS neurons have been described. (i) A neuronal enhancer
derived from the gata2 promoter was expressed at high levels in the
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Fig. 3. Phylogenetic conservation of kinases implicated in Tau phosphorylation. The sequence figure shows the striking degree of amino acid conservation between human and
zebrafish (A) GSK3β and (B) cdk5. Each panel shows the Human (upper) and zebrafish (lower) protein sequences, aligned using the ClustalW algorithm. Amino acids are shaded to
show identical, conserved and non-similar residues. The catalytic domain of each kinase is indicated by underlining, and important functional sites labeled as indicated.
developing CNS [108]. (ii) The zebrafish huc gene encodes an RNA
binding protein, HuC/D, commonly used as an early neuronal marker
[109] — a 2.8 kb fragment of the proximal 5′ flanking region was
sufficient to drive pan-neuronal expression in embryos [110]. (iii) The
eno2 gene, encoding the neuron-specific γ-enolase isoenzyme, is
expressed at low levels until 60–72 h post-fertilization, after which
high level expression persists into adulthood. The eno2 regulatory
region is complex, containing an untranslated first exon, and an intronic
CpG island that appears important for promoter activity. A 12 kb
fragment of the promoter, including the first intron, was active in
driving reporter gene expression in neurons, including many populations of relevance to disease, throughout the brain and spinal cord from
36–48 h post-fertilization through adulthood [111], and in the retina
and visual pathways [103] (Fig. 4). The optimal promoter for
construction of Tauopathy models is unclear, and each of these
promoters has been deployed in studies of transgenic Tau zebrafish
(see below). It is likely that further development of promoter resources
and other approaches to generation of transgenic animals will yield
other useful reagents for this application.
ductive potential or preventing survival to sexual maturity. One
possible solution would be deployment of a conditionally-expressing
system, for example the binary Gal4–UAS system previously
employed in Drosophila genetics. This system has been successfully
used in zebrafish, with recent modifications attempting to address
toxicity issues caused by the viral trans-activation domain usually
fused to Gal4, and inactivation of the UAS tandem repeats [112,113].
Isolation of useful driver lines for generation of neurodegenerative
disease models is ongoing, and the optimal strategy for deployment of
this technology is uncertain. Driver lines could be constructed by
expressing the Gal4-transactivator fusion protein using small promoter fragments similar to the approach for generating cDNA
transgenic animals; this has been used successfully with the huC
promoter in a larval Tau model [114]. In addition, the highly efficient
genomic integration of transgenes using the Tol2 transposon has
allowed the deployment of approaches in which random integration
of a gal4-expressing enhancer trap gives rise to robust stable
expression of Gal4 under the endogenous regulatory elements of
the gene of insertion [115]. Several such lines have been constructed
and are currently being further evaluated.
6.3. Special considerations for expression of transgenes that provoke
neurodegeneration in larvae
6.4. Assays for fully exploiting transgenic models
In order to fully exploit the potential of the zebrafish for highthroughput screening approaches, it may be necessary to drive Tau
expression at sufficiently high levels that disease pathogenesis occurs
during larval stages of development, when zebrafish can be
accommodated in 96-well plates. However, development of neurodegeneration at this early time point could provide strong selection
against establishment of transgenic lines, by compromising repro-
The power of screening paradigms might be enhanced by
deployment of assays that exploit unique properties of the zebrafish
model. First, direct visualization of neuronal populations of interest in
the zebrafish brain might allow non-invasive automated imaging to
ascertain the morphology, integrity or viability of target cells as an
assay end point. For example, the Tauopathies PSP and CBD are
associated with severe depletion of dopaminergic neurons causing a
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359
Fig. 4. The zebrafish eno2 promoter. The micrograph shows a single confocal plane through the head region of a Tg(eno2:egfp) zebrafish larva at 5 days post-fertilization, illustrating
widespread neuronal expression of the eno2 promoter. Rostral is to left, dorsal up. GFP expression appears green; the section was counterstained using a blue nuclear marker to
facilitate identification of anatomical landmarks. Key: Tel, telencephalon; TeO, optic tectum; Ce, cerebellum; MdO, medulla oblongata; SC, spinal cord; L, ocular lens; PRL,
photoreceptor layer of retina; IPL, inner plexiform layer of retina; GCL, ganglion cell layer of retina; TG, trigeminal ganglion; OC, otic capsule; PLLG, posterior lateral line ganglion;
LLN, axons of lateral line nerve.
Parkinsonian movement disorder that characterizes these conditions [116], and it would be of considerable interest to identify
compounds that protect dopaminergic neurons from abnormal forms
of Tau. Genetic labeling of dopamine neurons using fluorescent
reporters is ongoing, and has proved to be complicated. The promoter
regions of dopamine-neuron-specific genes are large and complex,
such that manageable fragments of the th [117,118] or slc6a3 [119]
promoters have not shown dopaminergic neuron-specific activity in
vivo. Alternative approaches are being undertaken, including identification of conserved enhancers, using large genomic constructs likely
to contain more cis-acting elements, or adopting an enhancer trap
approach to identify genomic integrants that recapitulate endogenous
gene expression patterns. The latter approach has yielded a single
transgenic line in which an integration event in the vmat gene yielded
zebrafish with GFP-expressing catecholaminergic neurons [67]. There
is a large literature on the development of promoter resources
allowing genetic labeling of other specific neuronal populations using
fluorescent reporters, which is outside the scope of the present
review.
Second, neuronal dysfunction presumably precedes cell death in
Tauopathy, and cells in earlier stages of pathological evolution might
present a more tractable therapeutic target than those close to
demise. It would be of considerable value to develop assays that
measure neuronal dysfunction, and which are sensitive to changes in
disease progression earlier in the evolution of pathology. Since genetic
and chemical screening against a zebrafish Tauopathy model would
be carried out in vivo, it might be possible to use automated motor
behavioral assays as a surrogate marker of neuronal function.
Consequently there is much current interest in establishing reproducible and well-characterized behavioral assays for neurodegenerative phenotypes. A recent report showed that an automated motor
behavioral test could be used to identify agents with neuropharmacological properties in a chemical screen [76]. Assays of motor
function have been previously deployed in order to detect changes in
behavior caused by alterations in neurochemistry and dopamine cell
integrity secondary to Parkinson's disease-associated toxins [81,120].
It may be possible to use a similar approach to investigate changes in
behavior preceding demonstrable cell loss in a zebrafish Tauopathy
model, in order to develop an assay to identify chemical modifiers of
early pathological progression.
7. Current transgenic zebrafish models of Tauopathy
Three recent publications have described the first proof-of-principle
experiments showing that expression of human MAPT transgenes can
provoke relevant phenotypes in zebrafish.
In the first publication, a 4R-Tau-GFP fusion protein was
transiently over-expressed in zebrafish larvae, using a gata2 promoter
element [121]. In cultured cells, the fusion protein was phosphorylated and associated with the cytoskeleton, similar to native Tau; in
vitro studies showed that the fusion protein aggregated, validating its
use in constructing an in vivo model of NFT formation. Zebrafish
embryos microinjected with the gata2:MAPT-egfp construct, showed
mosaic neuronal expression of the fusion protein. Some neurons
showed accumulation of fluorescent fibrillar structures resembling
neurofibrillary tangles, in the cell body and proximal axon. The human
Tau-GFP fusion protein was shown to be phosphorylated in the
zebrafish brain by western blot detection of a phospho-S396/S404
epitope, and expression of glycogen synthase kinase-3 in the zebrafish
embryo and adult brain was demonstrated by western blot. This initial
study validated the use of a GFP fusion protein to monitor evolution of
tangle pathology in vivo, and suggested that human Tau is phosphorylated in the larval zebrafish, confirming the prediction that there is
sufficient phylogenetic conservation of endogenous zebrafish kinases
to modify the human protein. No stable lines were derived in this
study.
Subsequently, stable transgenic zebrafish were constructed
expressing human 4R/0N Tau [111], the most abundant Tau species
deposited in progressive supranuclear palsy [43,44]. The transgene
was expressed under transcriptional control of the eno2 promoter
[111]. The full phenotype of these transgenic fish has not yet been
reported, since the initial paper focused on characterization of the
novel eno2 promoter. Abundant expression of human 4R/0N Tau was
found in the CNS, persisting into adulthood (Fig. 5A). In the adult
brain, refractile Tau accumulations resembling neurofibrillary tangles
were found within neuronal cell bodies and proximal axons in CNS
regions of pathological relevance to PSP, including the optic tectum
(Fig. 5B). Stable expression of the Tau transgene into adulthood in this
model will allow analyses designed to test whether age-related
pathology accumulates in these transgenic lines, similar to the
progressive changes seen in the human disease and recapitulated in
some murine models, a potentially important step in validating the
model system. Furthermore, widespread neuronal expression of the
transgene may allow examination of factors involved in cellular
vulnerability to Tauopathy.
More recently, exploitation of the Gal4–UAS system allowed
generation of a Tauopathy model showing a larval phenotype [114].
This is potentially an important advance, since the development of
phenotypic abnormalities at larval stages of development may allow
high-throughput screening, as discussed above. Human 4R/2N-Tau,
harboring a P301L mutation, was expressed from a novel bidirectional
UAS promoter, allowing simultaneous expression of a red fluorescent
reporter in Tau-expressing cells [114]. The high levels of mutant
P301L Tau expression provoked by the huc:gal4-vp16 driver induced a
transient motor phenotype during embryogenesis, likely caused by
peripheral motor axonal developmental abnormalities. At later time
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Fig. 5. Human Tau expression in Tg(eno2:MAPT) zebrafish neurons. A: A western blot was
made with protein lysate from wild-type zebrafish brain (lane 1), control post-mortem
human cortex (lane 2) and Tg(eno2:MAPT) brain (lane 3). The blot was probed using an
antibody to human Tau (upper panel) followed by an antibody to β-actin (lower panel).
Abundant expression of human 4R-Tau is seen in transgenic zebrafish compared with the
six isoforms in normal human brain [111]. B: Sections of Tg(eno2:MAPT)zebrafish brain
were labeled using the antibody to human Tau used in the western blot experiment shown
in panel A and a histochemical reaction yielding a red product. The micrograph shows a
brainstem neuron with dense Tau immunoreactivity in the cell body and proximal axon,
resembling neurofibrillary tangles shown in Fig. 1B [111].
points, after recovery of motor function, enhanced cell death was
observed in the spinal cord of transgenic animals. Abnormal Tau
conformers were detected using an antibody, MC1, which recognizes
a discontinuous epitope present in Tauopathy tissue [122], as early as
32 h post-fertilization; by 5 weeks of age, argyophilic material was
detected in the spinal cord, but the animals were otherwise
phenotypically unremarkable at this time point. Subsequent loss of
transgene expression prevented the examination of later pathological
changes. Tau phosphorylation was detected as early as 32 h postfertilization. Phospho-Tau specific antibodies labeled cells differentially; some phospho-epitopes, such as phospho-T231/S235 and
phospho-S396/S404 were detected widely in Tau-expressing neurons,
whereas others such as phospho-S422 and phospho-S202/T205 were
initially present in only a small subset of Tau-expressing neurons.
Between 2 and 7 days post-fertilization, however, expression of all of
these epitopes became widespread in Tau-expressing cells, suggesting
that Tau had accumulated multiple phosphorylations in a sequential
progression of biochemical changes. Tau phosphorylation was
reduced by application of novel high-potency GSK3β inhibitors that
were designed to target the human enzyme, confirming that
extensive homology between human and zebrafish kinases allows
small molecules to interact with orthologous targets from either
species. This important study was the first detailed phenotypic
description of a stable zebrafish Tauopathy model and showed proof
of principle that biochemical changes characteristic of human
Tauopathy can be recapitulated in larval zebrafish and modulated
by chemical inhibitors.
8. Conclusions — what have we learned and what next?
Examination of the first publications allows preliminary assessment
of the degree to which transgenic zebrafish models of Tauopathy seem
representative of human disorders. Evidence so far suggests that
processing of over-expressed human Tau in zebrafish CNS neurons
results in similar biochemical and pathological changes to those found in
human Tauopathies. These changes include: aberrant localization of Tau
to the somato-dendritic compartment; phosphorylation, with an
ordered (and possibly sequential) acquisition of phospho-epitopes;
adoption of abnormal conformations associated with Tauopathy; and
deposition as argyrophilic material. The availability of zebrafish lines
that express Tau in the brain through adulthood will allow more
detailed biochemical studies of Tau in the zebrafish CNS to characterize
solubility, fibril morphology and further clarify phosphorylation events.
However, these data are encouraging that relevant pathways are
sufficiently phylogenetically conserved between human and zebrafish
to allow recapitulation of cellular processing events relating to Tau in the
zebrafish model.
The pathophysiological consequences of human Tau transgene
expression in zebrafish CNS neurons are less clear. This is partly
because a detailed phenotype has only been reported for a single
transgenic Tau zebrafish line [114]. The motor axonal developmental
abnormality, motor phenotype and cell death reported in this model
have an uncertain relationship to the neuronal dysfunction and
neurodegeneration that characterizes human Tauopathy, on account
of the onset and subsequent resolution of abnormalities during early
development, and the atypical anatomical distribution in the spinal
cord and motor neurons. One recurrent finding from the murine
transgenic disease model literature is that phenotypes exhibited by
cDNA transgenic animals are critically dependent on the promoter
elements used to drive transgene expression. The temporal and
spatial expression pattern of the huC promoter used in this study
directed the atypical pattern of phenotypic abnormalities. However,
this may be unimportant for the purposes of drug and target discovery
relating to cellular mechanisms of Tau toxicity, provided that the
molecular mechanisms underlying neuronal dysfunction and death
are conserved between the model and the disorder. Further work,
including analysis of other zebrafish Tau lines constructed using
different promoter elements, will be necessary to clarify this point.
While detailed phenotypic analysis of the first cDNA transgenic Tau
models is in progress, it is interesting to consider future directions in the
generation of models and approaches for analysis. Use of models based
on over-expression of cDNA encoding human Tau would limit discovery
of novel molecular interventions to those targeting Tau modifications,
Tau deposition or other abnormal effects exerted by Tau on neuronal
function. However, there may be upstream targets related to transcription and splicing that would not be represented in cDNA models. For
example, changes in the cellular 3R-/4R-Tau ratio, caused by alteration
in the regulation of exon 10 splicing, underlie one form of FTDP17 and
may contribute to sporadic Tauopathies, where associated haplotypes
may alter 3R-/4R-Tau ratios [46]. It is not clear whether the human MAPT
locus would be appropriately regulated in transgenic Tau zebrafish,
allowing expression of physiological levels and patterns of alternatively
spliced products. Transgenic zebrafish lines harboring the entire MAPT
gene would therefore be of considerable interest. Furthermore, since
zebrafish evolution may have resulted in the functions of 3R- and
4R-Tau isoforms becoming distributed between two different genes, an
indication of whether alterations in the ratio of these forms can be
pathogenic in zebrafish might be accomplished by morpholino
knockdown experiments.
Initial evidence shows that the zebrafish will most likely be a
predictive platform for the discovery and assessment of Tau kinase
inhibitors, although arguably the greatest potential strength of the
model might be in discovering new therapeutic targets. In order to
gain novel insights from screening approaches, end points that are
independent of presumptions about underlying mechanisms, such
as neurobehavioral analyses or cell death assays, might be deployed.
Furthermore, experimental approaches that can be uniquely applied
in zebrafish larvae owing to their optical transparency, for example
physiological imaging modalities relating to cellular calcium levels,
have the potential to yield important insights from hypothesisdriven experiments that cannot be carried out using other available
models.
In conclusion, the first reports show that construction of
transgenic zebrafish expressing human Tau in CNS neurons is feasible
and suggest that the resulting models will be relevant and useful.
Consequently, there is cause for cautious optimism that novel
zebrafish Tauopathy models may provide important contributions to
ongoing efforts to address these common and devastating diseases.
Q. Bai, E.A. Burton / Biochimica et Biophysica Acta 1812 (2011) 353–363
Acknowledgements
We gratefully acknowledge research grant support from CurePSP —
The Society for Progressive Supranuclear Palsy (grants #441-05 and
#468-08) and NINDS (NS058369).
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Biochimica et Biophysica Acta 1812 (2011) 364–380
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Biochimica et Biophysica Acta
j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / b b a d i s
Review
Investigating regeneration and functional integration of CNS neurons: Lessons from
zebrafish genetics and other fish species☆
Valerie C. Fleisch a,b,⁎, Brittany Fraser b, W. Ted Allison a,b,c
a
b
c
Centre for Prions & Protein Folding Disease, University of Alberta, Edmonton, Alberta, T6G 2M8, Canada
Department of Biological Sciences, University of Alberta, Edmonton, Alberta, T6G 2J9, Canada
Department of Medical Genetics, University of Alberta, Edmonton, Alberta, T6G 2H7, Canada
a r t i c l e
i n f o
Article history:
Received 6 January 2010
Received in revised form 5 October 2010
Accepted 21 October 2010
Available online 28 October 2010
Keywords:
Zebrafish
Regeneration
Retina
Optic nerve
Spinal cord
Brain
Central nervous system
Stem cell
Glia
a b s t r a c t
Zebrafish possess a robust, innate CNS regenerative ability. Combined with their genetic tractability and
vertebrate CNS architecture, this ability makes zebrafish an attractive model to gain requisite knowledge for
clinical CNS regeneration. In treatment of neurological disorders, one can envisage replacing lost neurons
through stem cell therapy or through activation of latent stem cells in the CNS. Here we review the evidence
that radial glia are a major source of CNS stem cells in zebrafish and thus activation of radial glia is an
attractive therapeutic target. We discuss the regenerative potential and the molecular mechanisms thereof, in
the zebrafish spinal cord, retina, optic nerve and higher brain centres. We evaluate various cell ablation
paradigms developed to induce regeneration, with particular emphasis on the need for (high throughput)
indicators that neuronal regeneration has restored sensory or motor function. We also examine the potential
confound that regeneration imposes as the community develops zebrafish models of neurodegeneration. We
conclude that zebrafish combine several characters that make them a potent resource for testing hypotheses
and discovering therapeutic targets in functional CNS regeneration. This article is part of a Special Issue
entitled Zebrafish Models of Neurological Diseases.
© 2010 Elsevier B.V. All rights reserved.
1. Introduction
It had long been assumed that the mammalian central nervous
system (CNS) is incapable of regenerating neurons. However, today it
is widely accepted that sources of adult neurogenesis do exist, and
that these areas have the potential to underpin regeneration. Adult
neurogenesis is localized to the dentate gyrus of the hippocampus as
well as the lateral ventricles of the mammalian forebrain [1–3]. It has
been shown that injury to the CNS (caused by ischemia, seizures,
radiation or neurodegenerative disorders) triggers proliferation of
progenitor cells in the subgranular zone and the subventricular zone
in mammalian animal models and humans. However, in most cases
these newborn cells do not show long-term survival, fail to migrate or
integrate properly into the neuronal network (for a review see [4]).
This lack of regenerative ability seems to have arisen in the course
of evolution; most invertebrates (commonly used models are Hydra
and planarians) as well as a number of anamniotic vertebrates, such as
amphibians and fish, are capable of regenerating whole body parts and
organs (for a review see [5]). Although invertebrates probably show
☆ This article is part of a Special Issue entitled Zebrafish Models of Neurological
Diseases.
⁎ Corresponding author. Centre for Prions & Protein Folding Disease, University of
Alberta, Edmonton, Alberta, T6G 2M8, Canada. Tel.: +1 780 492 1087.
E-mail address: valerie.fl[email protected] (V.C. Fleisch).
0925-4439/$ – see front matter © 2010 Elsevier B.V. All rights reserved.
doi:10.1016/j.bbadis.2010.10.012
the highest degree of regenerative power, lack of genetic tractability as
well as the absence of higher level brain centres are major drawbacks
for their use as animal models in CNS regeneration research. Among
vertebrates, a similarly high regenerative potential is found in
amphibians, such as the newt or Xenopus, and in fish. Newts are
extremely difficult to maintain and breed under laboratory conditions,
and genetic tools have only been developed recently. Xenopus and
zebrafish are both popular animal models for CNS regeneration
research, but this review will focus on the teleost model zebrafish.
Zebrafish are very potent in regenerating a variety of tissues.
Traditionally, the zebrafish has been used as a model for studying the
regeneration of the fins [6]. Since the retina is the most easily
accessible part of the CNS, and morphology and function of the retina
are basically conserved among vertebrate species, zebrafish have also
been extensively used for studying the regeneration of the retina and
optic nerve (reviewed below). Moreover, zebrafish have also
increasingly been used to study regeneration of other nervous tissues,
such as brain nuclei and the spinal cord. In contrast to newts, zebrafish
are very easy and cost-effective to breed and produce high numbers of
offspring [7]. Moreover, the abundance of genetic tools, including
transgenic lines, knock-down (morpholino antisense technology [8]),
mutants and novel knock-out (zinc finger nuclease [9,10]) techniques
and microarrays, facilitate research aimed at identifying molecular
pathways underlying regenerative processes. Access to zebrafish
embryos is easy, as larvae develop outside the mother, and
V.C. Fleisch et al. / Biochimica et Biophysica Acta 1812 (2011) 364–380
transparency of larvae enables in vivo tracking of regenerating cells.
Zebrafish also offer very powerful behavioral and electrophysiological
tools to assess functional integration of regenerated neurons/axons
[11,12].
Studying the exact mechanisms of how anamniotes such as
zebrafish utilize stem cells to replace neurons of the CNS is valuable
for helping us to understand why humans/mammals mostly lack this
regenerative ability and more importantly, will provide us with ideas
of how human CNS tissue could be stimulated to regenerate after
injury. This type of knowledge will be crucial for advancing stem cell
therapeutics. As direct transplantation of stem cells has been proven
inefficient for restoring missing/damaged tissues, future approaches
might involve switching existing dormant progenitor cells or even
terminally differentiated cells back to a “stem cell mode”.
In order to address this scientific puzzle, a number of fundamental
questions immediately come to mind: What is the identity of the stem
cells giving rise to newborn neurons — are these cells the same ones
across species and across different tissues? Is the limitation on CNS
regeneration intrinsic to the neuron or do extrinsic signals control
regeneration (role of glia cell in inhibition versus promotion of
regeneration)? What are the molecular pathways underlying dedifferentiation of glial stem cells and specification of new neurons? Are
regenerated neurons functionally integrated into the CNS and how is
that achieved?
We are beginning to address these questions in zebrafish models
of CNS regeneration, and the present review aims to provide a
summary of the (partial) answers that have arisen from these studies.
2. Regeneration of various CNS tissues
Studies of neural regeneration in zebrafish have been carried out in
various CNS tissues. We review each in turn, dividing the topics into
retina, optic nerve, spinal cord, and brain nuclei.
2.1. Retina
Ocular disorders involving degeneration of cells in the neural
retina are exceedingly common. Death of rod and/or cone photoreceptors is found in retinal dystrophies, such as AMD (age-related
macular degeneration), Retinitis Pigmentosa, Usher Syndrome or
Leber Congenital Amaurosis. Death of retinal ganglion cells is the
largest contributor to vision loss in glaucoma, a condition affecting the
optic nerve. Cells lost from the retina cannot be replaced, as the
mammalian retina does not have intrinsic repair processes, rendering
the patient severely visually impaired or even completely blind.
In contrast to mammals, anamniotic vertebrates like zebrafish
have amazing regenerative capabilities, including the capacity to
regenerate any cell type in the retina. Cells are continuously added to
the zebrafish retina as it expands throughout the life of the fish. This
unique feature of teleost fish requires that sources of neurogenesis are
maintained even in the adult. In the intact retina, progenitor cells have
been classified as being localized to two distinct cellular compartments [13,14]. The CMZ (ciliary marginal zone) gives rise to all retinal
cell types, whereas rod photoreceptors arise from a separate lineage of
CMZ-derived Müller glia [13,15,16]. Neuronal progenitors of the CMZ
express a number of markers such as n-cadherin, components of the
Notch–Delta signalling pathway, rx1, vsx2/chx10 and pax6a [16].
Proliferating Müller glia cells in the INL (inner nuclear layer) produce
rod progenitors which subsequently migrate to the ONL (outer
nuclear layer), where they differentiate into mature rod photoreceptors [15,17,18]. Cells within both of these compartments show
characteristic features of stem cells, including asymmetric division
and self-renewal. An additional population of stem cells is activated
upon retinal injury and subsequently produces progenitors for
regenerating lost retinal cells (for a review see [16,19]). It has been
shown that in fish, damage to the retina induces the formation of
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columns of proliferating cells, termed “neurogenic clusters”, which
express a similar set of stem cell markers as progenitor cells residing
in the CMZ such as pax6, vsx1, notch-3 and n-cadherin [20–23]. Vihtelic
et al. showed that PCNA (proliferating cell nuclear antigen, a marker
for proliferation) positive cells can be detected as early as one day
after light-induced photoreceptor cell death in the INL (inner nuclear
layer) in albino zebrafish. These progenitor cells migrate to the ONL
(outer nuclear layer) within 96 h, where they differentiate into rod
and cone photoreceptors [24,25]. The identity of the cells comprising
these clusters was resolved only recently when several researchers
found evidence that Müller glia are the major source of stem cells in
the adult zebrafish retina.
2.1.1. Retinal astrocytes (Müller glia cells) function as stem cells in the
regenerating adult zebrafish retina
Studies in recent years have dramatically changed our view of
astrocytes as “mere support cells” or “neuronal glue”. Astrocytes are
now viewed as highly important cells with a number of complex
functions. Astrocytes are found in all tissues of the CNS (e.g. cerebellar
Bergmann glia, retinal Müller glia) and have a number of established
roles in metabolic support for neurons, K+ and neurotransmitter
uptake, synaptogenesis, angiogenesis and maintenance of the brain–
blood barrier (for a detailed recent review see [26]). Groundbreaking
studies of neuronal precursors in the CNS have elucidated the
existence of yet another type of glia cell [27,28]. These cells, exhibiting
neuronal stem cell features, are located in adult neurogenic regions
(the adult SVZ, subventricular zone and SGZ, subgranular zone). They
have been shown to give rise to newborn olfactory neurons [29–31]
and granule neurons [32–34].
The brain is not the only CNS tissue containing a large number of
astrocytes. Müller glia of the retina perform similar functions as other
glial cells in the brain, including structural and metabolic support of
neurons, ion buffering, and neurotransmitter homeostasis.
Studies in the chick retina [35,36] as well as experiments using
transgenic zebrafish lines in combination with lineage tracing have
revealed that, in addition to these basic support functions, Müller glia
cells act as retinal stem cells after injury. Following photoreceptor cell
death, they undergo morphological as well as gene expression changes.
Thummel et al. have shown that Müller glia cells dedifferentiate upon
injury as they lose expression of cell-type-specific markers such as GFAP
(glial fibrillary acidic protein) and GS (glutamine synthetase) [37]. Signs
of dedifferentiation include re-expression of BLBP (brain lipid binding
protein), up-regulation of components of the Notch–Delta signalling
pathway and redistribution of N-cadherin to the basolateral plasma
membrane [16].
In succession, several groups used lineage tracing via transgenic
expression of GFP in Müller cells in combination with immunocytological stainings and BrdU labelling to provide evidence that Müller cells
not only respond to injury, but also contribute to the stem cells of the
retina and give rise to neuronal progenitors (Fig. 1). Fausett and
Goldman used the α1T transgenic line, driving transgene expression in a
subpopulation of proliferative Müller glia cells after retinal injury [38].
BrdU labelling experiments revealed a dramatic increase of BrdU+ cells
in the INL at 2 dpi (days post-injury), preceding a rise in BrdU+ cells in
the ONL at 3 dpi. Most of these proliferating cells were GFP+, had a
Müller cell-like morphology and expressed the Müller cell markers
GFAP and GS. BrdU labelling at a later time point, 2 dpi, revealed staining
in the ONL and ganglion cell layer (GCL), suggesting migration of INL
precursors. The progenitors that had presumably arisen from reactive
Müller glia cells started to express neuron-specific markers after 7 dpi,
and were localized to the INL, IPL and GCL. BrdU addition at 4 dpi and
evaluation of cells at 60 and 180 dpi revealed cells expressing markers of
bipolar and amacrine cells, Müller glia and cone photoreceptors [39].
Bernardos et al. used a gfap:GFP transgenic zebrafish line
harbouring the Müller cell-specific gfap promoter driving GFP
expression specifically in Müller glia cells [40]. Following light
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V.C. Fleisch et al. / Biochimica et Biophysica Acta 1812 (2011) 364–380
Fig. 1. Radial glia throughout the CNS are a source of stem cells. Here we depict retinal Müller glia cells as an example of radial glial cells of the CNS. The retina of a 4 dpf zebrafish
larva with its three nuclear layers (GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer) is shown as a reference for the location of retinal progenitors (A).
Differentiated radial glia cells spanning the larval (and adult) zebrafish retina (B) become activated upon injury, dedifferentiate and start to proliferate (C). Neuronal progenitors of
all retinal cell lineages arise from these activated cells (D), and migrate to specific retinal layers where they differentiate into neurons (E).
damage, the number of GFP+ Müller cells increased 3 fold up to 5 dpi,
indicating a proliferative response. Coinciding with a peak in GFP+
cells, the expression of the progenitor cell markers pax6, pcna and crx
(a marker for photoreceptors progenitors) peaked between 1 and
5 dpi. The spatio-temporal distribution of pax6+ and crx+ cells was
consistent with a transition from pax6 to crx expression in progenitors
as they migrated from the INL to the ONL. After 1 month, cones and
rods regenerated completely and Müller cell density fell back to
unlesioned levels.
Morris et al. specifically induced death of cone photoreceptors by
using the pde6cw59 mutant zebrafish line, harbouring a mutation in the
cone-specific phosphodiesterase gene. BrdU exposure 4 h before
sacrifice and subsequent CAZ (carbonic anhydrase, a marker for
Müller glia cells) staining revealed a significant increase of BrdU+ cells
in the INL, partly co-localizing with CAZ. These results suggest that at
least a subset of Müller glia cells had re-entered the cell cycle [41].
Studies using morpholino-mediated knock-down of PCNA [42]
provided evidence that progenitor cell formation in zebrafish retina
after injury is dependent on proliferation of Müller glia cells. PCNA
knock-down prior to light damage inhibited proliferation of Müller
glia cells and resulted in decreased levels of GS expression as well as
Müller cell death. Importantly, rod photoreceptors and short and long
single cones could not be regenerated in morpholino-injected
zebrafish larvae [42]. Assuming the effects of PCNA knock-down are
primarily within the Müller glia, these data strongly supports the
hypothesis that these cells are the source of retina stem cells.
Several researchers showed that damage to photoreceptors
triggers a regenerative response through activation of Müller glia
cells. Initially, researchers had speculated that apoptotic photoreceptors would provide a molecular signal to Müller glia cells, thereby
activating dedifferentiation and proliferation. However, Fimbel et al.
showed that damage to the GCL and INL, without substantial damage
to photoreceptors, likewise causes Müller cells to re-enter the cell
cycle and generate retinal progenitors [43]. Low doses of intravitreal
ouabain injections caused GCL and INL death. At 1 day post-injury
(dpi), PCNA expression was seen in Müller glia cells, and by 5 dpi, its
expression shifted to neuronal progenitors expressing the olig2:egfp
transgene. After 60 dpi, 75% of ganglion and amacrine cells had
regenerated [43].
Various studies support Müller glia cells as one source, perhaps
even a major source, of progenitor cells in the zebrafish retina. Müller
glia cells are activated following retinal damage and subsequently
dedifferentiate, proliferate and give rise to different retinal cell types
(Fig. 1). As Müller glia cells are also found in the mammalian/human
retina, understanding which molecular cues are required to turn a
quiescent Müller glia cell into a retinal stem cell will be crucial for the
development of potential novel therapeutics which could be applied
to trigger regeneration in the human retina. Although under normal
physiological conditions, mammalian Müller glia cells do not re-enter
the cell cycle in response to injury [44–46], it has been speculated that
the human retina may have some regenerative potential. Several
studies have reported the presence of BrdU+ photoreceptors after
retina damage or after the addition of growth factors [47–51].
Moreover, the human retina may contain a compartment similar to
the zebrafish CMZ. In retinal explants, cells of the human retinal
margin as well as the INL re-enter the cell cycle when EGF (epidermal
V.C. Fleisch et al. / Biochimica et Biophysica Acta 1812 (2011) 364–380
growth factor) is added to the culture medium [52]. Thus the human
retina may harbour quiescent stem cells, which can be triggered to reenter the cell cycle by the addition of growth factors.
Unfortunately very little is known about the molecular pathways
leading to the cell-biological changes inducing the activation of Müller
glia cells. In recent years, a number of microarray studies focussing on
temporal expression changes during retina regeneration in zebrafish
have been conducted and have identified several candidate genes and
pathways presumably implicated in the regeneration process. Data
collected in these studies will be presented in the next section of this
review, and an overview of the molecular components that have been
identified can be found in Table 1.
2.1.2. Molecular pathways involved in retinal regeneration
In order to identify candidate genes involved in the regenerative
response in the zebrafish retina, a number of microarray-based
studies have been performed. While one study used surgical removal
of a small piece of the retina [53], mostly light damage paradigms have
been applied to cause retinal injury [54–57]. Microarray analysis
conducted on mRNA isolated from whole retina showed significant
changes in expression of genes implicated in the cell cycle, cell
proliferation, cell death, DNA replication, general metabolism and
axonal regeneration as expected [37,53,54,56]. Genes encoding the
opsin proteins were decreased in expression, reflecting death of
photoreceptors, whereas cell cycle genes increased their expression,
corresponding to progenitor proliferation [54]. As regenerationspecific responses are expected to occur in specific cell types (Müller
glia cells and emanating progenitor cells) expression analysis of RNA
extracted exclusively from those cells was expected to reveal novel
regeneration-specific candidate genes. Craig et al. examined the
expression changes in regenerating photoreceptor cells, by combining
light damage through continuous light of low intensity with lasercapturing of ONL tissue containing photoreceptors and their progenitors [55]. Samples collected at early time points after light damage
showed decreased expression of photoreceptor-specific genes (such
as the opsins) and up-regulation of stress response genes, indicative
of dying photoreceptors. In a second step, transcript changes at 24–
48 h post-injury were examined, as they were thought to represent
changes in progenitor cells migrating to the ONL. Differentially
expressed genes found in this group included the transcription
factors sox11b, fos, jun and pax6a as well as genes encoding secreted
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growth factors such as l-plastin, CC chemokine, galectin family
members, progranulin family members and midkines. In situ
hybridization and immunohistological experiments of selected
candidate genes showed that lgals9l1 (a member of the galectin
family) and progranulin-a were exclusively expressed in microglia,
and lgals1l2 in microglia as well as Müller glia cell, confirming the
specificity of the approach.
Whereas Craig et al. had isolated Muller glia cell-derived
progenitor cells from the ONL, Qin et al. used the transgenic zebrafish
reporter line Tg(gfap:GFP)mi2002 to directly isolate Müller glia cells
[57]. Gene expression profiles from intact and light-lesioned retinas at
8, 16, 24 and 36 h post-treatment were compared. A set of 953 genes
being differentially expressed was identified and subdivided into 3
clusters, depending on their temporal expression patterns. One set of
transcripts was up-regulated immediately after the lesion, and
included mainly genes implicated in translation and protein biosynthesis. A number of DNA replication and cell cycle genes were upregulated slightly delayed, reflecting re-entry of Müller cells into the
cell cycle. Among the transcripts down-regulated after lesion were
genes involved in chromatin assembly and ion homeostasis, consistent with dedifferentiation of Müller glia cells. As expected, an upregulation of cell cycle genes and growth factors such as pcna
and pdgfa (platelet-derived growth factor) was observed as well.
Interestingly, despite taking such a specific approach, Qin et al.
identified two candidate genes, hspd2 (heat shock 60-kDa protein 1)
and msp1 (monopolar spindle 1), which are not only essential for the
regeneration of the retina, but likewise important for fin and heart
tissue renewal in zebrafish [57].
Similar to hspd2 and msp1, the heparine-binding growth factors
midkines a and b seem to play a significant role in regeneration of a
variety of tissues in the zebrafish (retina, heart and fin) [54,58,59].
Photoreceptor apoptosis after light damage causes midkine expression
pattern changes in the zebrafish retina [54]. In addition to their
developmental expression (midkine-a in progenitors, Müller cells and
horizontal cells and midkine-b in retinal ganglion and amacrine cells),
both midkines are expressed in proliferating Müller glia cells and their
progeny, and midkine-b is additionally found in HCs [54].
Other candidate genes identified in microarray experiments and
hypothesized to be involved in the switch from differentiated Müller
glia cell to proliferating retinal stem cell are stat3 (signal transducer
and activator of transcription 3), ascl1a (ash1a) (achaete/scute
Table 1
List of molecular cues involved in retina regeneration in the zebrafish.
Gene
Classification
hspd1 (heat shock
60 kDa protein 1)
mps1 (monopolar spindle 1)
Mitochondrial protein chaperone involved
in stress response
Kinase involved in mitotic checkpoint
regulation
mdka (midkine-a)
(secreted) heparin binding growth factor
mdkb (midkine-b)
(secreted) heparin binding growth factor
ascl1a (ash1a) (achaete–scute Proneural gene
homolog 1a)
notch3
Component of Notch–Delta signalling
deltaA
Component of Notch–Delta signalling
(notch ligand)
pax6b
Transcription factor (neurogenesis,
regeneration)
stat3 (signal transducer and
Transcription factor
activator of transcription)
ngn1 (neurogenin 1)
Transcription factor
olig2 (oligodendrocyte
bHLH transcription factor
transcription factor 2)
lgals9l1-, 2 (galectin family)
Secreted growth factor
progranulin-a
Secreted growth factor
Action
Lesioning
paradigm
Reference
Formation of retinal stem cells from
dedifferentiating Müller cells
Proliferation of photoreceptor progenitors
Light damage
[57]
Light damage
[57]
Not clear yet
Not clear yet
Transition of Müller cell into proliferative stem cell
Light damage
Light damage
Surgical excision
[54]
[54]
[61]
Progenitor proliferation
Differentiation
Surgical excision
Surgical excision
[61]
[61]
Hypothesized role in proliferation and migration
of neuronal progenitor cells
Hypothesized to trigger Müller cell proliferation in the damaged
retina (has been shown to do so in the undamaged retina)
Hypothesized role in “re-differentiation” of Müller cells
Hypothesized role in maintaining Müller cells in a
dedifferentiated state
Up-regulated 24–48 h post-injury in microglia and Müller cells;
exact function unknown
Up-regulated 24–48 h post-injury in microglia; exact function
unknown
Light damage
[37]
Light damage
[56,60]
Light damage
Light damage
[37]
[37]
Light damage
[55]
Light damage
[55]
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homolog 1a) and members of the notch–delta family. Stat3 expression
is increased in photoreceptors and Müller glia cells following light
damage [56]. Interestingly, only a subset of Stat3+ Müller glia cells
co-label with PCNA, which led Kassen et al. to hypothesize that Stat3
might act as a molecular cue, triggering MCs to proliferate after injury.
In a recent publication, Kassen et al. identified a potential upstream
regulator of Stat3, CNTF (ciliary neurotrophic factor). CNTF activates
Stat3 to induce Müller cell proliferation in the undamaged retina [60];
whether or not it plays a similar role in the injured retina remains to
be investigated.
Yurco and Cameron found up-regulation of members of the notch–
delta family, as well as ascl1a (ash1a) in their microarray analysis of
differentially regulated genes after excision of a patch of adult
zebrafish retina. Importantly, ascl1a (ash1a) was up-regulated before
components of Notch–Delta signalling showed increased expression.
They proposed a model in which Müller glia cells start to express
ascl1a (ash1a) after injury and subsequently become divided into
two sub-populations, a notch3-expressing population consisting of
proliferating progenitors and a deltaA expressing population exiting
the cell cycle and differentiating into neurons [61]. Morpholino
knock-down of ascl1a (ash1a) inhibits Müller glia cell activation and
consequently regeneration [62].
Although ngn1 (neurogenin-1) is expressed in proliferating
progenitors and the transgene olig2:EGFP in dedifferentiated Müller
glia cells [37], nothing is yet known about their role in retina
regeneration.
2.1.3. Open questions
The unique capability to regenerate all retinal cell types, combined
with tractable genetics, makes zebrafish an ideal model for deciphering the molecular pathways underlying regeneration. Understanding
zebrafish biology is valuable for developing future therapeutic
interventions in human retinal disease. As evidence accumulates
that the molecular components and stem cells implicated in retinal
regeneration might be similar to those involved in regeneration of
other tissues, retinal regeneration research will help us build our
understanding of the general mechanisms of regenerative processes.
Despite our progress a number of open questions remain.
Differentiated Müller cells re-enter the cell cycle after injury to
produce retinal progenitors; however, little is known about the
extrinsic signals regulating lineage specification. In the undamaged
retina, Müller glia cell-derived progenitors in the INL primarily give
rise to rod photoreceptor progenitors; injury seems to induce a “fate
switch” for Müller glia cells, turning them into progenitor cells for
other retinal cell types. How this is achieved remains largely
unknown.
Another unresolved question is if all or only a subset of Müller glia
cells are multipotent and can give rise to retinal progenitors. pax6, a
gene associated with neurogenesis capacity and multipotency, is
expressed at low levels in all differentiated Müller cells in the
zebrafish retina; this may indicate that all Müller cells are able to
dedifferentiate. Additionally, Fimbel et al. showed that the majority of
MCs (90%) express PCNA at 3 dpi in the damaged retina [43].
Alternatively, there is compelling evidence indicating that it is only
a subset of MCs switching to the progenitor fate. Kassen et al. showed
that Stat3 is expressed only in a subset of Müller glia cells during
regeneration [56]. Similarly, only a subset of BrdU+ cells in the INL colocalized with the Müller cell-specific marker CAZ upon light damage
in the study conducted by Morris et al. [41]. Thummel et al. observed
two distinct groups of MCs in the gfap:EGFP transgenic zebrafish line
after damaging the zebrafish retina with intense light. While one
group of EGFP+ MCs maintained normal Müller cell morphology and
did not co-label with PCNA, another set of cells showed a reduced
amount of EGFP expression and did co-label with PCNA. The second
group likely represents MCs re-entering the cell cycle and dedifferentiating [42].
Although there is evidence that damage to photoreceptors and
other cell types of the retina, trigger the regeneration response, the
identity as well as the source of the actual “trigger signal” is not
known. Fausett and Goldman speculated that the molecular signal
arises from cells near the actual lesion site, as 90% of Müller glia cells
next to the lesion site are GFP+, whereas at sites far from lesion,
almost none are GFP+ [39]. Evidence in mouse models suggests that
photoreceptors signal damage to Müller glia through endothelin
receptors [63].
Finally, it is worth mentioning that Müller glia cells are not the
only glial cell population in the zebrafish retina. Microglia are
phagocytic cells that secrete pro- or contra-inflammatory signals to
either inhibit or promote neuronal repair and regeneration [64].
Microglia hypertrophy at 24 h after light damage to photoreceptors
has been shown. Subsequently, they become polymorphic and
migrate into the ONL [16,55]. Craig et al. found members of the
galectin and progranulin families up-regulated after light damage
[55]. Progranulin-a, lgals1l1 as well as lgals1l2 are expressed in
microglia thus suggesting that microglia likely carry out an important
role in retina regeneration and warrant further study.
2.2. Optic nerve
Injuries of the optic nerve have a highly detrimental effect on a
patient's life, severely impacting vision and ultimately leading to
complete blindness in a significant percentage of cases. Damage to the
optic nerve can occur by either trauma (e.g. head trauma) or can be
caused by diseases leading to death of retinal ganglion cells and
subsequent axon degeneration, such as general neurodegenerative
disorders (e.g. multiple sclerosis) or ocular disorders. The most
prominent of the latter is glaucoma in which increased intraocular
pressure is thought to lead to optic nerve damage. Glaucoma is the
cause of 15–20% of vision loss worldwide and thus treatment and cure
are major goals of public health research.
Successful therapeutic interventions aim to halt nerve degeneration and ideally, trigger regeneration of retinal ganglion cells and most
importantly, axon regeneration. However, mammals (including
humans) are unable to regenerate an injured optic nerve under
normal physiological circumstances. After injury, RGCs show only
transient sprouting and no long-distance regeneration. Compiling the
findings of a great number of studies in rodent models, we can
conclude that failure of RGC axons to regenerate can be attributed to
two major reasons:
1) Existence of a non-permissive/inhibitory environment and
2) Lack of growth-promoting molecules (in the environment or
intrinsic to RGCs).
Experimental therapeutic strategies typically target one of these two
causes, either by trying to counteract inhibitors of axon re-growth or by
activating the neuron-intrinsic capacity to regenerate (for a review see
[65]). Studies in several species have shown that macrophage activation
(e.g. by injuring the lens or using pro-inflammatory agents) can
promote (partial) axon regeneration [66–69]. More recently, researchers are trying to apply combinatorial treatments. Using a gene therapy
approach, Fischer et al. showed that activation of the RGC's intrinsic
growth state while simultaneously overcoming inhibitory signals,
significantly enhances axonal regeneration [70,71]. However, full
regeneration has not been achieved in any rodent animal models and
it is not clear if regenerated axons are able to correctly pathfind and
innervate targets in a precise fashion. The development of novel
therapeutic strategies certainly requires a thorough understanding of
the molecular basis of optic nerve degeneration and regeneration (or
the lack thereof).
Clearly, alternative vertebrate animal models with an intrinsic
capacity for regeneration will help us decipher how cell-intrinsic and
micro-environmental inhibitory and permissive signals/factors drive
V.C. Fleisch et al. / Biochimica et Biophysica Acta 1812 (2011) 364–380
RGC axon regeneration. Interestingly, the molecular and cellular
reactions to optic nerve injury observed in mammalian models
seem to be inversed relative to fish in many aspects. In zebrafish
we generally find an abundance of growth-promoting molecules.
Inhibitory molecules, if expressed, usually exhibit a different pattern
and/or timing of expression. This might partly be due to the fact that
the eye continues to grow throughout the lifespan and thus the visual
system is primed to continuously deal with newborn RGCs extending
their axons to the optic tectum. It seems logical that signalling cues
expressed during development will still be abundant in adulthood so
they can be read by newborn RGCs. It has been clearly shown that
after ON crush or lesioning, RGCs start to re-express molecules that
they also express during embryonic development. These include
cytoskeletal proteins, membrane-bound recognition molecules of the
immunoglobulin family (e.g. NCAM, L1 homologs) and GAP-43
(growth-associated protein 43) (for a review see [72–74]). GAP-43
is localized to growth cones and presynaptic terminals of developing
as well as regenerating axons [75]. Kaneda et al. examined the
expression patterns of GAP-43 and its phosphorylated isoform (which
is known to be active at growth cones) in the regenerating zebrafish
optic nerve and observed a biphasic increase of phospho-GAP-43.
Interestingly, the two distinct phases (the first one a steep increase in
expression after 7–14 days, the second one a long plateau increase
after 30–50 days) coincided with the recovery of two visuallymediated behaviors (OMR and chasing behavior) [76].
In the following section we will discuss recent studies aimed at
achieving an understanding of the molecular basics of optic nerve
regeneration in the zebrafish (see Table 2 for a list of genes).
2.2.1. Molecular signals/pathways implicated in optic nerve regeneration
2.2.1.1. Growth-promoting signals. Regenerating RGCs need two types
of “positive cues” enabling them to extend their axons to their
respective target regions: 1) molecular signals within the cell,
switching (or maintaining) the cell to (in) a growth state, and 2)
permissive and growth-promoting signals in the environment of the
optic tract and tectum, allowing axon extension, pathfinding and
correct target innervations.
2.2.1.1.1. Intrinsic growth-promoting factors. Whereas zebrafish
continue to express a variety of genes needed for RGC development
throughout their lifetime, mice often down-regulate their expression
prenatally.
In mice, several lines of evidence show that CNTF (ciliary
neurotrophic factor) acts as an RGC-intrinsic axon growth factor and
changes in CNTF expression levels during ON injury underlie RGC
apoptosis. In untreated mice, CNTF receptor is lost from RGC axons
after optic nerve crush and glial cells (astrocytes and oligodendrocygtes) start to express CNTF shortly after lesioning. It is thought that
the loss of CNTF receptor on RGCs renders them insusceptible, leading
to cell death, while the appearance of CNTF in glial cells promotes
scarring [77]. These processes can be stopped by inducing intraocular
inflammation, which induces retinal astrocytes to release CNTF,
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switching RGCs to a regenerative state [78] or intravitreal injections
of exogenous CNTF [79]. In cultured goldfish RGCs, CNTF in
combination with two molecules secreted by optic nerve glia,
axogenesis factor-1 (AF-1) and AF-2, drives axon regeneration [80].
In zebrafish, CNTF is known to play roles in photoreceptor
neuroprotection and induction of Müller glia cell proliferation in the
retina of undamaged zebrafish [60]. Its implication in ON regeneration
however has not been studied thus far.
Another class of proteins acting as RGC-intrinsic outgrowth
molecules are the reggies (or flotillins). Morpholino-mediated
combinatorial knock-down of the three zebrafish reggie orthologs
1a, 2a and 2b results in an overall reduction in RGC axon regeneration
[81,82]. Similar to the situation in zebrafish, knocking down reggies in
mouse hippocampal and N2a cells inhibits neuronal differentiation
and neurite extension in vitro [81]. These findings indicate that this
class of scaffolding proteins is essential for ON regeneration, most
likely by acting as microdomains for the assembly of multiprotein
signalling complexes recruited during regeneration.
Members of the KLF (krüppel-like factor) family are clearly
implicated in successful ON regeneration. They had been identified
initially by microarray analysis of changes in gene expression during
ON injury in zebrafish. Veldman et al. had used laser micro-dissection
to isolate RGCs from uninjured and 3-day post-injury zebrafish and
identified 347 up-regulated and 29 down-regulated genes. Out of six
genes chosen for further investigations using morpholino-mediated
knock-down, two out of those six, KLF6a and KLF7a, were necessary
for robust RGC axon re-growth [83]. The same research group recently
also identified KLF4 as a key player responsible for the loss of axon regrowth capacity in adult mice [84]. Mice with a tissue-specific loss of
KLF4 in RGC cells were subjected to ON crush and assessed for
regeneration after 2 weeks. Strikingly, knock-out mice showed an
increase in the number of regenerated axons, however axons did not
regenerate fully. The authors propose that different members of the
KLF family act in concert to regulate the regenerative capacity of CNS
neurons in mice.
2.2.1.1.2. Growth-promoting molecules secreted from a permissive
environment. In addition to RGC-intrinsic molecular signals, cues from
the ON microenvironment play a significant role in axon outgrowth.
Glial cells are major players in axonal regeneration, as they secrete a
variety of growth-promoting molecules. Examples are axogenesis
factors 1 and 2, which have been shown to activate the expression of
growth-related molecules via a purine-sensitive pathway in the
goldfish [80]. Goldfish and zebrafish oligodendrocytes up-regulate the
expression of members of the immunoglobulin family, such as L1, P0
and Contactin1a, after ON lesion [72,85,86], suggesting roles in axon
regeneration and re-myelination.
Interestingly, not only do glial cells seem to secrete permissive or
inhibitory factors into the optic nerve microenvironment, but so do
photoreceptors. Matsukawa et al. isolated purpurin as a candidate
gene important in ON regeneration in goldfish by screening a cDNA
library for genes up-regulated during regeneration. The site of
purpurin expression was shown to be goldfish photoreceptors [87].
Table 2
Molecular cues implicated in successful optic nerve regeneration in the zebrafish.
Gene
Classification
Action
Reference
reggie-1a, 2a, 2b
Microdomain scaffolding protein (assembly
of multiprotein complexes)
Growth factor
Transcription factors
Growth factors
Retinol-binding protein
Axon guidance molecule
Sulfated glycosaminoglycan
Repellent axon guidance molecule
Repellent axon guidance molecule
Regulate axon regeneration, axon outgrowth and neuronal differentiation
[81]
Drives axon regeneration in combination with axogenesis factors 1 and 2
Thought to regulate axon regeneration
Activate expression of growth-related molecules
Induces neurite outgrowth
Pathfinding
Pathfinding
Pathfinding
Establishes retinotopy during regeneration
[60,80]
[83,84]
[80]
[87,88,109]
[89]
[100]
[104,105,110]
[106–108]
CNTF (ciliary neurotrophic factor)
KLF 6a, 7a (krüppel-like factor)
axogenesis factor 1, 2
purpurin
netrin-1
Chondroitin sulfates
tenascin-R
ephrin-A
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Purpurin is a retinol-binding protein, and bound to retinol, induces
neurite outgrowth in retinal explant cultures. This outgrowth can be
inhibited by adding an RALDH2 (retinaldehyde dehydrogenase)
inhibitor (disulfiram). This has led to the speculation that the action
of purpurin might be mediated by retinoic acid synthesis. Nagashima
et al. tested this hypothesis by looking at the expression levels of
components of the retinoic acid synthesis pathway, such as RALDH2,
CYP26a1, CRABPs (cellular retinoic acid binding protein) and RARs
(retinoic acid receptor). RALDH2, CRABPs and RARS were significantly
up-regulated after optic nerve injury in goldfish, whereas CYP26a1
(an enzyme degrading retinoic acid) was down-regulated to about
50% of normal levels. These results indicate that retinoic acid
signalling might play a key role during the first stage of optic nerve
regeneration in fish [88].
The axon guidance molecule Netrin-1 is promoting RGC axon
regeneration in goldfish. Netrin-1 expression is maintained until
adulthood and receptors are up-regulated on newborn RGC axons
[89]. In contrast to zebrafish, its expression is developmentally downregulated in rats [89].
2.2.1.2. Inhibitors. Although initial sprouting of neurites is observed in
mice after optic nerve injury, RGCs die shortly thereafter through
apoptosis, which is thought to be triggered by the activation of
astrocytes initiating wound repair and scarring. Inhibitory signals can
be roughly subdivided into myelin, glial scar and repellent axon
guidance molecules.
Myelin proteins are major inhibitors for axon regeneration in
mammals [90]. Data on how myelin affects axonal re-growth in fish is
somewhat unclear. Whereas the role of myelin has not been studied in
zebrafish, findings from goldfish seem to argue that goldfish myelin is
not a strong inhibitor [91–93]. The myelin-associated Nogo protein is
a major inhibitor of axon re-growth in the optic nerve and spinal cord
of mammals [94]. Knocking out Nogo A,B or C or inhibiting the Nogo
receptor in mice abolishes this inhibitory effect and allows neurite
extension into the ON lesion [95]. Although zebrafish do possess a
Nogo A ortholog, the zebrafish isoform does lack the NogoA-specific
N-terminal domain that inhibits regeneration in mammals [96]. A
recent study examined the role of the second, C-terminal, inhibitory
domain (Nogo-66) in zebrafish axon regeneration [97]. Binding of
Nogo-66 to its receptor NgR has no inhibitory effect on zebrafish RGC
axon growth and in fact is growth promoting.
Another prominent source of inhibition in mammals is the glial
scar formed in response to optic never injury. A hallmark of the glial
scar is increased immunoreactivity for chondroitin sulfates, which has
been shown to inhibit axonal regeneration [98]. In an elegant
experiment, Charalambous et al. grafted chick neural tube stem cells
onto a lesioned rat optic nerve, and observed the induction of MMP
(matrix metalloproteinase)-2 and -14 in astrocytes of the optic nerve.
This in turn led to the degradation of matrix chondroitin sulfate
proteoglycans and allowed rat axons to migrate towards the thalamus
and mid-brain [99]. Chondroitin sulfates are also present in the
developing and adult non-retinorecipient pretectal brain nuclei of
zebrafish. However, they are not found before or after an optic nerve
crush in the optic nerve and tract in adult zebrafish [100]. Quite
different to the situation in rodents, where chondroitin sulfates seem
to play a direct role in inhibition of axon regeneration, Becker and
Becker postulate that in zebrafish, chondroitin sulfates play a role in
axon regeneration by acting as repellent axon guidance molecules
that help axons finding their correct path to their tectal targets [100].
As the zebrafish retina continues to grow throughout the life of the
fish, newborn RGCs are constantly added to the retina and their axons
need to find the correct path along the optic nerve. Developmentally
regulated axon guidance molecules thus might remain in the ON
microenvironment and play a key role in regeneration.
Semaphorin3a, a major repellent axon guidance cue, is upregulated after axotomy of the rat ON for 28 days [101]. Calander
et al. reported the expression of Sema3Aa in the retina, optic chiasm
and optic tectum in zebrafish. However, it does not seem to be present
during the time window when the axons extend [102]. Becker and
Becker state that neither of the two zebrafish 3A orthologs are
detectably expressed in the lesioned optic nerve tract in the adult
zebrafish (unpublished results reported in [73]). The lack of
expression of this repellent axon guidance molecule in zebrafish
might explain why axons are regenerated.
While the lack of repellent axon guidance molecules in zebrafish
might help newly generated axons to extend, the expression of
repellent guidance cues in specific patterns has been shown to
be essential for establishing correct pathfinding and retinotopy.
TenascinR is an extracellular matrix molecule acting as a repellent
guidance cue during development [103]. Areas of TenascinR expression
are also maintained in the adult zebrafish, bordering newly growing
axons from the retinal periphery [104], indicating that it also is
implicated in guiding regenerating axons to their respective brain
targets. In mice, TenascinR similarly seems to act as a repellent guidance
molecule. Outgrowth of embryonic and adult retinal ganglion cell axons
from mouse retinal explants is significantly reduced on homogeneous
substrates of TenascinR [105]. Becker et al. have observed an increase in
the number of cells expressing TenascinR mRNA in the lesioned optic
nerve and have hypothesized that the continued expression of the
protein after an optic nerve crush may contribute to the failure of adult
retinal ganglion cells to regenerate their axons in vivo.
Another class of repellent cues involved in establishing retinotopy
in the tectum are the Ephrin-As, acting via Eph receptors. Developmental gradients of Ephrin-A2 and -A5 are retained in unlesioned and
lesioned adult zebrafish [106], and blocking Eph receptors in goldfish
has a detrimental effect on establishing retinotopy during regeneration [107]. Interestingly, while EphA3 and A5 (receptors) have been
found to be re-expressed in a gradient in the retina of goldfish [108],
their expression is not recapitulated during optic nerve regeneration
in zebrafish [106].
2.2.2. Open questions
Data about molecular factors influencing optic nerve regeneration
are accumulating and provide starting points for new therapeutic
approaches. Recent gene therapy trials conducted in mouse models
have shown that combinatorial treatments can switch RGCs to a
growth state and lead to some extent of axon regeneration [111–113].
Despite this progress, several open questions remain: Are the newly
regenerated RGC axons going to innervate the appropriate pretectal
and tectal targets and re-establish a correct retinotopic map?
Moreover, ultimately, will regeneration of RGC axons lead to recovery
of visual function?
Strikingly, the answer seems to be “yes” in zebrafish and goldfish.
RGC axons not only re-grow, but also find their way back to their
original targets, leading to full recovery of visual function [72,114–
117]. Unfortunately, not much is known about the molecular pathways underlying restoration of vision, as research performed to date
has mainly focussed on the first steps in ON regeneration, axon regrowth and pathfinding. Integration of regenerated axons into
existing circuits is a prerequisite for restoration of vision in human
patients. Therefore, a thorough understanding of these late-step
processes in regeneration on a molecular level is indispensable for
successful future therapeutic approaches. The zebrafish model system
has great potential for uncovering the molecular pathways underlying
network integration of regenerated optic nerve fibers.
2.3. Spinal cord
Abundant research efforts to facilitate spinal cord regeneration in a
clinical setting have focussed largely on rodent models, providing
valuable insights into the cellular processes required for functional
recovery. Despite intense efforts, the limited ability of mammalian
V.C. Fleisch et al. / Biochimica et Biophysica Acta 1812 (2011) 364–380
spinal cord neurons to generate new cells and form new connections
remains a limiting factor. Thus the goals of basic research, to allow
translation into the clinical setting, include the enhancement of
regeneration by identifying interactions that alleviate these restrictions or activate latent cells. Anamniotic vertebrates exhibit substantial spinal cord regenerative capacity and can thus provide insight into
the events that underpin successful functional regeneration. Recent
reviews and volumes compare the utility and contributions of
anamniotic vertebrates to spinal cord regeneration [118,119].
Clinical sources of spinal cord injury typically include trauma and
neurodegenerative diseases. Principal issues in clinical spinal cord
regeneration include: i) protection of surviving cells from secondary
degeneration following injury; ii) proliferation of new cells; iii)
growth of axons through/around scar; iv) connection of axons to
original targets; and v) plasticity of the CNS to restore function despite
imperfect pathfinding. Spinal cord regeneration from stem cells will
require multimodal treatment to address neuron-intrinsic and
neuron-extrinsic limitations.
Following spinal cord injury in mammals, the number of new
axons generated is very limited. In sum, it appears that the plasticity of
spared axons is the principal substrate for the limited functional
recovery that is achieved [120]. Thus, studies of enhanced and
directed axonal sprouting will be valuable to implementing treatments. Regeneration of neurons will be especially important to
human corticospinal lesions and restoration of voluntary motor
control, because neuronal sprouting is limited in these areas [120].
An understanding of neuronal differentiation and functional integration is of high priority and a number of ongoing clinical trials using
stem cell therapy seek to address these issues [121–123].
Zebrafish, with their capacity to regenerate neurons and their
axons and regain function following experimentally induced spinal
cord injury, offer promise in addressing several key questions. Beyond
the various advantages of the model system, studying functional
regeneration of the spinal cord in zebrafish is expected to be
especially fruitful. Drug and gene discovery in regard to spinal cord
regeneration are very practical in zebrafish because measurements of
functional recovery can be automated in this highly active species
(reviewed below). Unfortunately, their small size will not permit
analysis of axon pathfinding to navigate large injuries. In the context
of spinal injuries observed clinically, larger rat models may be
considered too small. The size of an adult zebrafish is an order of
magnitude less than the size of a typical human spinal cord lesion.
However the small size of the organism is an advantage in many
regards, as in most model organisms, providing economy and
generation time efficiency not practicable in larger animals. In sum,
it appears that zebrafish will be invaluable to discovering targets for
functional regeneration of the spinal cord, serving as a very effective
compliment to murine and large-animal models.
Studies of the zebrafish spinal cord have demonstrated robust
regenerative capacity. Detailed research differentiates cell and injury
types that influence the regenerative response and delineates the
molecules that underpin such differences. Regeneration of zebrafish
adult spinal cord neurons from most of the spinal-projecting brain
nuclei is observed following spinal cord lesion [124]. Various nuclei
display different regenerative properties, including the molecular
pathways engaged, depending on the rostral–caudal position of the
lesion [125,126] and identifiable patterns within this heterogeneity
may be exploited to understand molecular pathways determining the
intrinsic ability to regenerate. Pathways underpinning regeneration
seem to be primarily those previously noted for similar functions in
mammals and almost exclusively focus upon cell-adhesion proteins.
The immunoglobulin superfamily cell-adhesion protein L1.1 contributes to spinal neuron growth, synapse formation and functional
recovery in zebrafish [127], as demonstrated by applying a morpholino directed against L1.1 to the spinal cord lesion. This effective gene
knock-down technology is an economical and informative loss-of-
371
function complement to tests in mammals, where L1 has been shown
to be up-regulated during regeneration in CNS nerve grafts and L1
fusion protein application can promote functional recovery of
locomotion [128,129]. Similarly, GAP-43 is up-regulated in zebrafish
spinal cord regeneration [125], as in mammals [130–132].
On the other hand, another cell-adhesion molecule, protein zero
(P0, a component of the myelin sheath) shows substantive difference
between anamniotes with regenerative capacity when compared to
mammals with limited spinal cord regenerative potential [85,133]:
This and difference to mammals allow studies in zebrafish to
contribute to understanding re-myelination following regeneration
[133]. Recent work has examined another cell-adhesion molecule,
Contactin1a, which is up-regulated in both neurons and oligodendrocytes during spinal cord lesion [86] and is discussed above in the
section on optic nerve regeneration.
Although many classes of neurons of the zebrafish regenerate
following spinal cord lesion, some do not. One example of cells with
limited ability to regenerate their axons is the Mauthner cell
[118,124]; a large and easily identifiable cell type that is amenable
to electrophysiological recording in larval zebrafish and controls
swimming [134–137]. Because other neurons consistently regenerate
via the glial scar following lesion, Bhatt et al. have suggested that the
Mauthner cell has intrinsic properties (a surface protein or a
component of its intracellular signalling cascade) that limit its
regenerative capacity [138]. It is noteworthy that if such an intrinsic
factor exists then it is presumably limiting regeneration through
detection of some property in the neuron's environment.
If this is the case, identification of molecules extrinsic to the
Mauthner cell environment that interact with Mauthner-specific
sensors is critically important. Although Mauthner cell regeneration is
rare, it can be bolstered by altering intracellular signalling, including
application of cAMP [138]. cAMP is known to affect neuronal
sprouting and regeneration in other systems although which of the
many pathways it may be modulating remains unclear. Mauthner cell
regeneration is an attractive target from a technical point-of-view, as
functional regeneration (reviewed below) can be assessed via trunk
movement [138] related to escape behavior, with the potential for
high-throughput screening in 96-well plates [139–141]. These large
cells can be recorded from or stimulated readily for electrophysiology
[138].
Heterogeneous glial reactions that are expected to affect regeneration of spinal cord axons in mammals also occur in zebrafish [125].
Macrophages/microglia increase in numbers and phagocytose myelin
debris, which may or may not be beneficial to axon regeneration
[142]. In mammals, such differences have been identified to underpin
the permissive or restrictive properties of glia towards regeneration
[143].
2.4. Brain nuclei
Clinical regeneration of neurons in higher brain centres is a
laudable, oft-described goal of stem cell biology. Ambitious targets of
stem cell treatment include neurodegenerative diseases including
Alzheimer Disease, Parkinson, and ALS [144–147], as well as stroke
and traumatic brain injury. Indeed, proliferating cells may not only be
expected to replace lost neurons, but may also be able to support the
survival of remaining cells.
Zebrafish are attracting considerable interest in understanding
cellular and molecular pathways directing radial glia to become stem
cells, and directing CNS stem cells to replace lost neurons. Adult
neurogenesis is substantial in the zebrafish brain [148–156] and
includes brain centres beyond the restricted forebrain proliferation
typically studied in mammalian models. Compared to mammals,
zebrafish adult neurogenesis is located in multiple centres, but is
fundamentally similar in process; subventricular proliferative zones
give rise to nascent neurons that migrate radially to their differentiated
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destination. There is a notable lack of published experimental paradigms
in which zebrafish brains are damaged, thus allowing the regenerative
response to be addressed. Nevertheless, these approaches are certainly
on the horizon [157], building on the substantial research in other fish
models [158]. Efforts to define zebrafish adult stem cell properties will
be very valuable in studies of functional regeneration.
Stem cells of the adult zebrafish brain have recently begun to be
defined, establishing zebrafish as a valuable model for adult brain
stem cell biology. In the tegmentum, her5 (orthologue of mammalian
Hes) cells meet the definitions of bona fide stem cells (including selfrenewal, slow proliferation and multipotency) and H/E(Spl) factors
generally have a role in maintaining a neural progenitor state. These
populations express markers of stem cells conserved with mammalian neural progenitors, including BLBP, GFAP, and Sox2. Presumptive
neurons arising also express conserved markers, including asha1,
her4, ngn1, and Delta [159,160].
3. Lesioning paradigms
Animal models of neuronal regeneration necessarily depend upon
the manner in which the neurons (and neighbouring cells) are
damaged. Various paradigms have been developed in an opportunistic
fashion, each with different goals and suite of advantages. In some
instances, experimentally induced damage may have a central goal of
trying to replicate a disease process. Although a plethora of animal
models of disease exist, few zebrafish models of disease have been
brought to bear on regeneration. Instead, most work have focussed on
an experimental intervention (surgery, neurotoxin application, etc.,
reviewed below) that strives for reproducibility. Traditionally such
studies have been opportunistic in that they ablate accessible cells. For
example the optic nerve and spinal cord are accessible to surgical
lesion, and the retina is accessible to neurotoxin delivery via
intraocular injection, or to toxic light lesion in albino animals. We
review here methods that have been informative for regeneration
studies in the past, and look ahead to transgenic paradigms that are
well suited to the zebrafish model and have the potential to allow cell
ablation/regeneration to be studied within a vast variety of neuronal
cell types. We end with a commentary that assessing the function of
regenerated neurons needs to become the benchmark of the field.
Thus physiological and behavioral measures of regeneration ought to
be a substantial component of assessing regeneration, and thus in the
design of CNS regeneration paradigms.
3.1. Advantages to refining methods
A variety of lesioning paradigms have been employed for the
induction of neuronal cell ablation with goals of learning more about
regeneration of the zebrafish CNS. Techniques range in the specificity
of lesioning (Fig. 2). Whereas surgical excisions of pieces of tissues are
not cell-specific, methods such as laser or light lesioning are potent to
target specific layers of the CNS. These techniques have allowed
research groups to observe the regeneration process, but a pitfall is
that many cell types are ablated and thus questions concerning
specific cell fate determination cannot be readily answered
[23,24,161]. Cell ablation using a laser can also be used for targeting
only specific neuronal cells of interest in a tissue [162,163]. The use of
chemical toxins, such as ouabain, injected into the eye also possesses a
certain level for controlled cell death. The concentration of the
injected toxin dictates the extent of penetration of the toxin into the
retinal layers, and therefore the amount of cell death [43,164]. Novel
Fig. 2. Multiple methods of neuronal ablation to study CNS regeneration in zebrafish. The method of cell ablation influences the complexity of regeneration and thus experimental
design. The specificity of the neurons targeted (ablation of only one or some neuronal cell types versus ablation of an entire section of retinal tissue) is dependent on the technique:
A) light/laser lesion, B) chemical ablation, C) surgical lesion or D) NTR transgenic ablation (see also Fig. 3).
V.C. Fleisch et al. / Biochimica et Biophysica Acta 1812 (2011) 364–380
techniques employing transgenic-chemical ablation of neurons are
proving to be the most specific means for targeted cell ablation [165].
Regeneration paradigms can be refined through the standardization of the lesioning paradigms. Studies will become more comparable
between treatments or between research groups when protocols are
performed with exacting rigor, eliminating some of the variables such
as the unwanted death of neighbouring neurons or the activation of
different cell death signalling pathways (i.e. apoptosis versus
necrosis) due to distinctive lesioning methods, both of which could
have an impact on the regeneration of the neurons of interest
[166,167]. Refinement of techniques also leads to simplifying the
ability to visualize cell ablation and regeneration in vivo [165].
With each investigation of regeneration, the possibility of applying
this knowledge towards therapeutics for the treatment of human
neurodegenerative diseases gets closer to becoming a reality.
Characterization of the biological mechanisms and signalling pathways involved in this process is crucial. Thus refined strategies for
neuron damage/ablation, each with its particular advantages, are a
worthwhile consideration.
373
paradigms in other tissues (e.g. surgical intervention in heart and tail
fins).
In studies of regeneration of the vertebrate spinal cord, researchers
have attempted to simulate human spinal cord injury in an effort to
discover potential therapies. Many investigations have employed
teleost fish including adult zebrafish, cichlids and goldfish, and similar
surgical techniques have been used to lesion their spinal cords
[124,170,179]. The vertebral column is exposed with a longitudinal
incision, then is transected in the region of interest [124]. A similar
technique to study spinal cord regeneration in mammals has been
performed using adult rats and mice [169,172], and regeneration
studies of Xenopus tadpoles have performed tail amputations to allow
for comparisons of tail regeneration with spinal cord regeneration
[173].
In an investigation by Becker et al., axonal regeneration of the
zebrafish spinal cord was studied. After transection of the cord, regrowth of projections from brain nuclei can be observed. Within six
weeks post lesion, most of the brain nuclei had axons that extended
past the transaction and into the distal spinal cord, indicative of
regeneration [118].
3.2. Methods of inducing neuronal death and damage
3.2.1. Surgical lesioning
Surgical lesioning of CNS tissues has been performed on the optic
nerve, spinal cord, brain and retinal neurons of many organisms including
teleost fish, mouse, rat and Xenopus [33,53,77,124,158,166–174].
Optic nerve crush is a surgical lesioning approach that is routinely
applied in studies using rat, mouse and teleost fish. The most common
surgical technique is to expose the optic nerve by gently pulling the
eyeball out of the socket then crushing it, taking care not to sever the
ophthalmic artery [77,100,106,166,167]. Optic nerve damage can
easily be visualized and confirmed. In zebrafish, the optic nerve
usually has a whitish opaque colour, but upon damage, a translucent
stripe across the optic nerve becomes visible where the forceps had
made contact [106].
Becker and Becker examined regeneration of the optic projection
in zebrafish after optic nerve crush to study the role of genes involved
in axonal growth, pathway selection, and target recognition [106]. The
axonal projection is retinotopic, meaning that it is formed from/
composed of retinal ganglion cells projecting to the optic tectum
[175]. After lesioning, certain tectal areas were observed to be primary
targets for the regenerating axons in that there was an increased
probability for functional integration of regenerated tissue. Becker
and Becker found that the pattern of expression of the recognition
molecules ephrin-A2 and ephrin-A5b correlated to the target areas,
suggesting that these molecules may play important roles in
functional regeneration of the optic nerve [106].
Although a major contributor to our understanding of nerve
regeneration, the relevance of optic nerve crush to human disease is
questionable. A significant advantage of this paradigm, however,
includes the ability to trace pathfinding and target accuracy of the
regenerated axons (reviewed above).
Retinal regeneration has been investigated by surgically removing
portions of the retina or the retina in its entirety [176,177]. Mensinger
and Powers investigated the effects of removing parts or the entire
retina on regeneration in the teleost models goldfish and sunfish [178].
While excision of the whole retina completely prevented regeneration,
leaving only 5% of retinal tissue intact led to restoration of functional
vision, as assayed by ERGs and dorsal light response (DLR). Surgical
lesioning of the retina has also been used as a means to study gene
expression profile changes during retinal regeneration [53].
Retinal damage via surgery has been useful for observing neuronal
regeneration, but is less powerful for studying only a specific subset of
retinal neurons because all neurons are regenerating at once [177]. It
is noteworthy that this type of lesion leads to formation of a blastema
during regeneration, and in this aspect may be comparable to lesion
3.2.2. Laser lesioning
Laser lesioning has been applied to induce ablation of neural cells
in various tissues and organisms [23,161,180]. Regeneration of the
retina of teleost fish has been investigated by ablating photoreceptors
with an argon laser. The argon laser promotes accurate placement of
the lesions and can be used to ablate photoreceptor cells in the outer
nuclear layer in the retina. In this procedure, the lens of the fish is
surgically removed in order to gain access to the retina for focussing
the laser. The resulting lesions appear as bleached spots on the retina
[23,161].
A particularly advantageous feature of laser lesioning is that it can
be employed on a cell-specific level to allow for regeneration studies
of subgroups of neurons. Moreover, it can be directed to ablation of
any part of the CNS. The targeted cells are labelled with an indicator
such as a fluorescent dye to allow for easy visualization of neurons. In
their study of the zebrafish hindbrain, Liu and Fetcho labelled cells
through retrograde uptake of the indicator CGD by injecting it into the
spinal cord of 4dpf fish [163]. After intense light exposure, the dye will
have adverse affects, and thus cause cell death. The targeted neurons
were identified by position and morphology, and subsequently
injured by exposing them to the maximum intensity of the laser.
Immediately after light treatment, fluorescence was observably
decreased, and by 24 h after treatment it was no longer detectable
in confocal images, suggesting cell death [163]. A similar approach
was used on optic tectum in zebrafish larvae, utilizing effective
measures of visual function [181]. Moreover, Kimmel et al. utilized
laser irradiation on trigeminal neurons to investigate if their input to
the hindbrain Mauthner cell affects its dendrite outgrowth [182].
These laser lesioning paradigms have not yet been used for
regeneration studies, but appear to be an attractive method for this
purpose.
Finally, it is noteworthy that femtosecond laser pulses have been
employed to ablate specific cells in live zebrafish [183]. Although this
has not yet been used to ablate neuronal cells of zebrafish, it has been
successful in studying neuronal regeneration in Caenorhabditis elegans
[184]. Developing this technology, and combining it with the optical
transparency and transgenesis of zebrafish have substantial potential
for specific ablation and neuronal regeneration.
3.2.3. Light lesioning
Light-induced lesioning is a popular method of cell ablation
because it targets photoreceptor cells while leaving the remainder of
the retina relatively unharmed, with less inflammatory response
compared to tissue injury [185]. This system has been shown to be
very effective for studies of the teleost retina, and is the most
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commonly used lesioning paradigm for assessing regeneration in both
albino and pigmented fish [186–188]. Mechanisms of cell death in
light lesion have been reviewed extensively elsewhere [187,189] and
likely include both generation of reactive oxygen species and
accumulation of heat leading to photoreceptor death.
Fish are initially dark adapted for a particular period of time,
followed by the application of constant light, which induces
photoreceptor cell death through apoptosis [24,40,185,190]. The
extent of retinal injury to the organism is affected by the fish's
history of light exposure, the period, quality and intensity of light
exposure, and the organism's pigmentation and genetic background
[24,187,191]. Regeneration of photoreceptors following such lesions
is substantial (please refer to the Retina section of this review).
3.2.4. Chemical ablation
Another technique used primarily for the study of teleost retinal
regeneration is to expose the tissue of interest to toxic chemicals.
Ouabain, a metabolic poison inhibiting the Na+/K+-ATPase, has
specifically been employed to induce degeneration of cells in the
retina [43,164,192]. Depolarization of the plasma membrane triggers
a cascade of enzymatic events, leading to cell death [193]. The extent
of cell death is proportional to the concentration of intravitreal toxin
[43,164,194]. High concentrations of ouabain have been shown to
rapidly cause death to all retinal nuclear layers, while the use of lower
concentrations can be useful in biasing ablation towards the inner
nuclear layers while leaving the photoreceptor layer relatively
unscathed. Fimbel et al. took advantage of this element and
demonstrated that selective damage to cells of the INL by injection
of low doses of ouabain could stimulate regeneration [43]. A zebrafish
model for whole retina regeneration has been presented by Sherpa et
al. who showed regeneration of RGCs after cytotoxin injection [194].
Intravitreal injection of 6-hydroxydopamine (6-OHDA) has been
used to ablate retinal dopaminergic cells in both goldfish and
zebrafish [195–199]. Almost 3 decades ago Negishi et al. had shown
that loss of dopaminergic neurons in goldfish retinas triggered
increased proliferation of retinal progenitors [198]. Li et al. have
used a behavioral test (escape resonse) to study the effect of loss of
dopaminergic neurons on visual sensitivity in the zebrafish [195].
3.2.5. Transgenic expression of toxic proteins
Ablation of particular cells has been achieved by expression of
disease genes or other toxic proteins in subsets of zebrafish
photoreceptors [200,201]. This genetic ablation of cones versus rods
has led to important insights into retinal progenitor pools in the
zebrafish retina [202]. In contrast to emerging transgenic methods
reviewed below, in which the toxicity is inducible and restricted to
the timeline of prodrug exposure, the continued fruitful nature of
these methods will be limited somewhat by the toxic protein being
abundant in the regenerated cell. Arguably, the early steps of cell
regeneration will be unperturbed in these models, and late-stage
molecular signatures and functional measures of regeneration will
need to consider whether the results are influenced by the toxic
proteins used for ablation.
3.2.6. Transgenic conditional cell-specific ablation
Genetic engineering techniques have become very practical in the
zebrafish, and with the advancement of these tools, mechanisms of
development and regeneration have become readily observable.
Recently, investigators have created conditional cell ablation techniques
by combining genetic and chemical tools to target the degeneration of
particular cell types (Fig. 3) [165,203,204]. By specifically directing
ablation of a particular neuronal cell class for a limited period of time,
rather than causing simultaneous death to many types of neurons, as
had been the standard procedure thus far, alterations and disruptions in
relevant genetic pathways can be analyzed for their potential roles in
CNS regeneration.
One recently developed method of transgenic cell-specific ablation
is nitroreductase-mediated conditional cell ablation [165]. Transgenic
zebrafish express the Escherichia coli gene nfsB encoding the
nitroreductase (NTR) enzyme. NTR is driven by a cell-specific
promoter, resulting in its expression in the targeted neuron type.
These neurons will function normally until the application of a
prodrug, metronidazole (MTZ). MTZ binds to NTR and is electrochemically reduced, converting it into a DNA cross-linking cytotoxin
[165] (Fig. 3B). The toxic form of MTZ remains isolated in the neurons
of interest and induces base transitions, transversion mutations, and
finally fragmentation of DNA [205]. This results in precise cell ablation
of the subset of neurons expressing NTR, while other neighbouring
cells appear to be unaffected by the toxin [165]. Ablation is
discontinued upon the removal of the prodrug, allowing for
regeneration to occur devoid of the influence of the toxin. The
method has recently been used to ablate specific classes of bipolar
cells in the zebrafish retina [204]. Based upon cell counts, the ablated
bipolar cells reappear in the retina, presumably regenerating from a
proliferating population. Another study has utilized a similar
approach to ablate rod photoreceptors and demonstrated a differential proliferative response based on the abundance of rods ablated
[241]. This study underlines the potential of the system, allowing
specific cell classes to regenerate among the complexity of cell types
in the retina. Certain questions remain to be tested, including whether
adjacent cells are damaged or killed by the method, and whether the
cell types regenerated are more or less diverse than those that are
ablated. The specificity of the system is tantalizing, as it ought to allow
a simpler system to define molecular mechanisms during regeneration of a single cell type rather than many cell types. Ideally, via
comparison of regeneration of multiple classes, the community will
learn triggers of cell-type-specific regeneration and functional
rewiring.
The methods used to date in zebrafish using nitroreductase cell
ablation also utilize the Gal4-VP16 and UAS combinatorial transgenic
system [165,204]. This allows the exciting potential for combination
with Gal4 enhancer trap lines [206–208] with many neuronal and
glial types now available for ablation in a variety of CNS tissues.
Although the toxicity and transcriptional effects of VP16 itself need to
be considered, this model system has enormous potential to
efficiently ablate single or multiple cell types to compare their
regeneration and functional integration.
3.3. Regeneration has limited utility if function is not restored
The regeneration of a neuronal cell is an impressive process, but
would be of limited utility if the cell does not functionally integrate
into the existing tissue system upon regeneration. This aspect is often
overlooked in regeneration studies. Neuronal regeneration in the
zebrafish can easily be observed, but the functionality of the
regenerated cells is often assumed but not experimentally tested.
Defining the molecular mechanisms that specify the regenerating
cell's fate will be an ongoing focus of the community, and will
certainly contribute to clinical specification of stem cells. On the other
hand, specifying cell fates alone will not be useful if the cells are not
directed to reconnect into the neuronal network. Here we review
regeneration paradigms that can be coupled with informative and/or
efficient methods to assess the regenerated function using electrophysiology and/or behavior.
3.3.1. Electrophysiology output
The ERG (electroretinogram) is a routinely used and extremely
valuable tool for studying degeneration and dysfunction of retinal
neurons in the zebrafish. This technique measures light-induced
changes of electrical activity in the eye [11,209,210]. The ERG is a
method for probing outer retinal function and can be adapted to larval
zebrafish and adult zebrafish, as well as multiple other organisms. In
V.C. Fleisch et al. / Biochimica et Biophysica Acta 1812 (2011) 364–380
375
this non-invasive procedure, a recording electrode is placed onto the
cornea of the zebrafish, and a response is induced by flashing light at
specific intensities and durations [11]. The observed response is
recorded by the instrumentation and interpreted into an ERG trace
(Fig. 4) that has a form very simlar to the ERG of mammals.
The vertebrate ERG output is expressed as three distinct waves
(Fig. 4). An initial low-amplitude negative a-wave reflecting photoreceptor activity is followed by the large positive b-wave, mainly
representing second-order (ON bipolar) neuron activity. The d-wave
appears at light offset and is indicative of postsynaptic activity
involved in the OFF response [11,211].
The ERG has been used in goldfish to assess functional recovery of
retinal neurons after cytotoxic lesioning and surgical extirpation of
the adult retina [178]. Recovery of the ERG a-wave up to 90% of
control levels coincided perfectly with the regeneration of photoreceptors, and more importantly, also the b-wave amplitude
recovered up to 50% in amplitude, demonstrating that regenerated
photoreceptors made proper synaptic connections with second-order
neurons [212]. The ERG has also been used to demonstrate functional
integration during regeneration of a specific cone class in rainbow
trout [191]. Restoration of function has also been established beyond
the retina during cone photoreceptor regeneration in trout [213].
These recordings from the optic nerve where it enters the tectum have
the advantage of demonstrating connectivity to higher brain centres,
but require surgery and thus preclude documenting regeneration
within the same individual.
Electropysiological tools can also be employed for detecting
electrical activity in the spinal cord of zebrafish. Patch-clamp
electrodes are placed extracellularly on the zebrafish in order to
record the locomotive response pattern [134,214].
3.3.2. Behavioral output
Behavioral responses are another mode to assess functional
integration of regenerated neurons into existing circuits. The loss of
certain behaviors upon the ablation of neurons, as well as the recovery
of those behaviors once the cells have regenerated, can be assessed by
a number of well-established paradigms in the zebrafish. Typically,
these have focussed on innate responses of fish to presented stimuli,
rather than operant conditioning, thus simplifying the methods
considerably. Indeed the simplicity of many of these assays should
make them accessible to most any research group.
3.3.2.1. Regeneration of motor behaviors. Regeneration after spinal cord
transection in adult zebrafish has been studied employing a simple
swimming performance assay [215]. 1 and 3 months after lesioning,
fish were transferred into a tunnel with water flowing at slow and fast
speed to test for swimming endurance as a function of fiber re-growth
[215].
Other examples of robust assays include the light-induced startle
response used in high-throughput screening for motor deficits [139–
141]. This assays certainly could be used to assess replacement of
functional connections during hindbrain or spinal cord regeneration.
Escape responses in zebrafish larvae can also be induced with a light
touch or puff of water directed at the trunk. This type of assay has
Fig. 3. Regeneration of a single neuronal class can be achieved following conditional
cell-specific ablation. To reduce the heterogeneity of the regenerative resonse, one can
reduce the complexity of the cell types that are ablated. This has recently been achieved
using neuron-specific transgenic expression of the bacterial gene nitroreductase (nfsB,
produces NTR protein), which kills cells upon application of the prodrug metronidazole
(MTZ) [204]. A tissue-specific promoter fused to green fluorescent protein (GFP) drives
expression of NTR in bipolar neurons of the retina (A). Cells expressing NTR develop
normally until MTZ prodrug application, e.g. in a bath treatment (B). Upon application,
MTZ is converted to toxin only in cells expressing NTR, leading to cross-linking of DNA
and cell death (C,D). Removal of MTZ permits cell-specific regeneration, including
expression of the fluorescent marker (E). This method can be combined efficiently with
enhancer traps, allowing regeneration to be studied in a variety of cell populations (see
text).
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Fig. 4. CNS regeneration studies should emphasize functional integration of the
regenerated cells. The electroretinogram (ERG) is an example of a non-invasive
technique that can be applied to measure neuronal function, thus allowing withinindividual examination of the regenerative process (e.g. measures before, during, and
after cell ablation). Analysis of the ERG response typically focusses on the amplitude of
the b-wave (bold, blue positive deflection) in response to light stimuli. The b-wave
primarily represents bipolar cell activity, and thus regeneration of this response can be
diagnostic for appropriate integration of photoreceptors and bipolar cells following
regeneration [191].
been used to assess functional regeneration of the Mauthner cell in
larval spinal cord [138].
3.3.2.2. Visually-evoked behaviors. Visually-evoked behaviors of zebrafish (and goldfish) can be easily stimulated and are readily accessible
for measurement and genetic analysis of functional vision [12,216].
The optokinetic response (OKR) is a robust assay for visual
function measuring zebrafish eye movements in response to a moving
stimulus (grating) [217–220]. Ablation of cone photoreceptors for
example, would be expected to impair colour detection in zebrafish
larvae. Consequently they would not be able to track the movements
of coloured objects. Cone photoreceptors' regeneration would lead to
recovery of functional colour vision and improved performance in the
OKR assay could be observed.
The OKR has been used to study retinal regeneration after ouabain
injection in the goldfish [221]. Cytotoxin injection resulted in lack or
greatly reduced OKR performance. Regeneration of retinal cell layers
resulted in partial recovery of several visually-evoked assays (ERG,
DRL and OKR), however visual sensitivity did not completely return to
control levels [221].
In the optomotor response (OMR), motion in a particular direction is
simulated and zebrafish will swim in the direction of the perceived
motion [222,223]. Failure of zebrafish to respond to the OMR stimulus
can be attributed to distinct/different underlying causes, and thus the
OMR can be used for studying regeneration of various CNS tissues.
Swimming arrest will occur following lesion, and functional regeneration should be able to (at least partially) restore the optomotor
response. As the OMR is dependent not only on a functional retina, but
also on the correct propagation of visual stimuli to the fish optic tectum,
regeneration of the optic nerve can also be tested. Kaneda et al. observed
that the expression increase of the growth cone marker GAP-43 after
optic nerve injury temporally concurred with the recovery of two
visually-mediated behaviors (OMR and chasing behavior) [76].
4. Regeneration as a potential confound when
studying neurodegeneration
International efforts are focussing on developing zebrafish as
models of neurodegenerative disease, by identifying zebrafish
mutants and deploying human versions of disease-associated genes
(see other papers in this volume). These efforts parallel those in mouse
and rat models, which have contributed much to understanding of
disease progression [224]. It is noteworthy that such models, where
neuron death is a benchmark outcome, are anticipated to exhibit
substantial regenerative response. Regeneration from intrinsic
stem cells during degeneration will represent both confounds and
opportunities to modelling neurodegenerative disease. In particular,
longitudinal studies assessing cell loss by quantifying neuron populations may be difficult if neurons are replaced at a rate similar to cell
death. Perhaps such regeneration underpins the lack of obvious
neuronal loss, despite neuropathological change in teleost models of
light-induced photoreceptor death [225] or prion infectivity [226]
(and other manuscripts in this volume). Instead, emphasis may be best
placed on methods that assay cell death via expression of available
cell death markers. Gene discovery approaches using zebrafish disease
models may also need to consider regeneration in the way experiments
are designed. For example proteomic, transcriptomic or metabolomic
studies of disease models will need to interpret changes in molecular
abundance in light of both degeneration and regeneration. One strategy
to address such issues may be to focus experiments on aged fish, e.g.
zebrafish more than two years old, where somatic growth and adult
neurogenesis have slowed and the regenerative response is expected to
be less robust.
Beyond experimental design, other fundamental processes in
neurodegeneration may be different in an organism that is capable of
replacing lost neurons. These may include the cellular and molecular
aspects of astrocyte reactions to injury. As reviewed above, populations of glia dedifferentiate to a progenitor cell state following
zebrafish neuronal injury. This is in marked contrast to mammalian
systems and thus the molecular and cellular responses of glia, or at
least a subset thereof, will be directed to a different biological process
than one expects in a clinical setting.
Similarly, declines in adult neurogenesis have been associated
with onset of dementia in AD [227]. This has led to hypotheses that
decreased neurogenesis is causal for learning deficits and cognitive
decline. Zebrafish represent an exciting opportunity to understand
adult neurogenesis, however their continued robust generation of
new neurons into adulthood directly opposes the disease process
observed during clinical neurodegenerative decline. It will be of
interest to examine if such neurogenesis is similarly affected in
various zebrafish models of human neurodegeneration. We expect
that it will be important to develop methods that allow adult
neurogenesis to be slowed in these zebrafish disease models.
A further complication to these disease models is that the diseaseassociated molecule being deployed in transgenic models is often
intimately linked to proliferation. One example is amyloid precursor
protein (APP), which is processed inappropriately in Alzheimer
Disease and overexpressed in various Alzheimer Disease animal
models. APP is enriched in neurogenic zones and acts to negatively
modulate neurogenesis via its intracellular domain [228,229] and
promotes neuritogenesis through its extracellular domains [230–
232]. Consistent with this, mouse models of Alzheimer Disease
integrating APP, Tau and Presenilin have reduced adult neurogenesis
[233–237]. Similarly, the prion protein (PrPC), often overexpressed in
animal models to allow cross-species infectivity and disease modelling, is also involved in modulating neurogenesis. PrPC is expressed in
adult neural precursors of mouse and positively modulates adult
neurogenesis [238]. Overall, the substantial adult neurogenesis in
zebrafish may be of great benefit to understanding the role for
proteins like APP and PrP in neurogenesis in healthy brains.
Degeneration models based on overexpression of disease-associated
molecules may be especially prone to adult neural regeneration
confounding data interpretations of described above. This will be
especially true when the products that recapitulate disease phenotypes also inadvertently affect a proliferative response.
We have briefly outlined potential confounds regarding interpretation and experimental design that are worth considering as zebrafish
models of neurodegeneration become available. Nevertheless, in
V.C. Fleisch et al. / Biochimica et Biophysica Acta 1812 (2011) 364–380
each case, these confounds can be viewed as an opportunity to examine
the differences across taxa that permit the adult brain to host
proliferation leading to replacement of new neurons. For example,
adult neurogenesis can be viewed as a therapeutic target for Alzheimer
Disease treatments [239]. It is also noteworthy that stem cell therapy of
degenerative diseases would need to be able to navigate these same
obstacles as well as be able to define mechanisms to limit proliferation
from becoming detrimental [146,240]. Thus it will be of great benefit to
define mechanisms to limit growth signals from inducing death and to
limit proliferation from becoming detrimental itself through hyperproliferation and cancer. Finally, in the same fashion as the retina, optic
nerve and spinal cord efforts reviewed above, regeneration from
intrinsic stem cells in the brain centres of zebrafish will offer
opportunity to understand cellular and molecular events requisite to
replacing neurons lost during disease progression.
5. Conclusion: zebrafish is a potent model of vertebrate
CNS regeneration
We have reviewed the intrinsic and impressive ability of zebrafish
to regenerate CNS cells and tissues. Combined with a substantial
repertoire of genetic tools and robust assays for functional integration
of regenerated cells, zebrafish are uniquely positioned to allow design
of sophisticated experiments to test hypotheses of CNS regeneration.
These approaches can be complemented by high-throughput in vivo
screens to explore for genetic and pharmacological modifiers of
regeneration. Transgenic techniques and availability of these fish from
stock centres are allowing refined cell ablation techniques that will
herald cell-specific investigations of regeneration at both the
molecular and functional levels. Finally, we note that several
amenable paradigms are available to assess function of the regenerated CNS tissue, which ought be utilized to assess the integration of
neurons into regenerated CNS circuits.
Acknowledgments
This work was funded by an NSERC Discovery Grant to WTA. VCF
was supported by an SNF Fellowship and BF by an NSERC Scholarship.
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Biochimica et Biophysica Acta 1812 (2011) 381–389
Contents lists available at ScienceDirect
Biochimica et Biophysica Acta
j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / b b a d i s
Review
Behavioural assessments of neurotoxic effects and neurodegeneration in zebrafish☆
Keith B. Tierney ⁎
Department of Biological Sciences, University of Alberta, Z 718 Biological Sciences Building, Edmonton, Alberta, Canada T6G 2E9
a r t i c l e
i n f o
Article history:
Received 2 December 2009
Received in revised form 27 September 2010
Accepted 21 October 2010
Available online 28 October 2010
Keywords:
Zebrafish
Behaviour
Activity
Sensorimotor
Learning endpoints
a b s t r a c t
Altered neurological function will generally be behaviourally apparent. Many of the behavioural models
pioneered in mammalian models are portable to zebrafish. Tests are available to capture alterations in basic
motor function, changes associated with exteroceptive and interoceptive sensory cues, and alterations in
learning and memory performance. Excepting some endpoints involving learning, behavioural tests can be
carried out at 4 days post fertilization. Given larvae can be reared quickly and in large numbers, and that
software solutions are readily available from multiple vendors to automatically test behavioural responses in
96 larvae simultaneously, zebrafish are a potent and rapid model for screening neurological impairments.
Coupling current and emerging behavioural endpoints with molecular techniques will permit and accelerate
the determination of the mechanisms behind neurotoxicity and degeneration, as well as provide numerous
means to test remedial drugs and other therapies. The emphasis of this review is to highlight unexplored/
underutilized behavioural assays for future studies. This article is part of a Special Issue entitled Zebrafish
Models of Neurological Diseases.
© 2010 Elsevier B.V. All rights reserved.
1. Introduction
1.1. The importance of behavioural endpoints
In animals, and arguably all life throughout the biological
kingdoms, behaviours are ways to integrate into the biotic and abiotic
environment. Behaviours are the actions and reactions taken to
internal and external cues that are intended to place organisms in a
preferable position with respect to fitness. Conditions and situations
that cause deviant or impaired behaviours therefore have clear
survival implications. The inclusion of a behavioural assessment endpoint
in studies of disease and neurotoxin exposure, especially those involving
non-lethal impairments, will help address whether sub-organismal
changes will affect survival. However, there is another reason to include
behavioural endpoints—they can be used to rapidly and effectively detect
changes of molecular to system level origins.
In vertebrates, all behaviours are achieved through nervous
control. However, not all behavioural modifications are of neurological origin. Non-neurological alterations can also affect behaviour—
some musculoskeletal issues, for example, such as those arising
Abbreviations: AChE, acetylcholinesterase; Anti-AChE, anticholinesterase; CPP,
conditioned place preference; CS, conditioned stimulus; dpf, days post fertilization;
OKR, optokinetic response; OMR, optomotor response; OP, organophosphorus agent;
OSN, olfactory sensory neuron; PEA, phenylethyl alcohol; PTZ, pentylenetetrazole; UCS,
unconditioned stimulus
☆ This article is part of a Special Issue entitled Zebrafish Models of Neurological
Diseases.
⁎ Tel.: +1 780 492 5339.
E-mail address: [email protected].
0925-4439/$ – see front matter © 2010 Elsevier B.V. All rights reserved.
doi:10.1016/j.bbadis.2010.10.011
through injury and developmental abnormalities, can cause altered
behaviours. Malformed limbs or other morphologically-based conditions may be associated with behaviours not apparent in normal
individuals. There are also a suite of developmental issues that are
related to neurological function, however, they are not related to
neurotoxins or neurodegeneration. Cerebral palsy, for instance, can
affect gross and/or fine motor control and have very apparent
behavioural abnormalities [1]. Additionally, in healthy animals,
internal, physiological processes can also modify neuron function
and these can result in behaviour phenotypes. For the purposes of this
review, only behavioural alterations arising though neurological
pathways and directly involving neurotoxin exposure or progressive
neurological pathologies will be given consideration.
Behavioural alterations arising from neurotoxin exposure and
neurodegeneration come about through very different means, even
though the behavioural “phenotype” (observable manifestation) may
appear similar in some instances. Neurotoxic agents affect the
functionality of “normal” neurons and may be reversible and of
limited duration. In contrast, neurodegeneration collectively refers to
a group of typically irreversible processes that work at the genetic to
system level, all of which result in the loss of neurons and/or their
functionality. Mechanistically, neurotoxic agents act in one of two
ways: in general, non-specific ways (e.g. polar or non-polar narcosis
[2]); or through affecting genetic and/or protein receptors. Either
mode of action can cause changes at molecular, cellular, system and
levels beyond. It must be noted that the first mode of action could
include disruption of cells in addition to neurons. With neurodegeneration, mechanisms of action include protein misfolding and
conformational disorders (proteopathies) and/or astrogliosis, i.e. an
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K.B. Tierney / Biochimica et Biophysica Acta 1812 (2011) 381–389
increase in astrocytes due to neuron death. For each mechanism of
neuron (or neural tissue) impairment, it should be possible to isolate
an individual behavioural phenotype for screening purposes.
All behaviours involve motion, and so given the appropriate methods,
all are quantifiable. The evolution of software solutions in the last decade
or so, including solutions from EthoVision (www.noldus.com), LocoScan
(www.cleversysinc.com), Loligo (www.loligosystems.com), and VideoTrack (www.viewpoint.fr) has greatly facilitated the accurate assessment
of subtle and directed movements. However, unlike the movement of
mammalian models, fish move in three dimensions. In studies of fish
motion, movement in the vertical plane is typically ignored or minimized
(i.e. by keeping the tank water shallow) [3–5], however, in at least one
case, a proprietary protocol was developed to include it [6]. Nevertheless,
with a model such as a zebrafish, the best solution may be to minimize
water depth, to perhaps twice body length. This depth does not appear to
result in overt stress and most fish settle down (acclimate) within 30 min
(personal observation). Current technology permits the rapid, and
potentially repeated, non-lethal testing of numerous fish (e.g. 4+ adults
to 96 larvae) [7,8]. Additionally, a method has been constructed to
calculate very subtle differences in the swimming mechanics of individual
fish [4]. Caveats exist in behavioural assessments. Organisms are designed
with systems that provide resilience and can act to retain higher level
responses [9]. Also, factors that affect responses at lower organisational
levels, e.g. protein level, may not necessarily have an obvious behavioural
phenotype. Behavioural studies clearly need a mechanistically-based
rationale for including a motion-based endpoint.
1.2. The relevance of zebrafish models
Ten years ago, zebrafish were identified as an up and coming model
for genetic disorders and developmental biology in humans [10]. Since
this time, the zebrafish genome has been fully sequenced and many
genes of high mammalian homology have been identified. Five years
ago, zebrafish were proposed as an untapped behaviour-genetic model
organism [11]. There are now high-throughput behavioural zebrafishbased screens able to detect specific neurological alterations/pathologies, some of which take advantage of protocols similar in function and
purpose to those used on rodents (e.g. olfactory-based endpoints).
Furthermore, zebrafish are more amenable to manipulation and
behavioural testing during early development. Their transparent larvae
facilitate localization of proteins and the use of morpholino manipulation. For these reasons, and given that rodents are more expensive and
require greater growth periods than zebrafish, the adoption of zebrafish
models continues in typically rodent dominated fields. This review is
intended to communicate the current use of behavioural endpoints in
zebrafish, and how protocols used with other animals may be adapted.
Numerous behavioural assessment avenues remain unexplored.
2. Types of behavioural endpoints
As with most animals, fishes exhibit intricate behaviours that
depend on locomotion and may be the result of complex decisions
[12]. Behavioural responses must be viewed in a three-part hierarchical
manner: (1) basic motor responses, which underlie (2) sensorimotor
responses, that facilitate and/or integrate with (3) learning and memory
(Fig. 1). Although the first “behavioural level,” arguably does not involve
true behaviour since it does not necessarily include sensory responses,
many studies have included locomotory (activity) changes as a
behavioural endpoint [13–15]. While this endpoint may appear
simplistic, it does not mean it is not meaningful, in fact, quite the
opposite: all behaviours are predicated on the ability to move.
Furthermore, in tests of higher-level behaviours, such as those involving
learning, it may be difficult to rule out potential neurological issues of
the lower founding “levels.” For example, if feeding response decreased
in fish exposed to a dissolved neurotoxin such as an organophosphorus
(OP) insecticide, the effect could be the result of impaired locomotory
Fig. 1. Behavioural responses can be evoked by internal and external signals and/or be
altered by impaired neurological condition. With zebrafish, assessment methodologies
exist to test neurological performance in absence of signals through to the ability of the
brain to retain and integrate sensory signals of diverse origins, which makes zebrafish a
powerful model for testing compounds that affect neurons over brief to life-spanning
timeframes. CPP = conditioned place preference.
ability, and/or impaired olfactory sensory neuron (OSN) function, and/
or cognitive ability.
Implicit in neurodegeneration and neurotoxin exposure is that
organism condition, and so likely behavioural responses, will change
with time. With neurotoxins, there are four temporal phases to
describe their effects: direct, secondary, tertiary and quaternary
effects. The first two phases deal with the presence and actions of an
agent within the organism, and the latter two, without. Direct effects
are those local or systemic effects that occur over the short run and
would typically be the “intended” effects, such as the actions of an
anticholinesterase (anti-AChE) drug. Secondary effects consist of
adaptations made over longer periods to restore equilibrium from
drug actions, such as upregulation of acetylcholinesterase (AChE)
expression. Tertiary effects are those that follow from the discontinuation of administration of an agent that has become physically
depended, i.e. involve withdrawal and stress. Over extended periods,
tertiary effects may give way to quaternary effects, such as
malnutrition. The majority of behavioural assays focus on direct and
secondary effects, however, there are studies of tertiary effects, e.g.
withdrawal from cocaine [16].
In the following, a diverse array of endpoints using apparatus from
simple, one chambered tests, to complex multi-chamber, decisionbased challenges are discussed (Table 1). The majority of the
behavioural endpoints can be conducted on zebrafish 3–4 days post
fertilization [17,18], excepting learning/memory-based endpoints.
2.1. Basic motor response endpoints
Compounds that modulate neuron firing rate have potential to
affect locomotory activity. In fishes, locomotory activity endpoints
include swimming speed [19], distance covered [14], and turning rate
(angular velocity) [20]. Changes in zebrafish activity have been noted
in studies investigating the mechanisms of addiction to amphetamines [21], cocaine [16], ethanol [11,22], and nicotine [23], as well as
the effects of pesticides [24,25].
Whether any given drug/neurotoxin increases or decreases
activity is related to concentration/dose and time. For example,
exposure to a low concentrations of D-amphetamine caused hyperactivity while higher concentrations caused hypoactivity [21].
Cholinesterase inhibiting drugs/pesticides are well known to have
similar effects [26–29]. Clearly, agonizing stimulatory receptors or
inhibiting neurotransmitter degradation will potentially lead to
activity increases in the short run that are not sustainable in the
long. In fact, persistent stimulation may result in excitotoxicity and
neuron apoptosis. Behavioural manifestations of neurotoxin exposure
may not appear until later in life. Additionally, some activity changes
K.B. Tierney / Biochimica et Biophysica Acta 1812 (2011) 381–389
383
Table 1
Behavioural test endpoints and their associated stimuli available to fishes, including zebrafish. Some endpoints that are underutilized or hold promise in toxicity or degenerative
studies are in bold.
Apparatus
Stimulus
Stim. type
Type of test
References
Open arena
None
Space
Food, dead
Food, alive
Tap
Glass bead
Predator
Predator scent
Simulated conspecific
Water flow
Social
Simulated object
None
Vis
Vis; Olf
Vis; Olf
Aud
Aud; Vis
Vis
Olf
Social
Motion
Vis; Motion
Vis
Vis
Vis; Olf
Olf
Vis
Chemo
Drug
Vis
Pain
Vis
Vis; Stress
Vis
Activity levelA
Searching behaviourA
Appetitive behaviourA
Prey captureA
Startle responseA
Startle responseA
Alarm responseA
Alarm responseB
AggressivenessB
UcritB
SchoolingB
OMRA
OKRA
Attraction; avoidanceB
AttractionB
CPPB
CPPB
CPPB
CPPB
Aversive CPAB
CPPB
CPP, CPAB
SchoolingB
[14,16,20–22,24,25,30]
[22,23,70,90]
[37]
[40,42,43]
[7]
[58]
[39,40]
[68–70]
[11,22]
[83,84]
[58,59]
[47,49,50]
[51,52]
[15,74,75]
[76–79]
[93]
[97]
[97,98]
[22]
[16]
[99]
[100]
[22]
Confined tube, or flume
Moving background (striped drum)
Counter-current olfactometer
Y-maze
T-maze
Two-chamber tank
Three-chamber, gated
Three-chamber, separated
Odours⁎
Odours⁎
Preferred location
Food
Location
Shade
Electric field
Colour
Confinement
Social
Abbreviations: Aud, auditory; CPA, conditioned place aversion; CPP, conditioned place preference; OKR, optokinetic response; Olf, olfactory; OMR, optomotor response; Ucrit, critical
swimming speed; Vis, visual. Note: Zebrafish age may affect whether specific endpoints are appropriate, as the endpoints may depend on the development of swimming ability,
sensory responses, and memory. In general, assays requiring swimming or sensory-evoked swimming are available as soon as 48 post hatch (~4 dpf; endpoints marked A) [37,106].
Swimming intensive and memory based endpoints have mostly been conducted on adults (approx. ≥3 months), however many of these assays could likely be conducted on
juveniles (endpoints marked B). An exception: OMR relies on immobilizing fish, and so fish cannot be N 7 dpf [51]. A caveat: olfactory-based alarm responses may be most robust and
least variable at 50 dpf [73].
⁎ Odours include food, conspecific, pheromones, predator, and synthetic compounds.
may only be evident after the removal of the drug, i.e. during
withdrawal [16].
Zebrafish activity has proven a viable endpoint for detecting
neurological impairments received during development. For example,
embryonic zebrafish exposed to chlorpyrifos, an organophosphorus
insecticide and ubiquitous environmental pollutant, had reduced
swimming activity 6+ days later [24]. If persisting neurological
impairment or neurodegeneration are not readily apparent, a drug
can be used to evoke activity. Altered neurology will be apparent in a
lower response threshold to the drug. The convulsant (seizure
inducing drug) pentylenetetrazole (PTZ) has been used in recent
studies in zebrafish [30], including high throughput 24 and 96 well
plate assays [3,31,32], for just such a purpose. Specifically, zebrafish
embryos injected with domoic acid (DA), a neurotoxin of diatom
origin, reduced the concentration of PTZ required to evoke stage I and
II seizure activity [33]. Activity endpoints can also be used to detect
neurodegeneration. Specifically, to model the effects of Parkinson
disease, neurotoxic drugs can be injected directly into specific brain
regions (using microinjection), although this has historically not
carried out on fish [34]. A recent study demonstrated that unilateral
injection of the drugs 1,2,3,6-tetrahydropyridine (MPTP) and 6hydroxydopamine (6-OHDA), two neurotoxins with specific targets,
reduced the speed and distance zebrafish travelled [14]. Microinjection techniques make the zebrafish model portable to a variety of
neural ablation studies.
A consideration for activity-based sensory endpoints is that they
may not actually be stimulus-free. When a fish or other animal is
introduced into a new environment, such as a test tank, they will
likely begin searching and forming a cognitive map of their new
surroundings [35]. This process will draw upon one or more senses
and involve synaptic plasticity (discussed below). Even with elimination of searching behaviour, perhaps accomplished through tank
acclimation, activity changes may be based on input from the
interoceptive senses, i.e. the perception of internal movement and/
or pain (aspects of the somatosensory system). Activity-based
endpoints clearly have application, but they may be more of an
umbrella for other neurological impairments isolatable through
sense-specific tests.
2.2. Sensorimotor endpoints
Fishes have a suite of exteroceptive senses analogous to those of
mammals. These include vision (photoreceptor-based), audition
(mechanoreceptor-), olfaction, gustatory (both chemosensor-), balance
and somatosensory, which includes touch (mechanoreceptor-), pressure, temperature (thermoreceptor-) and pain (nociceptor-). Fishes also
posses senses that do not have mammalian analogs, e.g. magnetoreception. Furthermore, some of the analogous senses, e.g. gustation, are
morphologically dissimilar—fishes, including zebrafish, have solitary
chemosensory cells (SCCs), which are externally-located taste buds
[36,37]. And unlike mammals, sound and motion are not only perceived
by the ear and vestibular (inertial detection) system, but by an
additional system, the lateral line. In this system, neuromast cells
structurally similar to inner ear mechanoreceptor “hair” cells, detect
changes in water pressure and motion relative to themselves. These
cells can also detect sounds and provide information regarding
acceleration and velocity [38]. In the below, known and potential
sensorimotor endpoints will be discussed.
The senses enable reflexive, innate responses to “unconditioned
stimuli” (UCS), as well as learned responses to “conditioned stimuli”
(CS). Both UCS and CS can be used to test sensorimotor responses, and
can be positive (attractive) or aversive. UCS evoked behavioural
responses stimuli have been included in this section, but their
modification is included in the proceeding section on learning and
memory. Positive UCS or CS stimuli may arrive via various senses but
uniformly enable the organism to place itself in an actual or perceived
improved fitness position that otherwise offers some benefit or
reward; example stimuli include food, cover, mate call or odour.
Aversive stimuli do the opposite; examples stimuli include electric
shocks, abrupt and severe changes in lighting, temperature, sound,
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(unpleasant) odours, or other sensory assaults causing discomfort.
Stimuli can also be neutral, i.e. perceptible but without an associated
behaviour [8]. For behavioural endpoints, both CS and UCS stimuli are
routinely used in behavioural studies in fishes, including those with
and without conditioning.
Changes in sensory responses can be due to impaired sensory
perception or due to impaired motor performance. Separating
perceptual vs. motor impairment in sensory studies may involve
using an additional endpoint, such as cell or tissue level assessment of
neuron function. Another challenge is that most behaviours are
multisensory, and isolating specific impairments may be difficult or
impossible. Nevertheless, behavioural endpoints exist to evoke
responses through each of the senses.
2.2.1. Visual endpoints
Unless experiments are conducted under darkness or with visually
occluded fish, all behavioural endpoints arguably have some visual
component. In fish, vision specific endpoints include predator
(simulated or actual) avoidance, prey capture, optomotor and
optokinetic responses. All of these methods have clearly defined
endpoints and involve coordination, except the last, which uses
immobilized fish.
One of the most basic assays draws upon phototaxis, i.e. light
seeking behaviour. Zebrafish are active during the day, and so should
spend time in light unless they perceive a risky (predation-prone)
condition. When given a choice between a lighted or shaded area in an
open arena, zebrafish did indeed spend less time under shade (~40%)
[22]. Ethanol exposure caused a concentration-dependent decline in
the proportion of time spent under shade. With exposures to ethanol,
zebrafish sheltering was reduced to ~ 10–15%, i.e. exposure evoked
risky behaviour. Such a robust and easily quantifiable behavioural
contrast could be exploited to test other neurological impairments.
Alarm (antipredator) behaviours are often used in fish research,
although they are usually investigated using olfactory cues. Nevertheless, visual cues can be used to evoke alarm [39,40]. Specifically,
presenting fish with a large shape modelled after a predator or an
actual predatory fish, such as in a neighbouring tank, can evoke
dashing, freezing and shelter seeking. Fast start responses (s-starts)
have been shown to increase in response to the presentation of a
simulated predator (painted falcon tube), and be sensitive to the effects
of ethanol exposure [22]. A recent paper suggested that alarm response
could be a very valuable tool for mechanistically understanding the
roles of signalling molecules, specific receptors and neurons in neural
circuitry [41]. One of the advantages to aversive responses is that they
are easy to score either manually or through video analysis. However,
in some stocks of lab reared fish, innate responses may have been lost.
In this case, conditioning would need to be carried out.
Visually guided prey capture (as opposed to chemosensory-guided)
has been used in a variety of fish species, although not typically on such a
small one. Nevertheless, visually guided appetitive behaviours can be
carried out even in 7-day-old zebrafish using paramecia [42]. Other prey
items such as daphnia, hyalella and brine shrimp have been used in
other species, and there is no reason why they cannot also be used with
zebrafish. Assay endpoints include time to initiate attack (latency),
number of attacked or captured prey, capture efficiency, and time to
cessation [40,42,43]. For example, a recent study on striped bass noted
that time to prey capture was increased 6 days following exposure to an
anti-AChE pesticide, the OP diazinon [44]. Another used zebrafish larvae
with laser ablated premotor neurons in the retinotectum and
reticulospinal neurons to isolate neural circuitry associated with visually
directed prey capture [42]. Tracking software currently uses contrast
(i.e. a dark object, the animal, against a light background) to track as
many as ten objects in one open arena (EthoVision; www.noldus.com).
However, prey may not be easily discernable, and their removal from an
arena may not yet be easily quantifiable. A high throughput version of
this assays remains for development.
A test of optomotor response (OMR) was pioneered over 80 years
ago (see [45]) and has been frequently used on a variety of animals
since, including mice [46] and zebrafish [47]. The test is one of
tracking: animals reflexively keep pace with a rotating a striped or
“grated” drum. At a high enough rotation speed, the lines will blur and
fish will cease to follow. The parameter of interest is the point at
which the animal ceases to track. Excitatory and inhibitory drugs
should increase and decrease this point, respectively, and neurodegeneration will obviously affect it. Another way to conduct the test is
to hold the drum speed constant but vary the illumination [48]. This
method was capable of resolving optomotor differences between
strains. A different version of the test replaces the uniform stripes
with one large stripe. The large stripe represents a threatening object,
and so fish will swim away from it. This test proved capable of
detecting retinal degeneration in a “night blind” mutant [49].
A variation of the OMR assay includes using competing visual
grating [50]. Specifically, a background reference grating is kept at a
constant speed in one direction while a test grating of variable speed
is moved in the other. Fish will move in the direction of the more
strongly perceived cue. Three scenarios are possible: the fish will track
the reference cue when the test cue is of lower contrast, or they will
track the test cue when it is of higher contrast, or when there is
insufficient contrast between the two, they will stop moving. The null
motion point provides an index of visual guided response, with acuity
inversely related to contrast. This test identified performance
differences from a mutation in a vesicular glutamate transporter.
The OMR has been adapted to isolate eye tracking ability in static
larval zebrafish [51,52]. In this “optokinetic response” (OKR) assay,
larvae are immobilized in methylcellulose gel, placed in the same
visual scenario as adults (a circular arena with a rotating striped
perimeter), and smooth tracking and rapid saccades with stripe
sweep are recorded. Impaired altered visual performance will be
evident in decreased saccadic movement [52–54]. The output of eye
velocity in degrees/s can be determined using computer software (a
protocol is available [55]). A consideration is that fish cannot be N 7
dpf because gel immobilization becomes problematic [51]. The OKR
test is functionally equivalent to the finger tracking-based field
sobriety test, where the degree at which eye motion switches from
smooth tracking to saccadic movement is indicative of intoxication.
Zebrafish are a schooling species that exhibits territoriality [56,57].
Although schooling and aggressive behaviour are not solely driven by
vision–they can rely on sensory input from the octavolateralis (sound/
motion), etc.–they are predicated on it. The propensity of a small group
of zebrafish that was physically but not visually isolated from a larger
school to swim near the larger group, i.e. their “social preference,” was
inhibited in a concentration-specific manner by ethanol [22]. School
cohesion, apparent in “nearest neighbour distance” (NND), may also be
used as a sensitive indicator of intoxication [58] and genetic-based
differences [59]. Aggressive behaviour (agonistic behaviour) consists of
alternating and/or coincident fin displays and attack behaviours.
Displays consist of erection of dorsal, pectoral or anal fins, and/or
slapping of the caudal fin; attack behaviours include biting motions and
directed swimming at the attacker [22]. An inclined mirror can be used
to simulate a conspecific, and assess aggression [11,22]. In an
“aggressiveness test,” ethanol exposure was correlated with increased
time near the mirror and aggressive displays [22]. Automatic video
analysis of fin motions obviously poses a challenge, but changes in speed
and distance near the mirror would be easy to determine.
2.2.2. Olfactory endpoints
In fish, olfaction is so important to so many behaviours that it cannot
be done without (reviewed in [60]). This is the reason numerous studies
have focused on its impairment through exposure to a variety of
neurotoxic contaminants (reviewed in [61]). In humans, olfaction is not
so indispensable, however, its loss does correlate with neurodegenerative diseases such as Parkinson's and Alzheimer's [62]. The use of
K.B. Tierney / Biochimica et Biophysica Acta 1812 (2011) 381–389
zebrafish olfactory-based behavioural endpoints of neurodegeneration
remains largely unexplored. A recent publication suggests olfactory
endpoints may also be viable in prion research [63].
In fishes, odorants and odorant classes are associated with specific
behavioural responses, which facilitate isolating specific neuronal
impairments. Odorants include amino acids, bile salts and pheromones,
which evoke feeding and antipredator, social (assumed), alarm and
mating-based responses, respectively [60]. Amino acids associated with
food, such as L-alanine [64], evoked appetitive behaviours such attraction
(directed movement up a concentration gradient) [65]. A mixture of ten
amino acids, including L-alanine, evoked increases in turning rate and
activity level at 4 dpf [37], suggesting the possibility of using amino acid
responses in high throughput assays. Bile salts remain untested as
behavioural modulators in zebrafish, but given their efficacy at evoking
OSN responses [66], they may be of use in olfactory-based assays.
Pheromone-based behavioural endpoints of mating have been restricted
to other species (e.g. F-type prostaglandins evoke substrate probing in
salmonids [67]), however, the behaviours evoked by alarm pheromone
are well studied.
Alarm pheromone is anxiogenic and makes for a very reliable
endpoint since it is an innate reflex. Alarm responses involve a set of
stereotypical behaviours, which include dashing, freezing, jumping and
erratic movements [68–70]. All the actions are “fight or flight” in nature,
and are associated with increases in stress hormone concentrations [70].
Many fish studies have included alarm response as an endpoint of
olfactory neuron function [61]. Numerous neurotoxic agents, including
anti-AChE pesticides, alter alarm response, possibly through impairing
both peripheral (OSNs) and central neurons [61,71]. Alarm behaviour
endpoints are highly sensitive, with some OPs impairing responses with
exposures to just 1 ppb [28,71]. Studies wishing to test the functionality
of the hypothalamic-pituitary-interrenal (HPI) axis (analogous to the
hypothalamic-pituitary-adrenal (HPA) axis in mammals) may benefit
from the inclusion of an alarm assay [41].
A recent study found that hypoxanthine 3-N-oxide (H3NO)
evoked alarm response in zebrafish [72]. Whether this compound is
alarm pheromone or a component thereof, this discovery has great
potential to improve the standardization of alarm response testing,
since a molecularly based behavioural concentration-response curve
can now be constructed. The previous alarm response testing methods
involved the use of a skin extract or filtrate derived from the skin of
conspecifics. These methods suffer by not being able to standardize
the unknown(s) pheromone, however some did endeavour to do so
by measuring total protein [6]. In zebrafish, the alarm response may
be most robust and least variable 50 dpf [73].
One of the simplest tests for olfactory-evoked motion involves the
use of an avoidance trough or “counter current olfactometer.” In this
device, water/odour sources flow from either end of rectangular
trough and exit in the center without mixing appreciably. In this way,
there are effectively two odour zones in the tank, and fish can choose
to spend time in either one. As long as a fish is swimming back and
forth prior to odorant introduction (i.e. has not preferred an end
before stimulus introduction), both attractive and aversive responses
can be determined [15,74,75]. This test is not unlike a Y-maze, where
odours flow down two arms to a null or mixing zone. Y-maze can be
problematic in some cases because fish may elect to make no decision,
i.e. not explore the arms. Nevertheless, both Y-mazes and avoidance
troughs have been useful in fish studies of the attractive or aversive
qualities of amino acids, pheromones and neurotoxic contaminants
[76–79].
2.2.3. Auditory endpoints
Even though the anatomy of the zebrafish ear is dissimilar to that
of mammals (i.e. no cochlea), the neuron hair cell structure is highly
conserved. Additionally, the lateral line cells are structurally and
functionally the same as ear hair cells. Both ear and later line cells can
be disturbed by compounds of human origin (i.e. “ototoxic” agents).
385
For example, the antibiotic streptomycin can negatively affect fish
behaviour [80]. For these reasons, hair cell alteration has mammalian
parallel (reviewed in [17]).
An auditory-evoked startle response has recently been adapted
from rodents to zebrafish [7]. In the study, adult zebrafish were placed
individually in an eight tank array and video was recorded from
overhead. The stimulus consisted of an automated, mechanical “tap”
to the tank bottom. Fish swimming distance was determined over the
following 5 s, and the taps were repeated every minute for a total of
ten times. This session was then subsequently repeated. This design is
particularly interesting because its neurological assessment is twodimensional: it tests an innate, behavioural reflex (which includes
integration of sound and space, and depends on physiological
condition), as well as the ability to adapt (habituate) rapidly to a
stimulus. In their study, they found that both endpoints could show
changes due to larval exposure to chlorpyrifos. However, it was
arguably the ability to adapt that highlighted the biggest neurological
alteration. The difference in startle response magnitude was greater
and more significant with repetition. The purpose of this high
throughput auditory-based test was to assess the persisting effects
of chlorpyrifos exposure, however there is no reason this assay could
not be used to screen/assess a variety of neurological conditions/
pathologies. Furthermore, given their findings regarding habituation,
studies including other stimuli (such as odorants), could benefit from
including such a parameter.
2.2.4. Interoceptive sensory endpoints
Fish rely on balance and experience pain. However, unlike
mammals, equilibrioception (balance) is accomplished through the
octavolateralis system, which includes neurons that perceive sound
and motion. Additionally, fish do not rely upon their “limbs” (i.e. fins)
for balance. In fact, removing two or more fins has frequently been
used as a non-invasive, low impact means of identifying fish [81,82].
Nevertheless, neurotoxins affecting the vestibular portion (i.e. inertial
sense) of the inner ear, or neurodegeneration in general, may
conceivably affect balance, and so may impair rheotactic ability.
Performance deficiencies could be gauged through measuring the
time to current orientation.
Decades of research on fish swimming performance has relied on
mild electrical shock to evoke swimming [83,84]. Detecting altered
neurological performance through nociceptor-based response is not
common in fish, but it does show promise, particularly with agents/
conditions which modify anxiety. A surprising result of a recent study
was that cocaine did not alter a behavioural avoidance of a mild
electrical field [16]. Given that establishing a local electric field in an
open arena is not difficult, and that fish will instinctively avoid it, this
method has promise for screening neurotoxins with anaesthetic
effects.
2.3. Learning and memory based endpoints
In mammals, the hippocampus is involved with cognitive
plasticity. Zebrafish lack a hippocampus, but they do have a
developmentally similar region, the lateral pallium, that appears
functionally equivalent [85]. Reference memory (i.e. acquired memories) and working memory (i.e. the attentional component of shortterm memory), and their interplay (i.e. cognitive plasticity) can be
tested in a variety of ways, including both classical (Pavlovian) and
operant (instrumental) conditioning. Both conditioning methods
evoke response(s) using a pre-existing behaviour (evoked by either
CS or UCS), but for classical conditioning, a neutral stimulus is converted
into a positive CS (through their repeated pairing), and in operant
conditioning consequences are used to reinforce or extinguish a CS. In the
below, endpoints are discussed for (1) attention, for (2) searching
(map building) behaviour, for (3) the acquisition of responses to
neutral stimuli (classical conditioning), as well as for (4) the
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K.B. Tierney / Biochimica et Biophysica Acta 1812 (2011) 381–389
reinforcement or extinguishment of positive and negative stimuli
(operant conditioning).
2.3.1. Attentional endpoints
Behaviours generally result from the integration of multiple
signals. What is important is the ability to discern important signals
amidst background noise. Neurological filtering in vertebrates has
been referred to as the “cocktail party effect,” coined to describe the
ability of a person to hear a signal such as their name spoken in a room
full of chatting people [86]. Implicit in “selective attention” is that
distracting signals be ignored. One assessment methodology used in
non-human primates involves coupling a positive stimulus, such as
food, with the occurrence of another stimulus, such as a particular
visual signal, and varying delay between the two [87]. Distractability
can be assessed though inserting irrelevant stimuli in the delay
period. Since filtering draws upon reference memory and working
memory, it is a useful correlate of neurological function. While
numerous paradigms exist to test selective attention-based tests in
rodents and primates [87,88], no obvious correlate currently exist for
zebrafish. However, a functionally similar test might be carried out by
challenging zebrafish with competing cues of fitness relevance. For
example, fish could be exposed to a food odorant, which would
initiate appetitive behavioural response, then interrupted with an
alarm cue, which should evoke anti-predator behaviour. The degree
and rate at which the appetitive behaviour is replaced and antipredator responses exhibited (i.e. evokes attention) could prove
meaningful endpoints of sensorimotor function and cognition. To
explicitly test memory function, the UCS food and/or predator cues
could be replaced with CS cues, such as behaviourally neutral amino
acids or PEA. An attentional endpoint could be especially useful in
testing cholinergic system functionality, since working memory is
dependent on it [89], and since many drugs agonize or antagonize
cholinergic receptors and neurodegenerative diseases such as Alzheimer's
impair it.
2.3.2. Searching (spatial) endpoints
Introduction to new surroundings will stimulate searching behaviour and map building [35]. The formation of a new cognitive map is
another form of neural plasticity and therefore a possible performance
metric for neurological impairment. Numerous studies have already
validated that searching behaviour in an open arena is a meaningful
endpoint in zebrafish [22,70,90]. Searching behaviour was recently used
in combination with two anxiogenic factors, alarm pheromone or
caffeine, and two axiolytic factors, ethanol and fluoxetine (i.e. the SSRI
Prozac), as an endpoint in “a novel tank test” [70]. Release into a new
tank evoked a bottom seeking in control fish, which was followed by
subsequent upper tank exploration. With alarm pheromone and
caffeine exposure, the number of upper tank explorations was reduced,
and the time taken to explore (latency) increased. With exposure to
ethanol or fluoxetine, the reverse pattern was noted: transitions to the
upper tank increased, and the latency to upper tank searching was
greatly diminished. Nicotine had the opposite effect, fish were not as
inclined to dive to the tank bottom [23].
Spatial memory is mediated through the lateral pallium. Studies
have found that lesioning the functionally equivalent region in rats
(hippocampus), abolished searching behaviour [91]. Given the ability
to focally lesion larval zebrafish brain tissue and neurons using laser
ablation [42], studies of searching hold promise for cognitive
endpoints. A tried and true test of spatial memory in rats is the
Morris water navigation task, which is essentially map building under
stress and with visual cue reference. The rate of learning of the
platform location can be gauged by the latency to platform discovery
and distance covered in successive trials. Learning under stress in fish
could be accomplished in a variety of means, such as by the ability to
find an escape portal (to another potion of a test tank), under the
threat of predation, evoked by visual or olfactory stimuli. One
zebrafish study using a net as a freight stimulus showed that the
latency to discover an escape portal decreased significantly over six
training sessions [92].
2.3.3. Classical conditioning endpoints
Zebrafish studies have demonstrated that both visual [93] and
olfactory [8] cues unassociated with behavioural response(s) can be
converted to behavioural response cues. For example, over a few
(b10) trials, zebrafish were able to visibly discriminate and select
coloured arms of a T-maze where they would receive a food reward
[93]. As for odorant-based learning, a recent study demonstrated that
by pairing PEA (a neutral stimulus) with food odours (L-alanine and Lvaline) over a number of training sessions, appetitive swimming
behaviour (turning rate) could be evoked by the addition of PEA alone
(i.e. it was converted to a CS) [8]. Classical conditioning endpoints
may be increasingly adopted since conditioning can be achieved
rapidly. For example, just one pairing of a neutral stimulus (red light)
with alarm pheromone (UCS) evoked significant conditioning in a
related species (fathead minnow) [94]. These data suggest that the
strength of the innate response determines the rate of learning.
In many species of vertebrates, shaping is used to evoke a desired
behaviour. This method consists of reinforcing successive steps
towards a target behaviour. This method of reference memory
creation may be unreasonable for fish; however autoshaping may
be a viable option. Autoshaping was developed in pigeons and
consisted of the pairing of a visual cue (light) with food. Initially the
pigeons will respond (peck) to the UCS, but over time will respond to
the CS alone [95]. There is no reason why this model would not also
work in fish. Zebrafish will swim into odour sources and sometimes
“bite” at water inflows including food odour (personal observation).
With repeated trials, they could be expected to bite at a water flow
source alone.
2.3.4. Operant conditioning endpoints
A variety of methods using open arena to multi-chambered arenas
exist to test stimulus reinforcement or extinguishment. Open arenas
have been used for tests involving food odour and predator scent. In
the first, an appetitive response to food flake was enhanced by pairing
it with amino acids food odours [8]. In the second, a study of a closely
related species (fathead minnow) found that it took 6 to 8 days and
two to four days to visually and olfactorily acquire a predator,
presumably by the pairing of a UCS (alarm pheromone) with visual
cues and scent, respectively [39]. Visual and odorant cues clearly have
potential for assessing cognition, and the differential time course
could provide a valuable diagnostic tool.
A common method of operant conditioning involves presenting an
animal with one or more choices between arena arms or areas, and
based on the decision, either rewarding the animal with food or
punishing it with pain or stress. A popular paradigm is “conditioned
place preference” (CPP) or sometimes just “place preference” (PP), in
which animals are rewarded with a drug after selecting a particular
location [96]. The reinforcing stimulus needs to be a positive
psychoactive stimulant, such as cocaine [97] or amphetamine [98].
A version of CPP is “conditioned place avoidance” (CPA), which is
where the conditioned area was initially avoided [99]. For example, by
“addicting” zebrafish to D-amphetamine, preference for a side of the
tank with two large freight inducing black dots could be evoked [99].
In general, CPP and CPA are good models for testing the neurological
bases for addiction, but they remain largely unadopted but available
in toxicity or degenerative studies.
At least two methods exist to test vision-based learning in fishes,
the T-maze and the three chambered arena. Both methods share some
similarities to the radial arm maze (RAM), frequently used in
mammalian models. In the T-maze, fish are introduced into the long
arm (base of the T) and they will generally swim to one or both of the
sort arms. However, as many as 5% fish may not move from the base
K.B. Tierney / Biochimica et Biophysica Acta 1812 (2011) 381–389
arm unless provoked [97]. The intended outcome is for a fish to favour
one arm based on the desirability of the settings (“spatial preference”)
[97], or a reward coupled to a visual cue [93]. In tests using the three
chambered arena, fish are introduced to a center area that is flanked
on either side by gated chambers [100]. To generate avoidance
behaviour of one of the side chamber, fish are penalized (using
confinement) for making repeated selections of one tank side (in
general, to condition an animal to avoid an area based on a visual cue,
nociceptor and stress based methods are used [101]). The three
chamber confinement test demonstrated that cholinergic agonization
can improve the number of correct decisions [100].
In all operant models discussed above, a conditioned CS will lose
its behavioural stimulatory efficacy as its repeated presentation goes
unpaired with the initial pre-existing, positive stimulus [8]. As with
acquisitional endpoints, the rate of change in the reinforced decision
can provide a correlate of neurological function [102].
3. Problems associated with behavioural endpoints
Variation in responses/traits/parameters increases with organisational level, and so behaviours are obviously the most variable of
organismal responses. Additionally, in fish, as other animals, their
state will affect memory recall and motivation, and they are not
without personality. Furthermore, with aquatic species, special
consideration must be made for the water source. Municipal water
is often rife with neurotoxic contaminants, which may vary in
concentration considerably even over brief time periods. Water
needs to be carbon filtered and tested to remove/account for any
confounding chemical effects.
An appreciable amount of individual variation can be accounted
for by classifying behavioural phenotypes, such as low or high activity
subgroups. Doing so will increase the power of the test; however, not
all traits that appear obviously related to behaviour may be so. In
juvenile brook trout, for example, swimming performance ability was
not associated with actual (conative) swimming activity [103]. Some
temporal issues can be exploited; food responsiveness and willingness to take risks, for example, are enhanced with brief periods of food
deprivation [65,103]. Social status can also affect stress level which
will in turn modulate activity and stimuli responses [103]. If social
endpoint was to be used, accounting for dominance behaviour in fish
may present a challenge. Careful observation and selective removal of
individuals of stronger resource utilization (e.g. food consumption)
could help resolve variation.
4. Conclusions
Vertebrates have been proposed as the best models for elucidating
mechanisms of neurodegeneration [104]. Among vertebrate models,
zebrafish are finding ever increasing application. In studies of drugs,
zebrafish make for excellent models, since delivery can be achieved in
many cases by simple addition to the tank water. Furthermore,
microinjection techniques are now available to deliver agents directly
to specific cells/tissues, even within larval fish [105]. This review has
highlighted behavioural endpoints available to zebrafish that probe
basic neurological function, that test behaviours predicated on the
function of diverse types of sensory neurons, and that investigate
cognitive performance. Some behavioural endpoints test multiple
neurologically-based abilities; this review is intended to provide a
template by which to link and/or apportion specific physiological,
perceptual and cognitive impairments. Most methodologies described
herein have very tight parallel with mammalian models. Furthermore,
many behavioural tests, especially those using high throughput
screens, remain in evolution. The future holds promise for the
development of a suite of standardized behavioural assays that can
be used to determine the mechanisms by which neurotoxic effects
and neurodegenerative diseases develop and progress.
387
Acknowledgments
The author is grateful to W. Ted Allison for motivation in doing this
work, and for grants to KBT from the Canadian Wildlife Federation
(CWF) and the University of Alberta.
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Contents lists available at ScienceDirect
Biochimica et Biophysica Acta
j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / b b a d i s
Review
Aberrant forebrain signaling during early development underlies the generation of
holoprosencephaly and coloboma☆
Patricia A. Gongal a,1, Curtis R. French a, Andrew J. Waskiewicz a,b,c,⁎
a
b
c
Department of Biological Sciences, University of Alberta, Edmonton, Canada T6G 2E9
Centre for Neuroscience, University of Alberta, Edmonton, Canada T6G 2E9
Women & Children's Health Research Institute, University of Alberta, Edmonton, Canada T6G 2E9
a r t i c l e
i n f o
Article history:
Received 1 December 2009
Accepted 8 September 2010
Available online 16 September 2010
Keywords:
Holoprosencephaly
Coloboma
Sonic hedgehog
Gdf6
Retinoic acid
Retina
a b s t r a c t
In this review, we highlight recent literature concerning the signaling mechanisms underlying the development of two neural birth defects, holoprosencephaly and coloboma. Holoprosencephaly, the most common
forebrain defect, occurs when the cerebral hemispheres fail to separate and is typically associated with
mispatterning of embryonic midline tissue. Coloboma results when the choroid fissure in the eye fails to close.
It is clear that Sonic hedgehog (Shh) signaling regulates both forebrain and eye development, with defects in
Shh, or components of the Shh signaling cascade leading to the generation of both birth defects. In addition,
other intercellular signaling pathways are known factors in the incidence of holoprosencephaly and
coloboma. This review will outline recent advances in our understanding of forebrain and eye embryonic
pattern formation, with a focus on zebrafish studies of Shh and retinoic acid pathways. Given the clear overlap
in the mechanisms that generate both diseases, we propose that holoprosencephaly and coloboma can
represent mild and severe aspects of single phenotypic spectrum resulting from aberrant forebrain
development. This article is part of a Special Issue entitled Zebrafish Models of Neurological Diseases.
© 2010 Elsevier B.V. All rights reserved.
1. Introduction to holoprosencephaly and coloboma
2. Part I: Holoprosencephaly
As the vertebrate embryo develops, the ventral forebrain and retina
are subject to similar extracellular cues that drive their development. A
combination of human genetic, animal model, and cell biochemical
studies has shed light on the signaling pathways that are essential for
patterning this region of the embryo. Within the forebrain, researchers
have identified deficiencies in Nodal, Shh, and retinoic acid (RA)
pathways, as causes of the ventral forebrain birth defect holoprosencephaly. In the retina, mutations in components of the Shh, RA, and
BMP signaling pathways prevent proper closure of the choroid fissure,
and result in coloboma. The overlap in genetic causes for these two
distinct birth defects implies that they may be more accurately
considered as components of a common phenotypic spectrum. This
review will define the recent advances in our understanding of
Holoprosencephaly (Part I) and Coloboma (Part II), and propose the
commonality of their causality.
Holoprosencephaly (HPE) is the most common forebrain birth
defect in humans [1] and encompasses a broad range of craniofacial
and neural defects. Traditionally, HPE has been defined as the
failure of the forebrain to divide into two distinct cerebral hemispheres during development. In the most profound cases, the fetus
displays cyclopia and the complete absence of lobar divisions in the
cerebrum [2–7]. The HPE spectrum also includes ethmocephaly,
where the fetus displays a proboscis (an abnormal tubular nose-like
structure) and closely-set eyes, and cebocephaly, where the fetus
has closely-set eyes and single nostril [8]. More subtle defects have
also been classified as falling within the HPE spectrum, including
median cleft lip, absence of the olfactory bulb, agenesis of the
corpus callosum, and a single, centrally located maxillary incisor
[2,8]. While HPE is rare in live births (1/16,000), approximately 1/
250 conceptuses are thought to be affected by HPE [1,9]. This large
difference in frequency likely reflects an incompatibility of
severe HPE with life, and a high rate of miscarriage of affected
embryos.
HPE can be caused by both genetic abnormalities and exposure to
teratogens, including alcohol [10,11] and retinoic acid [12,13].
Approximately one-quarter of cases are due to detectable genetic
anomalies [5]. Mapping chromosomal deletions in affected patients
facilitated the identification of the first genes linked to HPE [14–20],
☆ This article is part of a Special Issue entitled Zebrafish Models of Neurological
Diseases.
⁎ Corresponding author. CW405, Biological Sciences Building, Biological Sciences
Department, University of Alberta, Edmonton, Alberta, Canada T6G 2E9. Tel.: +1 780
492 4403; fax: +1 780 492 9234.
E-mail address: [email protected] (A.J. Waskiewicz).
1
Present address: Laboratoire de Génétique Moléculaire du Développement, U784
INSERM, Ecole Normale Supérieure, 46 rue d'Ulm, 75230 Paris cedex 05, France.
0925-4439/$ – see front matter © 2010 Elsevier B.V. All rights reserved.
doi:10.1016/j.bbadis.2010.09.005
P.A. Gongal et al. / Biochimica et Biophysica Acta 1812 (2011) 390–401
and later work has demonstrated a high frequency of microdeletions
and point mutations in these genes [21].
Research in zebrafish over the last decade has yielded many
insights into the normal function of HPE genes, and how their
disruption can result in human disease. The eight genes linked to
HPE have been shown to play important roles in three major
developmental signaling pathways: Nodal, Sonic hedgehog, and
retinoic acid (RA). The forkhead box transcription factor FOXH1 (also
known as FAST1 and schmalspur in zebrafish) [22] and EGF-CFC family
protein CRIPTO (also known as TDGF1 in mammals and one-eyed
pinhead in zebrafish) [23] are components of the Nodal pathway. The
signaling ligand SONIC HEDGEHOG (SHH), transmembrane receptor
PATCHED (PTC) and transmembrane sterol-sensing protein DISPATCHED-1 (DISP1) are core members of the Sonic hedgehog signaling
pathway, while the transcription factor SINE OCULIS HOMEOBOX-3
(SIX3) directly regulates shh expression [24–27]. The atypical homeodomain transcription factor TG-INTERACTING FACTOR (TGIF) regulates
retinoic acid signaling during development [28–30], while ZINC
FINGER OF CEREBELLUM (ZIC) transcription factors have recently
been proposed as a regulator of Nodal, Sonic hedgehog, and retinoic
acid signaling in the forebrain [31]. There is significant crossregulation among these three pathways, which has important
implications for early brain development. This cross-regulation is an
important consideration in interpreting phenotypes associated with
an alteration of gene function within the three pathways, such as in
HPE patients (see Fig. 1 for an anatomical overview of forebrain
signaling relevant to HPE).
2.1. Specification of the prechordal plate
The prechordal plate is the anterior-most axial mesoderm, and a
critical ventral patterning center that underlies the developing
forebrain. Research in zebrafish has delineated a clear role for Nodal
391
signaling in the formation of the vertebrate prechordal plate.
Disruption of this tissue results in the fusion of the eye fields and
deletion of ventral forebrain tissue, a phenotype that strongly
resembles the most severe cases of HPE [32–36]. The prechordal
plate is the anterior-most derivative of Spemann's organizer (the
shield in zebrafish), which, during gastrulation, acts as a signaling
center to induce dorsal and anterior structures. The shield is specified
very early in zebrafish development (the 128 cell-stage) and arises
from the cells of the dorsal margin. The transcription factor β-catenin
is stabilized in the nucleus of prospective shield cells, and when
zygotic transcription initiates, activates transcription of the Nodal
ligand and morphogen squint [37]. Within the shield, levels of Nodal
signaling are asymmetric. The most vegetal cells are subject to the
highest levels of Nodal signaling, and become the prechordal plate,
while cells positioned nearer the animal pole, exposed to lower levels
of Nodal signaling, become notochord [38]. The prechordal plate, after
the cell movements of gastrulation, comes to underlie the ventral
forebrain, while the notochord underlies the more posterior neural
tube.
2.2. Nodal signaling and HPE
Nodal is a member of the TGF-β superfamily of signaling proteins.
Members of this protein superfamily exist as dimers, and activate
type I and type II transmembrane serine/threonine kinase receptors.
Nodals bind the type I receptor protein ALK4 or the complex of ALK7
(type I) and ActRIIB (type II) [39]. The HPE gene CRIPTO/oep physically
interacts with the ligand–receptor complex, and is required for Nodal
signal transduction, positioning this protein as a Nodal co-receptor
[40–43]. Ligand binding results in the phosphorylation of the type I
receptors, which in turn phosphorylate the transcription factor
Smad2. Phosphorylated Smad2 is imported into the nucleus, where
it binds Smad4 and other cofactors, including bonnie and clyde (bon;
mixer) and the HPE gene FOXH1 (schmalspur) [44,45]. The Smad
complex then activates the transcription of target genes, such as the
prechordal-plate-specific transcription factor goosecoid (gsc) [46] and
the morphogen shha [47].
Forward genetic screens in zebrafish have identified mutations in
the Nodal pathway components linked to HPE: the co-receptor Cripto/
oep, and the Smad2 cofactor FOXH1/sur [48,49]. Loss of function of
these genes causes the loss or reduction of the shield and the
elimination of the prechordal plate, causing severe forebrain deficits
within the HPE spectrum, including cyclopia.
2.3. Cripto/one-eyed pinhead in HPE
Fig. 1. Ventral forebrain patterning is regulated by signaling between the pectoral plate
and overlying neural tube. Holoprosencephaly (HPE) is caused by a complex set of
molecular genetic signaling pathways. Retinoic acid (RA, gradient shown in red) plays a
key role in this process and is synthesized in the posterior paraxial mesoderm by
Aldh1a2 and degraded anteriorly by Cyp26a1. Tgif, which causes HPE, regulates
expression of both cyp26a1 and aldh1a2. Within the prechordal plate (PCP, blue),
signaling among Zic, Nodal (Cyclops or Cyc) and Sonic hedgehog (Shh) creates a
signaling center underlying the ventral diencephalon. Shh signaling activates both ptc1
and nkx2 genes.
oep transcript is maternally deposited in 1-cell zebrafish embryos,
and embryos lacking both maternal and zygotic oep (MZoep) have a
much more severe phenotype than zygotic mutants. MZoep mutants
fail to develop a morphologically distinguishable shield, and lack all
shield-specific gene expression. As a consequence, embryos lack most
mesodermal cell types, including the prechordal plate and notochord.
This in turn causes a failure to specify ventral neurectoderm, and
embryos display a cyclopic phenotype [40].
In zygotic oep mutants that retain maternal sources of this protein,
shield-specific gene expression is initiated normally, but fails to be
maintained. The notochord is specified correctly, and expresses shha,
suggesting that low levels of Nodal signaling are sufficient for
notochord development. However, zygotic oep mutants lack prechordal plate, and shha is not expressed in forebrain regions,
indicating that the specification of prechordal plate is particularly
sensitive to a reduction in Nodal signaling [36].
Two human patients with mutations in CRIPTO have been
described [23]. One patient displays several HPE-like microforms,
including a hypoplastic corpus callosum. This patient has a heterozygous P125L mutation in the CFC domain of the oep orthologue
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TDGF1, which is required for the protein to bind type I TGF-β
receptors. While wild type TDGF1 will rescue the zebrafish oep mutant
phenotype, a P125L mutant version does not, suggesting that this
protein is nonfunctional, and the malformations in this patient may be
due to TDGF1 haploinsufficiency. A second patient displays semi-lobar
HPE, and a V159M mutation in CRIPTO, which is in a domain thought
to mediate the protein's association with the cell membrane [50].
However, this mutant version of the protein can still rescue an oep
mutant, suggesting that the basis of HPE in this case may involve
additional genetic or environmental factors. Indeed, within most
patient families, the penetrance of HPE is highly variable. It has been
widely speculated that interaction between multiple genetic deficiencies or environmental insults are responsible for this variability,
and underlie most cases of HPE [4–6].
2.4. The transcription factor Foxh1 and HPE
FOXH1 is a Smad-binding protein which targets activated Smad
complexes to specific DNA sequences [51]. It is required for
transduction of a subset of Nodal signals, and is partially redundant
with the paired-like homeobox transcription factor Bon/Mixer [52].
Zebrafish lacking maternal and zygotic Sur (MZsur) have reduced, but
not eliminated, shield-specific gene expression. MZsur embryos also
lacking bon have abolished shield-specific gene expression, indicating
that these genes are partially redundant. Foxh1 mouse mutants
entirely lack specification of the node, prechordal plate, and
notochord, and display a severely hypoplastic forebrain [53].
Similarly, MZsur zebrafish mutants also show the elimination of the
prechordal plate, although notochord-specific gene expression is still
present. Interestingly, notochord-specific genes are activated even in
the absence of both bon and sur, suggesting that these two are not the
only Smad cofactors that can transduce Nodal signal to specify
notochord in zebrafish. Besides its role as a Nodal signal transducer,
Sur is also required for the maintenance of cyclops and squint
expression during gastrulation [44], however, expression of neither
cyclops nor squint can rescue prechordal plate specification, cyclopia,
or other phenotypes in MZsur embryos. Therefore, it is likely that
these defects mostly result from an inability to transduce Nodal
signals within the axial mesoderm.
A number of mutations in FOXH1 have been identified in HPE
patients [22], including those that disrupt the forkhead DNA binding
domain and Smad-interaction domain. The activity of these mutant
versions has been assessed by measuring the activity of RNA to rescue
zebrafish sur mutants. These studies demonstrate that mutations in
the Smad-interacting domain eliminate protein activity, while
variants in the forkhead domain reduce protein activity by approximately 20% [22].
Overall, in live births, the occurrence of Nodal pathway mutations is
rare [22]. Work in animal models suggests that this is likely due to the
essential nature of this signal pathway during the very early stages of
development. Indeed, in patients suffering from recurrent miscarriage, a
very high proportion of embryos lost in the first trimester show severe
neural and midline defects [54]. Embryos with severe loss of Nodal
function are likely not viable, and it is plausible that Nodal pathway
mutations contribute to a proportion of early pregnancy losses.
2.5. Sonic hedgehog signaling and HPE
Defects in the Sonic hedgehog pathway are the most frequent
cause of HPE [18], and this pathway has long been known to be
essential for forebrain patterning. An essential role for Nodal signaling
is the initiation of shh transcription in the prechordal plate and
notochord [38]. Shh secreted from the axial mesoderm initiates shh
transcription in the floor plate, the most ventral and medial cells of the
neural tube. A graded hedgehog signal emanating from both the axial
mesoderm and floor plate is required for diencephalon specification,
ventral neuronal identities, and the correct separation of the eye field
into two distinct domains [55].
The mechanism of Shh signal transduction has been the subject of
intense research. The protein undergoes extensive post-translational
modifications, including autocatalytic cleavage and cholesterylation of
the N-terminus, which becomes the mature ligand [56]. Subsequently,
a palmitate moiety is added to the N-terminus of the ligand by the
enzyme known as Hedgehog acyltransferase or Skinny hedgehog [57].
The N-terminal signaling ligand processed with both lipid modifications is denoted as ShhNp. Cleavage and lipid modification is required
for secretion outside the cell. A series of proteins, including Dispatched
and You/Scube2 are required for its export. ShhNp then binds to
transmembrane proteins (Interference hedgehog; IHOG and Brother
of IHOG; BOI) that facilitate its interaction with the Patched receptor
[55]. In the absence of ligand, Ptc inhibits the function of Smoothened
(Smo). Upon ligand binding, this inhibition is relieved; Smo becomes
activated, and induces a signaling cascade that leads to the regulation
of the localization or cleavage of Gli transcription factors. In Drosophila,
a single Gli protein, named Cubitus interruptus (Ci), is present. Upon
ligand binding, the cleavage of Ci into a repressor isoform is inhibited,
and the activator isoform transcriptionally upregulates target genes
[58].
Sixty-four unique mutations in the SHH gene have been linked to
holoprosencephaly [59]. These include frameshift and nonsense
mutations, missense mutations in functional domains or uncharacterized but conserved domains, or changes in residues important for
processing, such as the post-translational addition of palmitate and
cholesterol moieties. Two orthologues of SHH exist in zebrafish, shha
and shhb (formerly known as tiggy-winkle hedgehog). This complicates
analysis of mutants for these two genes, as each can compensate for
the other's function. Mutants lacking shha have a mild phenotype,
including abnormal fin and somite patterning, and axonal pathfinding
defects, while knockdown of shhb alone causes no detectable
phenotype [60–62]. Because of redundancy, shh loss of function has
mostly been accomplished in zebrafish by mutations in or pharmacological inhibition of Smoothened (Smo) [63]. The compound
cyclopamine directly binds and inhibits Smo signal transduction
[64]. Unlike the Shh ligand itself, smo does not appear to be duplicated
in the zebrafish genome, and smo mutants show a severe phenotype,
although not as severe as cyclopamine-treated embryos. Smo is
present maternally, and maternal transcript is likely to allow for some
early Shh signal transduction, which cyclopamine would block. smo
mutants have a suite of defects, including synopthalmia and improper
motor neuron specification in the spinal cord. Moreover, mutants
show a dorsalized forebrain and a complete lack of hypothalamic
tissue. smo mutants also have a lack of anterior pituitary cell fates, and
instead form an ectopic lens at this site. Interestingly, an optic stalk
marker, pax2a, is ectopically expressed in the forebrain, suggesting
that hedgehog signals play a key role in distinguishing forebrain from
eye identities [63].
The HPE gene PATCHED is the receptor for Shh, and is in fact a
negative regulator of the pathway. Unliganded Ptc represses Smo, and
ligand binding prevents Ptc repression activity. Therefore, loss of Ptc
function results in constitutive activation of the Shh pathway [65–67].
Zebrafish have two ptc homologues, and mutants for both ptc1 and
ptc2 have a strong upregulation of hedgehog signaling, concomitant
with a ventralization of the neural tube and mispatterning of the
somites [68]. Conversely, gain of Ptc function results in constitutive
repression of the pathway, and also results in phenotypes within the
spectrum of HPE, including midline defects, such as malformation of
the corpus callosum [66,69].
Several PTC mutations have been linked to HPE. Two of these are in
the extracellular domain in a position thought to interact with the
SHH ligand. Two others are located within intracellular domains of the
protein, and it is hypothesized that these domains might be important
for PTC's interaction with SMO [70]. It has been proposed that these
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mutations represent a gain of PTC inhibitory function of the SHH
pathway, as HPE phenotypes are well-known to be linked to SHH loss
of function. However, it is possible that a gain of SHH function could
cause HPE-like phenotypes as well, since in zebrafish, a loss of Ptc1
and consequential increase in signaling results in narrow-set eyes and
moderate forebrain patterning defects [68].
The function of the HPE gene DISPATCHED has also been modeled
in zebrafish, where there is a single described orthologue [71].
Although disp1 expression is broad, it is enriched in regions of high
Shh signaling: the ventral forebrain, notochord, and floor plate. In
Drosophila, disp has been shown to be required for export of processed
Shh ligand [72]. Suggesting that this function is conserved, disp lossof-function in mice and zebrafish causes phenotypes typical of Shh
deficiency [73–76]. Zebrafish embryos with mutations in disp1
(chameleon) have phenotypes typical of a reduction in Shh signaling:
strongly reduced expression of shh target genes, and a dorsalized
neural tube. Further supporting the model that disp regulates Shh
export, overexpressing shh RNA in disp1 zebrafish mutants causes an
attenuated transcriptional response compared to shh overexpression
in wild type embryos [76].
2.6. The transcription factor Six3 and HPE
Zebrafish models of the function of the HPE gene SIX3 are
complicated by the presence of three SIX3-related genes, six3a,
six3b, and six7, which are all expressed in the prechordal plate during
gastrulation, and then in the anterior neurectoderm during somitogenesis [77]. Interestingly, however, six3b/six7 loss of function
indicates that Six3 proteins are required in the neurectoderm to
repress Nodal target gene expression, which contributes to the proper
establishment of left/right asymmetry [77]. Complete loss of six3 gene
function may reveal additional roles for six3 regulation of Nodal
signaling.
Zebrafish rescue studies indicate that the vast majority of HPEassociated SIX3 mutations act as hypomorphs [78]. Work in mice
suggests that defects in a direct interaction between SIX3 and the Shh
signaling pathway underlie the development of HPE [24]. Geng and
colleagues generated mice with a HPE mutant version of Six3
knocked-in to the endogenous Six3 locus. While heterozygous mutant
mice display HPE-like phenotypes at a low frequency, removing one
copy of Shh results in severe cyclopia and loss of ventral diencephalic
gene expression. Further, mice heterozygous for the mutant Six3 and
with one copy of Shh deleted have a loss of both Shh and Six3
expression in the ventral diencephalon, indicating that these two
genes form a critical positive feedback loop. The regulation of Shh by
Six3 is direct, and the Six3-binding site in the Shh forebrain enhancer
is mutated in a patient with semi-lobar HPE [79], indicating the
importance of the genetic interaction between these two proteins for
forebrain development.
2.7. Retinoic acid signaling and HPE
Retinoic acid has a critical role in early neural patterning. This small,
diffusible molecule is synthesized from vitamin A (retinol) precursors
through a series of enzymatic reactions. The retinol dehydrogenase
Rdh10 catalyzes the conversion of retinol to retinaldehyde during
development [80], and aldehyde dehydrogenases (raldh or aldh1a
gene family) convert retinal to retinoic acid [81]. There are two
characterized aldh1a genes in zebrafish: aldh1a2, which is expressed at
high levels in paraxial mesoderm and in the dorsal retina [82,83], and
aldh1a3, which is expressed in the ventral retina flanking the optic
stalk [84]. Recently, an additional enzyme, cyp1b1, has been demonstrated to synthesize RA in multiple tissues during chick development
[85]. This gene is expressed in a much more restricted domain in
zebrafish than chick, and is present in the anterior telencephalon and
eye, beginning mid-somitogenesis (Gongal et al., submitted). Its
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contribution to zebrafish neural or eye patterning has yet to be
established. However, mutations in mouse and human Cyp1b1 are
linked to the development of juvenile-onset glaucoma, suggesting an
important role in eye development [86–88].
The best characterized role of RA during development is in the
hindbrain, where it acts as a potent posteriorizing signal on neural
tissue. High levels of RA signaling are required for specification of
caudal hindbrain fates [82,83,89–91]. During gastrulation, the
forebrain is insulated from the posteriorizing influence of RA through
the activity of cyp26 (cytochrome P450, subfamily XXVI) genes, which
hydroxylate RA, a modification thought to target the molecule for
degradation [92]. cyp26a1 is the earliest expressed cyp26, and is
expressed in the anterior neurectoderm, initiating during gastrulation
[93]. Slightly later, cyp26b1 and cyp26c1 transcription is initiated in a
subset of telencephalic cells (cyp26c1) and the diencephalon (cyp26b1
and cyp26c1) [94,95], where they limit RA activity. Both cyp26b1 and
cyp26c1 are also expressed in dynamic, rhombomere-specific patterns
in the hindbrain, where they are essential for proper anterior–
posterior patterning [94–97]. Together, RA synthesis and degradation
genes set up a highly dynamic pattern of RA levels in the developing
CNS.
Abnormal RA levels have severe phenotypic consequences,
particularly for the forebrain. Exposure to exogenous RA during
development causes severe craniofacial defects, eye patterning
anomalies, and ectopic forebrain expression of posterior CNS genes
[11,98,99]. RA levels become abnormally high when Cyp26 activity is
reduced, which has been modeled in mice, chick, and zebrafish.
Overall, embryos with reduced Cyp26 function display defects in
patterning of the diencephalon, eye, hindbrain and neural tube
closure defects [30,96,100–102]. Further, without Cyp26 activity,
embryos are highly sensitive to environmental changes in retinoids,
and the entire neural tube takes on posterior hindbrain identity upon
exposure to doses of retinal that cause no defects in wild type
embryos [96].
High levels of retinoic acid have recently been linked to DiGeorge
syndrome (also known as velo-cardio-facial syndrome), a suite of
multiple congenital defects, including craniofacial abnormalities such
as cleft palate [103,104]. A critical region mapped in patients with
chromosomal deletions encompasses the gene tbx1, which regulates
expression of all three cyp26 genes during somitogenesis [102]. Taken
together with the large body of evidence that increased developmental RA levels can cause forebrain patterning defects, it is plausible that
changes in RA signaling underlie some of the phenotypes observed in
DiGeorge syndrome.
RA deficiency also causes severe developmental defects. Loss of RA
has been modeled in vitamin A-deficient quail and swine [105–107],
mouse and zebrafish mutants for RA-synthesizing genes
[82,83,90,108], and chick and zebrafish by application of pharmacological inhibitors [89]. Although defects observed vary slightly
depending on the organism, common phenotypes include the
formation of a single telencephalic vesicle, mispatterning of the
diencephalon, failure to specify the caudal hindbrain, and eye
abnormalities. Thus, both too much and too little RA cause defects
in similar tissues, and many of these phenotypes are reminiscent of
HPE. Interestingly, similar defects are observed in a gain or loss of RA,
such as the mispatterning of the diencephalon and failure of the
neural tube to close.
2.8. The transcription factor TGIF and HPE
Until recently, little was known about the role of the HPE gene TGIF
(5′-TG-3′ interacting factor) in forebrain development. Work in cell
culture systems demonstrates that TGIF can transcriptionally regulate
both retinoid and TGF-β signaling. Tgif can bind Smad2 and Smad3,
and represses the transcription of a TGF-β-dependent reporter [109],
and mutant versions of TGIF that occur in human patients do not show
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this same repression activity [110]. As alterations of Nodal signaling
are well-known to cause forebrain defects in animal models, and
Nodal is a TGF-β pathway, it was widely assumed that misregulation
of Nodal function explained the incidence of HPE in human patients
deficient in TGIF [5,7,110–113]. However, there has been a notable
absence of evidence from animal models for this hypothesis.
In fact, TGIF was originally identified because of its involvement
with retinoid signaling. This atypical homeodomain transcription
factor was discovered in a search for proteins that bind to a retinoid X
response element (RXRE) [29]. Bartholin and colleagues provide
further evidence that TGIF can regulate retinoid signaling [28]. They
convincingly show that TGIF binds RXRs (most robustly RXRα and
RXRγ, and RXRβ to a lesser extent), and that TGIF can form a complex
both with RXR and its nuclear receptor binding partners, including
RARs and PPARs (peroxisome proliferator activated receptor). Using a
reporter assay, Tgif can repress RA-dependent transcription under the
control of a DR5 element. However, this element is activated by RXR
expression, regardless of the presence or absence of ligand. Further, it
isn't clear whether this repression activity is specific for RXR or
generally acts on other nuclear receptors. Therefore, the in vivo
relevance of Tgif's function in this assay remains to be established.
Four independent groups have generated Tgif-null mice [28,114–
116]. None of these mutants has neural patterning phenotypes. Mice
have multiple copies of Tgif-like genes, including Tgif2, Tgif-like-onthe-X and Tgif-like-on-the-Y, whose functions are poorly understood,
and which may compensate for the loss of Tgif activity in these
mutants. However, depending on genetic background, heterozygous
and homozygous mutants do have an increased rate of exencephaly
when treated with RA [28,116], suggesting that Tgif plays a role in the
regulation of RA signaling.
A fifth group interested in Tgif function noted that some patients
had a nonsense mutation that left the first repressor domain and
homeodomain intact, but eliminated the second and third repressor
domains. Kuang and colleagues generated a mouse with an equivalent
mutation by deleting Tgif's third exon. Interestingly, mice with this
mutation do show neural patterning defects, although these are
strain-specific [117]. TgifΔexon3 mice show severe hypoplasia of the
forebrain and exencephaly. Shh protein levels are reduced, and the
ventral forebrain marker Nkx2.2 has reduced expression. Because of
these phenotypes, Kuang and colleagues propose that this version of
Tgif may function as a dominant negative. However, defects in this
mutant are still highly dependent on genetic background, which
suggests that there are unknown and essential genetic modifiers that
exist in this strain.
In contrast to mice, zebrafish have only two tgif family genes, tgif
and tgif2, and therefore genetic redundancy may be less of an issue for
functional analyses. Studies in zebrafish have revealed that Tgif is an
essential regulator of retinoic acid metabolism gene expression [30].
Tgif is required for the initiation of both cyp26a1 and aldh1a2,
indicating that this gene regulates both RA degradation and synthesis.
tgif morphants have reduced diencephalic gene expression, a
phenotype that resembles that of cyp26a1 mutants, suggesting that
excess RA in the forebrain is responsible for this mispatterning. These
results suggest a molecular basis for Tgif-dependent holoprosencephaly; embryos lacking TGIF function may be highly sensitive to
teratogenesis from environmental fluctuations in vitamin A [30].
2.9. Genetic interactions between Sonic hedgehog and retinoic acid
signaling in the forebrain
While deleterious for the forebrain during gastrulation, RA is in
fact required for proper forebrain development at later stages. One of
its important roles is facilitating Sonic hedgehog signaling. The genetic
interactions between Shh and RA signaling are complex, and are quite
different between species, as well as between different tissues and
developmental stages (see below). RA regulates Shh signaling at
multiple points in the pathway, including transcription of the ligand,
response to the signal, and post-translational processing. The
mechanisms of these interactions are unclear, and identifying the
precise molecular bases that underlie RA-Shh interactions are key
topics for future work.
Interpreting the effects of changes in shh expression can be
challenging, as transcription of this gene is regulated by an
autoregulatory loop. When Shh signaling is reduced, shh transcription
is upregulated [118], so both upregulation and downregulation of the
pathway may be correlated with an increase in shh transcription.
Nevertheless, shh expression is critical for activity of the pathway, and
RA regulates signaling at this level.
Aldh1a2 mouse mutants have normal Shh transcription (and
protein levels, detected by immunohistochemistry) in the axial
mesoderm during early stages of forebrain patterning. Slightly later
in mouse development (by the 16 somite stage), however, RA does
regulate the transcription of Shh itself. This regulation is tissuespecific, however, as in a Aldh1a2 mutant, Shh is decreased in some
tissues (ventral diencephalon), and increased in others (infundibulum) [108]. The functional consequences of this loss of transcript are
unclear, but occur too late to explain early loss of Shh target gene
expression in aldh1a2 mutants.
In contrast, transcriptional regulation of shh by RA seems to be
more important in bird development. In vitamin A-deficient quail, shh
expression is reduced in prechordal plate at early stages [106].
Further, locally applying RAR and RXR inhibitors in the chick forebrain
abolishes shh expression. Treated embryos display cyclopia and loss of
forebrain tissues [119]. This forebrain phenotype is more severe than
an Aldh1a2 mouse mutant, perhaps because receptor inhibition causes
a more profound loss retinoid activity. Aldh1a2 and Aldh1a3 are
partially redundant in the mouse forebrain patterning, but double
mutants cannot be analyzed (without maternal supplementation with
RA), due to very early lethality caused by cardiac defects.
A second method of RA regulation of Shh signaling has recently
been revealed in mouse. Aldh1a2 mutant mice have a reduction in Shh
target gene expression, such as Gli3 and Olig2, while no change in Shh
transcription or protein levels is detectable [108]. Importantly, Ribes
and colleagues demonstrated that RA deficiency causes a failure to
appropriately respond to the Shh signal [120]. Although the
mechanism of this interaction is not yet understood, treating
Aldh1a2 mutants in vitro with Shh ligand does not result in high
levels of Shh target gene expression, as does treating wild type
embryos. These results could reflect a role for RA in regulating the
activity of Shh protein activity, localization, processing, degradation,
or RA signaling could be required for the transcription or function of
a Shh pathway component. Future experiments will reveal the myriad
of mechanisms used in regulating Shh activity, such as modulation of
palmitoylation [121,122].
2.10. Zic genes and the coordinated regulation of Shh and RA signaling
While cross-talk between the Shh and RA pathways is likely essential
to coordinate their activities during neural patterning, these two
pathways are also connected by common regulators. A transcription
factor that regulates both the RA and Shh pathways has been identified:
zic1. The precise effect of a single factor transcriptionally regulating
components of multiple pathways is not well understood. However, it is
plausible that this method of regulation functions to maintain a precise
balance between the activities of the two pathways.
zic1 (whose related gene zic2 has been linked to HPE) has been
linked to disruptions to both the retinoic acid and Shh signaling
pathways [31]. zic1 morphants show a loss of Shh target gene expression
(ptc1), concomitant with a mild reduction in shha expression and a
strong reduction in shhb expression. zic1 loss-of-function results in
higher levels of RA signaling in the eye and forebrain mid-somitogenesis. zic1 morphants have an expansion of optic stalk in the eye and
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forebrain, a tissue that strongly expresses the RA-synthesizing genes
aldh1a3 and cyp1b1, which might cause the high RA levels observed in
the eye and forebrain. Overexpressing cyp26a1 makes some zic1
morphant phenotypes more severe, suggesting that RA deficiency,
rather than excess, contributes to these phenotypes. Indeed, RA levels in
the somites are reduced, as assayed by RARE-yfp reporter expression.
Nevertheless, although the detailed mechanisms underlying Zic1
function are not yet clear, its overall function is clearly to regulate
both RA and Shh signaling, which may help facilitate the coordinated
actions of these two pathways during neural development.
Zebrafish zic2 has also been shown to play a key role in regulating
forebrain development. Morphant embryos that lack zic2a display a
pronounced reduction in arx and dlx2 gene expression, indicating a
defect in specification of the prethalamic region of the forebrain [123].
Though the changes in ptc1 gene expression are mild, it is possible
that other zic genes are compensating for a loss of zic2a [124]. Recent
research on zic2a has shown that it plays an essential role in
regulating proper formation of the eye. Indeed many of the genes
discussed in this section function at the same developmental stage to
regulate formation of the eye. Given the striking similarities between
forebrain and eye patterning mechanisms, the second component of
this review will deal with eye formation birth defects, most notably
colobomata, and the known signaling pathways that regulate ocular
morphogenesis.
3. Part II: Coloboma
During early development, the vertebrate eye contains a transient
ventral fissure that is essential for the entry of mesenchyme cells into
the retina, where they will give rise to blood vesicles that will nourish
the eye throughout the life of the organism [125]. Failure of the optic
fissure to close results in coloboma, an important cause of visual
impairment worldwide. Depending on the size and location, this
congenital malformation can lead to blind spots, lazy eye, and reduced
visual acuity in patients [126].
Coloboma can affect numerous tissues in the posterior eye including
the choroid, and optic nerve, as well as tissues of the anterior eye such as
the iris, cornea, and lens [127]. Ocular colobomata have been observed
as an independent defect, or as a component of syndromes that often
include other ocular abnormalities as well as craniofacial and neurological phenotypes [128]. These include microphthalmia [127,129,130],
cataracts [131], and CHARGE syndrome (coloboma, heart defects,
choanal atresia, retarded growth, and genital and ear anomalies)
[132]. Colobomata can be sporadic, or heritable with autosomal
dominant, recessive, and X-linked inheritance cases being reported.
The vertebrate eye is patterned along proximal–distal axis early in
development. The distal eye cells give rise to neural retina and retinal
pigmented epithelium, while more proximal cells give rise to the optic
stalk and choroid. Although transient, the choroid fissure acts as an
entryway into the eye, allowing vascularization to occur. Before the
onset of cellular differentiation in the retina, the fissure begins to close
using a contact-dependent dissolution of the basal lamina at the
contacting neuroepithelial on each side of the optic fissure [133].
Thus, genetic abnormalities or environmental toxins that disrupt this
process, likely present as coloboma.
A number of genetic lesions and chromosomal rearrangements
have been identified that lead to coloboma in human patients, many
of which involve the loss of Sonic hedgehog signaling or one of its
downstream target genes [134,135]. As aberrant Shh signaling is one
of the leading causes of holoprosencephaly, it is important to note
that many individuals suffering from mild forms of HPE
also display coloboma. In addition, defects in the RA [136,137], and
Bmp [127] signaling pathways have been implicated in the
generation of coloboma or syndromes involving colobomataneous
phenotypes.
395
3.1. Sonic hedgehog signaling is required for closure of the optic fissure
To date, only one family has been identified with coloboma due to
mutation in the sonic hedgehog (shh) gene itself [135]. It is likely that
null mutations, or even strong hypomorphic mutations in the Shh
gene are incompatible with life as Hedgehog signaling is required
forebrain patterning and development. Thus, most knowledge
concerning Shh and its role in ocular coloboma comes from
experiments in animal models. Both the zebrafish and mouse have
proven to be useful models for the study of the eye, and have provided
valuable insight as to the role of Shh and its downstream target genes
in the development of coloboma.
In zebrafish, like most other vertebrates, there are two tissues that
act as sources of Sonic hedgehog ligand that are relevant for the
development of the eye: first, the floorplate in the ventral midline of
the diencephalon, and second, the neural retina itself. It is the former
that is responsible for eye patterning and closure of the optic fissure
[138], while Shh signaling derived from within the retina is required
later in development for the timely cell cycle exit and differentiation
of neural lineages [139].
3.2. Sonic hedgehog regulates genes required for eye patterning
The zebrafish eyecup is partitioned early in development, establishing the retina, optic stalk, and retinal pigmented epithelium into
molecularly distinct domains. Midline diencephalic Sonic hedgehog is
required for this partition by maintaining the expression boundary of
spatially restricted transcription factors [138]. Loss of shh in zebrafish
leads to severe coloboma, and the loss of ventral-anterior homeobox 1
and 2 (vax1 and vax2), which delineate the optic stalk and ventral
neural retina regions [140]. Inactivation of both vax genes in zebrafish
results in severe coloboma [140], supporting the model that shh acts
genetically upstream of vax genes to regulate closure of the optic
fissure (Fig. 2).
In addition to regulating vax transcription factors, Shh is required
for the proper expression of members of the paired box family of
transcription factors, with pax2 and pax6 being important for eye
development and closure of the optic fissure. Shh is required for
expression of pax2, while negatively regulating the levels of pax6
[138,141]. Mutations in both genes have been mapped in patients
with coloboma or colobomataneous syndromes; mutations in pax2
are linked to renal-coloboma syndromes [142,143] while mutations in
pax6 tend to be found in patients with coloboma of the optic nerve
(along with other retinal phenotypes including aniridia). pax2
expression is restricted to proximal eye structures such as the optic
stalk and fissure, while pax6 is restricted to more distal structures. The
expression of pax2 in cells surrounding the choroid fissure requires
Shh, and overexpression of shh leads to pax2 expression in more distal
structures such as the neural retina [138]. Conversely, loss of Shh
signaling results in reduced expression of pax6 neural retina. Thus, it
appears that control of pax gene expression via shh is required for the
proper partition of early eye to define and maintain the partition of
optic stalk and neural retina. It is clear from studies involving both Pax
and Vax proteins, that early embryonic patterning of the eye into
distinct optic stalk and neural retinal lineages is essential for
subsequent fusion of the optic fissure.
Recent evidence from zebrafish model studies suggests that the
regulation of pax genes via Sonic hedgehog signaling is indirect, and
involves the regulation of other factors that modulate pax gene
expression. One such factor, Zinc finger protein of the cerebellum 2a
(Zic2a), has been shown to negatively regulate the transcriptional
output of Sonic hedgehog signaling, while itself being transcriptionally regulated by Hedgehog signaling cascades [124]. Loss of zic2a
results in severe coloboma [124,144,145] likely due to mispatterning
of the optic stalk and retina, as optic stalk markers are ectopically
expressed in the retina in Zic2a-depleted embryos [124].
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P.A. Gongal et al. / Biochimica et Biophysica Acta 1812 (2011) 390–401
tion of the retinoblastoma gene (Rb), leading to abnormal cell cycle
progression and increased proliferation [147]. Work in mice demonstrates that accurate regulation of the cell cycle acts upstream of Sonic
Hedgehog and causes abnormalities in the eye. Loss of function studies
of involving the cell cycle regulator E2F4 result in coloboma, and
aberrant expression of Sonic Hedgehog ligand at the ventral midline
[148]. E2F4 is a transcription factor downstream of the Rb gene,
further linking Rb cell cycle control to the development of coloboma.
In addition, Shh effector genes are abnormally expressed upon loss of
E2F4, with Pax2 expression extending ectopically to the distal optic
cup, and reduced Pax6 expression in the ventral optic cup [148].
3.3. Retinoic acid plays a key role in closure of the optic fissure
Fig. 2. Sonic hedgehog signaling is required for eye patterning the eye along the mediolateral axis. A complex transcriptional interaction between Sonic hedgehog (Shh) and
Zic pathways specifies the identity of the midline of the ventral diencephalon. The Shh
diffusible ligand is required for specification of ventral eye structures, including the
optic stalk and ventral neural retina. Shh is required for expression of vax2 and pax2
transcription factors, both of which are expressed in the optic stalk and ventral retina.
This signaling cascade is opposed in the dorsal retina through the action of Bmp ligands,
such as Gdf6. Bmp signaling cascades are required for the expression of dorsal retinal
specific transcription factors such as Tbx5 and Tbx2. Loss of Shh signaling, or any of its
downstream signaling components can lead to coloboma. Loss of Bmp signaling in the
dorsal retina can also lead to coloboma, as well as ectopic fissure formation in the dorsal
retina.
Although numerous candidate genes have been identified downstream of Shh for the development of coloboma, there is little data
concerning the mechanism by which fusion of the optic fissure is
prevented in shh mutants. In wild type animals, fusion is facilitated by
contact between the epithelial tissue on either side of the optic fissure
leading to a degradation of the basal lamina. Inhibition of Shh
signaling and the consequential mispatterning along the proximal–
distal axis of the eyecup have been shown in some cases to physically
prevent contact between epithelium on either side of the optic fissure.
For example, in the zebrafish blowout mutant where there is a
mutation in ptc1, (a gene that functions as a Shh receptor and negative
regulator of Shh signaling; see Section I of this review), ectopic
expression of optic stalk markers such as pax2, is observed, and the
stalk grows ectopically into the retina, physically preventing contact
between the epithelial tissue on both sides of the optic fissure [146].
Conversely, when pax2 is lost in zebrafish, contact between the
epithelial tissues on either side of the optic fissure still occurs, yet
there is no dissolution of the basal lamina and the fissure persists.
Thus, pax2 must be required for fissure closure subsequent to contact
between the two sides of the fissure.
It has recently been demonstrated that cell cycle regulation may
also play a key role in regulation of optic fissure closure, and is linked
to Shh signaling. For example, mouse humpty dumpty mutants display
severe coloboma and cell cycle defects. This transgenic mouse
contains a mutation in Phactr4, which results in hyperphosphoryla-
Retinoic acid has long been implicated in the development of the
eye in humans and animal models. Like Sonic hedgehog, mutations in
components of the core RA pathway are rarely found in human
patients with coloboma due to the wide array of functions retinoic
acid performs in the developing embryo. However mutations in
Retinol Binding Protein (rbp) have been described in a sibling-pair that
presented with iris coloboma [149]. In both siblings, no serum Rbp
was detected, and patients had approximately 17% of normal retinol
levels. In addition to iris coloboma, these patients also suffered from
night blindness but were otherwise healthy. Furthermore, there is
evidence that suggests that in some populations, vitamin A-deficient
(VAD) diets are correlated with the incidence of coloboma [136],
reiterating the link between aberrant retinoid levels to the development of coloboma.
Animal models have yielded major insights into the role of retinoic
acid during eye development. For example, mice that receive
teratogenic doses of retinoic acid while pregnant give birth to pups
with eye defects, including coloboma [150,151]. In zebrafish, local
delivery of RA to the eye via implanted RA soaked beads not only
results in coloboma, but can also induce ectopic fissure formation
anywhere in the retina [152]. Furthermore, failure to close the optic
fissure in RA treated mice correlates with changes in gene expression
along the proximal–distal axis of the developing eye [151] in a
manner comparable to that seen with a loss of Sonic hedgehog
signaling. Epistasis analysis indicates that sonic hedgehog itself is
downregulated by teratogenic doses of RA [153], although this
relationship was only tested in tissues other than the floorplate (the
critical source of Shh ligand for optic fissure closure). Nevertheless,
this demonstrates that RA can act upstream of Shh in a living
organism.
Although teratogenic doses of RA clearly regulate Shh signaling
and cause coloboma, little work has been done involving manipulation of RA at physiologically relevant levels. In most studies, embryos
are incubated in a concentration of RA that far exceeds what an
embryo would naturally be exposed to during development. In the
developing embryo, RA levels are higher in the ventral retina than in
the dorsal retina, thus creating a gradient of retinoic acid signaling
across the dorsal–ventral axis of the eye [154,155]. This gradient
cannot be mimicked by simply exposing the whole embryo to RA.
Thus, loss of function studies are required to determine if endogenous
RA is required for closure of the optic fissure. These studies have been
fewer in number, and the role of endogenous RA in fissure closure
appears to differ among different model systems. It has been noted,
for example, that treatment of developing mice with citral, a
competitive inhibitor of aldehyde dehydrogenases [156–158] caused
a delay in (although did not prevent) the closure of the optic fissure
[151]. Vitamin A depletion in mice also leads to coloboma that occurs
with a reduction in ventral retinal cell number and a loss of cell
adhesion molecules surrounding the optic fissure [159]. This
phenotype may be due to abnormal morphogenesis of the early
eyecup, as opposed to later patterning events, as when aldehyde
dehydrogenase genes are knocked out in mice, the dorsal eyecup
P.A. Gongal et al. / Biochimica et Biophysica Acta 1812 (2011) 390–401
invaginates, but the ventral eyecup completely fails to form [160].
Similarly, in zebrafish, treatment of embryos with citral results in the
failure of the ventral eye to form, while the dorsal half remains
relatively normal [154]. Knockout of the dorsal-specific aldh1a2 does
not result in coloboma, although it is likely that compensation by the
ventral-specific aldh1a3, for which there is currently no mutant
zebrafish strain available. Thus it appears in both mice and zebrafish,
endogenous RA may affect closure of the optic fissure not because of
improper patterning of ocular tissues, but due to its requirement for
earlier morphological events leading to morphologically normal eyes.
3.4. Bmp signaling and coloboma
Mutations or chromosomal aberrations involving members of the
bone morphogenetic protein (Bmp) family have also been described
in human patients with coloboma. Asai-Coakwell and colleagues
demonstrated that a deletion encompassing GROWTH AND DIFFERENTIATION FACTOR 6 (GDF6/BMP13) is likely responsible for bilateral
retinal coloboma and unilateral iris coloboma [127]. Further studies
revealed that mutations in the prodomain of GDF6 account for 1.6% of
retinal colobomata in the human population [161]. This mutation
results in reduced Gdf6 protein secretion from the cell, which is
mechanistically consistent with Gdf6's non-cell autonomous effects
[162,163]. Mutations or chromosomal aberrations involving the
closely-related bmp4 can also cause coloboma in human patients
[164].
Animal models have confirmed the association of gdf6 and bmp4
with coloboma, and have provided insights as to the mechanism by
which each gene can affect closure of the optic fissure. Both genes are
expressed in the retina during early stages of development, and
knockdown of gdf6a (one of two gdf6 paralogues in the teleost
genome) in zebrafish results in retinal coloboma [127,162]. Similarly
to exogenous RA application, loss of Gdf6a signaling in zebrafish leads
to ectopic fissure formation [162,163]. Studies of axial patterning
molecules expressed in the retina indicate that gdf6a is responsible for
limiting ventral eye marker expression, as loss of this protein results
in expansion of vax2 expression to include both the ventral and dorsal
retina. This may explain the ectopic fissure in the dorsal eye, which is
respecified to a ventral fate in the absence of Gdf6a. In addition, Gdf6a
is required for proper establishment of RA signaling in the eye. For
example, the dorsal-specific aldh1a2 is not expressed in embryos
lacking Gdf6a, while the ventral-specific aldh1a3 is upregulated [162].
While details regarding the mechanism by which bmp4 can cause
coloboma are not as well understood, it has been noted that bmp4
expression overlaps with both sonic hedgehog and patched1 expression. Further suggesting a genetic interaction between BMPs and
Sonic hedgehog signaling is that an increased phenotypic severity is
observed in human patients with low-penetrance mutations of both
bmp4 and shh than with either single mutation [164]. Supporting this
model, studies in mice have implicated Bmp4 as essential for
Hedgehog induced coloboma. In a conditional knockout of the
Smoothened gene, which is required to transduce the Hedgehog
signal, Bmp4 expression is strongly expanded throughout the ventral
retina [165]. As Bmp4 expression is normally confined to the dorsal
retina, the authors speculate that coloboma in these embryos may be
caused by ectopic BMP signaling. In addition, the expression of ventral
retinal and optic stalk markers, including Vax2 and Pax2, show
reduced expression in Smoothened-null embryos, however only at
developmental time points after ectopic expression of Bmp4 is
observed. Thus, loss of ventral retinal and optic stalk gene expression,
and the resulting coloboma in embryos suggest that the lack of
Hedgehog signaling results in the inability to limit Bmp4 signaling.
Furthermore, overexpression of Bmp4 in the developing optic vesicles
can inhibit Hedgehog expression itself [166], supporting a model
whereby localized expression of Sonic hedgehog and Bmp ligands
397
constitute antagonizing signaling centers required for patterning the
eye along the dorsal–ventral axis and closure of the optic fissure.
In many model systems, Bmp4 and Gdf6 have been proposed to
function redundantly. As loss of gdf6a results in coloboma and has
been shown to regulate retinal expression of bmp4 [162], it is likely
that Gdf6a also plays a role in the formation of coloboma in embryos
lacking Smoothened function. Surprisingly, loss of Smoothened does
not result in any change of expression retinoic acid synthesizing genes
such as Aldh1a2 or Aldh1a3 [165]. Gdf6 has been shown to regulate the
expression of both of these genes, demonstrating that although Bmp4
and Gdf6a act redundantly for some developmental processes, at least
some independent functions for each molecule are required for
closing the optic fissure.
3.5. Coloboma may be linked to overall growth of the eye
Mutations in human BMP4 and GDF6 result in both coloboma and
microphthalmia, indicating that closure of the optic fissure may be
genetically linked with the overall growth of the eye in utero. A
number of other genes have been reported to cause both microphthalmia and coloboma, including transcription factors such as
orthodenticle homeobox 2 (otx2) and Sex determining region Y-box 2
(sox2). Whole gene deletions in Otx2 tend to result in severe
microphthalmia, while milder mutations tend to result in either
unilateral or bilateral coloboma [167–169], suggesting that the level of
gene activity influences both growth of the eye and closure of the
optic fissure. Although required for differentiation of the retinal
pigmented epithelium [170,171], otx2 expression is also observed in
the early proliferating cells of the eyecup [172], and could thus affect
growth of the eye before it is required for specification of RPE.
However to date, there is no data that sheds light on the mechanism
by which otx2 influences both growth of the eye and closure of the
optic fissure.
Mutations in the SOX2 gene have also been identified in human
patients [173]. Similar to Otx2, the severity of loss of function in Sox2
correlates with the severity of disease, whereby mutations causing
complete loss of function result in severe microphthalmia, while
milder variants tend to manifest as coloboma [167,174]. Although it is
not known whether Sox2 is directly involved in closure of the optic
fissure, the product of this gene is required for the proliferation of
early retinal progenitor cells [174], reiterating the potential genetic
link between eye growth and closure of the optic fissure.
4. Conclusions
The genetic pathways that influence the formation of HPE and
coloboma, as outlined in this review, have considerable overlap.
Holoprosencephaly and colobomata represent birth defects that can
arise from aberrant Sonic hedgehog signaling. Shh is required in the
forebrain for the formation of ventral neural structures and division of
the early eye field into two distinct hemispheres. Interference with
this signaling pathway, either through genetic mutation or via
environmental factors can thus cause holoprosencephaly. Forebrain
Shh signaling is also needed for early eye patterning, a process that is
required for closure of the optic fissure. Given the overlap in
mutational etiology, holoprosencephaly and coloboma are likely to
represent severe and mild ends of the same phenotypic spectrum.
Retinoic Acid signaling pathway can also play a role in the
development of HPE and coloboma. RA is required for proper
forebrain morphogenesis and influences Shh signaling through a yet
unknown mechanism. In the eye, RA is required for morphological
events required for proper eye morphogenesis, and possibly for
patterning eye tissues. Nodal signaling is also important, as it is
required for the initiation of shh expression in the ventral forebrain. In
the eye, Bmp signaling is required for eye patterning, and thus closure
of the optic fissure, possibly through an antagonistic signaling
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relationship with Shh. It is thus clear that alteration of genetic
pathways that subsequently influence Shh signaling, in addition to the
Shh pathway itself, can result in the generation of HPE or coloboma.
[26]
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Contents lists available at ScienceDirect
Biochimica et Biophysica Acta
j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / b b a d i s
Review
Fish models in prion biology: Underwater issues☆
Edward Málaga-Trillo a,⁎,1, Evgenia Salta b,1, Antonio Figueras c, Cynthia Panagiotidis d, Theodoros Sklaviadis b,⁎
a
Department of Biology, University of Konstanz, 78457 Konstanz, Germany
Department of Pharmacology, Aristotle University of Thessaloniki, 54124, Thessaloniki, Greece
c
Instituto de Investigaciones Marinas, CSIC, 36208, Vigo, Spain
d
Centre for Research and Technology-Hellas, Institute of Agrobiotechnology, 57001, Thessaloniki, Greece
b
a r t i c l e
i n f o
Article history:
Received 8 December 2009
Received in revised form 11 September 2010
Accepted 21 September 2010
Available online 7 October 2010
Keywords:
Prion protein
Transmissible spongiform encephalopathy
Neurodegeneration
Fish
Zebrafish
a b s t r a c t
Transmissible spongiform encephalopathies (TSEs), otherwise known as prion disorders, are fatal diseases
causing neurodegeneration in a wide range of mammalian hosts, including humans. The causative agents –
prions – are thought to be composed of a rogue isoform of the endogenous prion protein (PrP). Beyond these
and other basic concepts, fundamental questions in prion biology remain unanswered, such as the
physiological function of PrP, the molecular mechanisms underlying prion pathogenesis, and the origin of
prions. To date, the occurrence of TSEs in lower vertebrates like fish and birds has received only limited
attention, despite the fact that these animals possess bona fide PrPs. Recent findings, however, have brought
fish before the footlights of prion research. Fish models are beginning to provide useful insights into the roles
of PrP in health and disease, as well as the potential risk of prion transmission between fish and mammals.
Although still in its infancy, the use of fish models in TSE research could significantly improve our basic
understanding of prion diseases, and also help anticipate risks to public health. This article is part of a Special
Issue entitled Zebrafish Models of Neurological Diseases.
© 2010 Elsevier B.V. All rights reserved.
1. Introduction
1.1. Prion disorders
Prion diseases or TSEs are a group of rare but fatal neurological
disorders that affect humans and animals. Whether sporadic, inherited
or acquired, these illnesses generally correlate with the accumulation of
misfolded PrP in the brain and the appearance of widespread
neurodegeneration after long incubation times [1]. The classical
histopathological landmarks of TSEs are spongiform vacuolation,
neuronal loss and astrocytic gliosis, whereas the main clinical
manifestations in humans include progressive dementia, cerebellar
ataxia and myoclonus [2]. Interestingly, although such symptoms are
also observed in more common neurodegenerative disorders like
Abbreviations: TSEs, transmissible spongiform encephalopathies; PrP, prion
protein; BSE, bovine spongiform encephalopathy; CJD, Creutzfeldt–Jakob disease;
GPI, glycosylphospatidylinositol; MBM, meat and bone meal; p.i., post inoculation; i.c.,
intracerebrally; PK, proteinase K; GI, gastrointestinal
☆ This article is part of a Special Issue entitled Zebrafish Models of Neurological
Diseases.
⁎ Corresponding authors. E. Málaga-Trillo is to be contacted at Department of
Biology, University of Konstanz, Universitätstrasse 10, 78457, Konstanz, Germany. Tel.:
+49 7531 884581; fax: +49 7531 884863. T. Sklaviadis, Department of Pharmacology,
School of Pharmacy, Aristotle University of Thessaloniki (AUTH), AUTH Campus, 54214
Thessaloniki, Greece. Tel.: +30 2310 997615; fax: +30 2310 997720.
E-mail addresses: [email protected] (E. Málaga-Trillo),
[email protected] (T. Sklaviadis).
1
Equally contributing authors.
0925-4439/$ – see front matter © 2010 Elsevier B.V. All rights reserved.
doi:10.1016/j.bbadis.2010.09.013
Alzheimer's disease (AD) and Parkinson's disease (PD), prion diseases
have received special attention because of their infectious nature and
the associated risk of epidemics.
In fact, prion diseases gained considerable notoriety in the 1990s
because of the massive outbreak of mad cow disease (bovine spongiform encephalopathy, BSE) in Europe and the negative impact it had on
public health and the food industry. TSEs are, however, not a recent
phenomenon. Their history dates back to the 1700s when scrapie, a
related neurological condition among sheep and goats, was first
described in England [3]. Other TSEs in animals include chronic wasting
disease (CWD) in deer and elk, transmissible mink encephalopathy and
feline spongiform encephalopathy (FSE) [4]. The most common TSEs in
humans, Creutzfeldt–Jacob disease (CJD) and Gerstmann Sträussler
Scheinker syndrome (GSS), were discovered much later (1920s and
1930s, respectively [5]), and it was not until the 1950s that the study of
kuru, another human prion disease, set off a chain of revolutionary
discoveries that would redefine the meaning of “pathogen” and push a
biological dogma against the ropes.
1.2. The nature of the infectious TSE agent
Kuru was originally described as a neurological epidemic of the Fore
linguistic group in Papua New Guinea [2]. For some time, the disease
was believed to be infectious, and possibly transmitted during
cannibalistic rituals. Nevertheless, failure to transmit kuru to laboratory
animals led to the erroneous assumption that it had a genetic basis.
Towards 1959, remarkable similarities between kuru, CJD and scrapie
E. Málaga-Trillo et al. / Biochimica et Biophysica Acta 1812 (2011) 402–414
were recognized at the neuropathological, clinical and epidemiological
levels [6,7]. Since scrapie had been already shown to be transmissible
[8], it became apparent that kuru and CJD were also of infectious
etiology. Indeed, by 1968, both human diseases had been successfully
transmitted to chimpanzees [9,10]. Despite these advances, the nature
of the scrapie agent continued to be the subject of intense debate for
many years. Because TSEs exhibit strain variation and long incubation
periods, it was argued that the agent could be a slow-virus [11].
However, direct proof for this “viral hypothesis” has been difficult to
obtain [12]. Instead, it has been shown that the transmissible agent is
resistant to formalin (a chemical that inactivates viruses) and to various
other treatments that destroy nucleic acids, but not to proteolytic
digestion [13–15]. These and other developments finally led to the
enunciation of the “protein-only hypothesis” by Stanley Prusiner
(1982), according to which TSEs are caused by prions, small proteinaceous particles capable of replicating in the absence of nucleic acids
[14,16].
The protein-only hypothesis has not remained uncontested. While
some continue to advocate the existence of an unidentified scrapie
agent [17–19], others see the notion of an infectious, self-replicating
protein as a clear violation of Crick's central dogma of molecular biology
[20], which excludes the flow of biological information from protein to
protein. Notwithstanding the controversy, Prusiner's theory reasonably
accounts for the complex etiology of TSEs, the physicochemical
properties of the agent, and the existence of scrapie strains [3]. Today,
it is widely accepted that prions are largely – if not entirely – composed
of PrPSc, an abnormally folded isoform of the cellular prion protein
(PrPC) [21]. The molecular mechanism by which normal PrPC “converts”
into infectious PrPSc is, however, poorly understood. It has been
proposed that PrPSc can associate with PrPC and induce its conversion
into more PrPSc, triggering a chain reaction [22]. Alternatively, PrPSc and
PrPC may coexist in equilibrium until mutation or sporadic events favor
the formation of oligomeric PrPSc seeds, which undergo repeated
aggregation and fragmentation cycles [23]. In either case, the concept of
PrP conversion is central to the protein-only hypothesis because it
explains the ability of prions to replicate and propagate disease.
1.3. The prion protein
In 1982, PrPSc was identified as the major constituent of infective
fractions purified from hamster brain homogenates [24]. Subsequent
characterization revealed that the pathogenic protein was host-encoded
and not the product of a viral gene, as it had been assumed previously
[25,26]. It was also established that PrPSc is posttranslationally derived
from PrPC [27]. Thus, although the two isoforms differ greatly in their
spatial conformation [28], they have the same amino acid sequence and
are encoded by the same single-copy gene, Prnp [29]. Under normal
conditions, PrPC is a glycoprotein tethered to the outer plasma
membrane by a glycosylphosphatidylinositol (GPI) anchor. Its unique
molecular structure, studied by nuclear magnetic resonance (NMR) and
crystallographic techniques, can be roughly divided into two halves: a
flexible N-terminal domain rich in repetitive motifs, and a C-terminal
globular domain containing a characteristic array of three α-helices and
two β-sheets. At the center of the polypeptide chain, a short
hydrophobic stretch connects the two major domains [30,31]. In
contrast, attempts to resolve the molecular structure of PrPSc have not
been successful.
Almost 30 years after the discovery of PrP, there is still no consensus
about what its physiological role might be. Since PrP has no obvious
similarities to known protein families, functional affinities cannot be
inferred from amino acid sequence comparisons. Neither can organ- or
tissue-specific functions be hypothesized because PrP is widely
expressed in most adult and embryonic cell types [32–35]. Astonishingly, the generation and analysis of PrP knockout mice failed to provide
conclusive information about the physiological role of PrP, as these
animals develop and behave rather normally. Aside from a few subtle
403
abnormalities, their only clear “phenotype” is their resistance to prion
infection [36]. It is probably not exaggerated to say that as many PrP
functions have been proposed as methods have been employed to
address this question. Thus, the list of putative PrP roles in vitro and in
vivo includes activities as diverse as cytoprotection from apoptosis [37]
and oxidative stress [38], olfactory behavior [39], copper metabolism
[40], neurogenesis [41], neurite outgrowth and neuronal survival [42],
lymphocyte activation [43], hematopoietic stem cell self-renewal [44],
synaptic function [45], activation of signal transduction [46], and
binding to cell adhesion molecules [47]. Unfortunately, it is not clear
how many of these findings may be of actual physiological relevance. In
addition, it seems unlikely that a single molecular function of PrP could
account for such diverse biological roles.
The clear correlation between PrP misfolding and neurodegeneration may suggest that PrPSc is the direct cause of prion disease.
Nevertheless, solid experimental evidence indicates that prions
cannot induce neuronal damage in the absence of PrPC. For instance,
PrP knockout mice are resistant to scrapie inoculation [48], even if
they carry PrPC-expressing neurografts [49], or if PrPC depletion is
induced postnatally [50]. Notably, transgenic mice expressing only a
GPI-anchorless version of PrP (thus unable to attach to the plasma
membrane) can efficiently replicate prions without developing prion
disease [51]. Altogether, these studies demonstrate that prion
neurotoxicity is not triggered by PrPSc itself, but by an activity of
PrPC at the cell surface. Accordingly, it has been proposed that prion
replication might cause PrPC to either lose a neuroprotective role, gain
a neurotoxic one, or subvert a neuroprotective role into a neurotoxic
one [52]. Therefore, a thorough characterization of PrPC function will
likely be instrumental in ascertaining the molecular basis of prioninduced neurodegeneration.
1.4. Open challenges in prion biology and pathogenesis
The past five decades have seen remarkable achievements that
shaped our current understanding of TSEs. These include the
transmissibility of kuru and CJD to laboratory animals, the isolation
of the scrapie agent, the development of the protein-only hypothesis,
the identification of PrP and its isoforms, and the study of prion
propagation in genetically modified mice. Yet, very little has been
learned about basic aspects of prion pathogenesis, such as the
mechanisms of PrP misfolding and prion replication, the physiological
function of PrP, or the cellular pathways through which prions induce
neurodegeneration. To this day, prion disorders remain incurable, and
neither early diagnostic methods nor anti-prion drugs have successfully been developed. Of special concern is the possibility that animal
prion diseases may be transmitted to humans via the food chain. In
fact, experimental evidence indicates that transmission of BSE to
humans was the likeliest cause for the appearance of a new variant
form of CJD (vCJD) [53,54]. These findings illustrate the need to ensure
food safety, not only through efficient quality control but also by
experimentally assessing the risk of prion transmission across a wide
range of animal hosts.
So far, only beef and lamb have been identified as potential sources of
dietary prion infections. Other major animal food sources like fish and
poultry appear to have remained free of prion contamination. However,
it is not clear whether these organisms are susceptible to prion
pathogenesis because TSE research has been carried out predominantly
in mice and other mammals. Therefore, the study of prion biology in
non-mammals, particularly in fish, is of great relevance to food safety.
Fish constitute a vital food resource for humans and animals worldwide
(Table 1), with direct impact on important economic activities like
fishing and aquaculture.
In addition, several fish species have become valuable tools in
biomedical research. One of them, the zebrafish (Danio rerio), is an
excellent model to study vertebrate development and the biology of
human disease. It has many advantages as a genetic system, such as its
404
E. Málaga-Trillo et al. / Biochimica et Biophysica Acta 1812 (2011) 402–414
Table 1
Commercially important fish species worldwide.
Source: Food and Agriculture Organization of the United Nations, Fisheries and
Aquaculture Department (http://www.fao.org/fishery/en).
Common
name
Scientific
name
Main producers
Salmon
Salmo salar
Japan, Europe,
North America
Austria, Germany,
Hungary, Poland
Africa, China, Papua New Guinea, USA, China
Philippines, Thailand, Indonesia
Carp
Tilapia
Main consumers
Chile, Norway, Scotland, USA,
Canada
Cyprinus carpio Central Europe, China
Oreochromis
niloticus
niloticus
Tuna
Thunnus
albacores
Catfish
Ictalutus
punctatus
Trout
Oncorhynchus
mykiss
Seabream Sparus aurata
Sea bass
Dicentrarchus
labrax
Japan, Australia
Japan, Australia
USA, China
USA, China
Europe, USA, Chile
Europe, USA,
Japan
Europe
Europe
Mediterranean countries
Mediterranean countries
divergent regions of the protein, respectively. Remarkably, and despite
having undergone substantial sequence variation, the globular domain
retained its structural fold throughout evolution [67,69]. These observations strongly suggest that the globular domain carries out an important
function, which was already present in the last common ancestor of all
vertebrates. Whether prion behavior was already an intrinsic attribute of
such primitive PrPs, or whether it appeared late in evolution remains to
be clarified.
So far, TSEs have only been reported in mammals. This is not
surprising, since prion biology is a field primarily focused on the study of
human diseases. Nevertheless, if PrP is intrinsically able to form
infectious prions, then its presence in non-mammals implies that
these animals would – at least in theory – be susceptible to some form of
prion disease. Evaluating this scenario is particularly difficult without
basic knowledge about PrP conversion and prion replication in nonmammalian species. Moreover, it is not even clear what the eventual
manifestations of TSEs in fish or birds might be. Clearly, extensive
experimentation will be required to establish whether these organisms
can acquire and transmit prion disease.
2.2. Prion transmission and the species barrier
large offspring size and short generation time [55]. Furthermore,
zebrafish embryos are transparent and develop externally, making
them amenable to detailed cellular analyses and genetic manipulations. Moreover, the zebrafish is rapidly emerging as a valuable tool in
clinical and pharmaceutical research aimed at drug discovery and
validation [56,57].
In this article, we review current knowledge about the occurrence of
PrPs, prions, and TSEs among vertebrates, focusing on recent advances
using fish models to characterize the physiological function of PrP and
the possible risk of prion transmission between fish and mammals.
2. Prion disease: a mammalian attribute?
2.1. PrP and prion-like “behavior”
Besides PrP, a handful of mammalian proteins appear to be able to
replicate in a prion-like manner. Among them are the molecular culprits
of known neurodegenerative disorders, such as amyloid-β (Alzheimer's
disease), α-synuclein (Parkinson's disease), tau (Tauopathies) and
amyloid A (AA amyloidosis) [58]. Such observations are undoubtedly
intriguing, yet it still needs to be established whether the rogue versions
of these proteins are indeed infectious and, like bona fide prions, capable
of spreading disease between individuals or even across species. In
fungi, at least six different proteins exhibit prion-like behavior, as
judged by their ability to propagate phenotypic traits between cells
without the need for nucleic acids [59]. However, these proteins have
neither structural nor functional similarities to PrP, or to one another.
Thus, while the study of fungal prions has provided valuable information
about the molecular basis of prion conversion, it is not clear to what
extent this knowledge may apply to mammalian prion disease [60].
In addition to mammals, PrP homologues have also been identified in
birds [61], reptiles [62], amphibians [63] and fish [64], but not in
invertebrates. Comparison of amino acid sequences indicates that PrPs
have evolved differently in each vertebrate class. For instance, although
mammalian and avian sequences are strongly conserved, with similarity
scores of ~90% in each group, conservation between the two groups is
rather low (~30% similarity) [65,66]. Likewise, PrP sequences among
bony fish are relatively well conserved (50–60% similarity), but exhibit
only 20–25% similarity to mammalian sequences [67]. PrPs also exhibit
considerable variation in size, ranging from ~260 amino acids in
mammals and birds to ~600 amino acids in fish. Despite these large
differences in length and sequence, all vertebrate PrPs share the same
protein domains (Fig. 1) [67,68]. The degree of conservation varies
significantly along the polypeptide, with the short hydrophobic stretch
and the repetitive domain being the most conserved and the most
Prion transmission across different species is limited by the so-called
species barrier. Following primary inoculation between two species,
only some animals develop disease, with long incubation periods that
may reach the normal life span of the species. This phenomenon is
clearly illustrated by the resistance to TSE observed in rabbits, even after
intracranial inoculation with TSE agents of sheep, mouse and human
origin [70]. It has been suggested that the unique structural characteristics of rabbit PrP make it less prone to pathogenic conversion [70,71].
Typically, species barriers become attenuated after serial passaging of a
prion strain, resulting in shortened incubation times and the consequent
adaptation of the host to the specific strain [72]. At the molecular level,
the efficiency of interspecies prion transmission appears to depend on
the degree of sequence and structural identity between the PrPs of the
two species involved [73]. For instance, wild type mice do not become
infected with hamster prions, whereas transgenic mice overexpressing
hamster PrP do [74]. However, BSE can effectively be transmitted to a
wide range of species and maintain its pathogenic characteristics, even
when passaged through an intermediate species carrying a different PrP
coding gene [75].
It has been proposed that every species harbors a number of possible
PrPSc conformational variants, with a thermodynamically “preferred”
conformation (strain) being the decisive factor for interspecies
transmission. According to this “conformational selection model” [76],
a significant overlap between the preferred PrPSc configurations of two
species is likely to result in a relatively easy transmission of prions
between them [77]. On the contrary, two species not sharing common
PrPSc structures would have to overcome a considerable barrier to
transmission, and pathogenesis would not ensue without modification
of the strain type. Therefore, evaluation of the prion transmission from
one species to another should also be based on the structural identity
between the host and donor PrP molecules and not exclusively on their
amino acid sequence [78].
The route of infection is another parameter affecting the magnitude
of species barriers. Most inoculation studies rely on the use of
intracranial injection because “natural” per os infection is usually less
effective [79,80]. Notably, even after homologous oral transmission of
BSE to cattle, animals may remain presymptomatic for 28 months,
despite the evident PrPSc deposition in their brains [81]. Although the
relative importance of the aforementioned factors has been extensively
studied, the outcome of an intra-species prion transmission cannot be
predicted in advance. It is noteworthy, that two different TSE strains that
can effectively infect one species may present completely modified
transmissibility potentials when transmitted to a second species. Thus, it
has been proposed that a more suitable term to describe these
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405
Fig. 1. Fish and mammalian prion proteins. Upper panel: Fish possess two prion proteins, PrP-1 and PrP-2. Despite differences in sequence and length, fish and mammalian PrPs share
the same protein domains (drawn at scale). Lower panel: Recent studies indicate that a common involvement of vertebrate PrPs in cell–cell communication. In cultured cells, PrPs
elicit cell–cell contact formation. In the zebrafish embryo, PrP-1 and PrP-2 are deployed during gastrulation and neural development, respectively. L = leader peptide, R = repetitive
domains, H = hydrophobic stretch, G = globular domain; orange triangles indicate the ends of the mature polypeptides. PrP localization at cell contacts is depicted in red.
phenomena would be “transmission barriers” instead of “species
barriers” [76].
Recent data have led to the reconsideration of many parameters that
were thought to be indicative of a species barrier, such as the lack of
clinical symptoms. Interestingly, cases of subclinical infection in
mammals displaying neuropathological and biochemical abnormalities,
often including PrPSc accumulation, have been reported [82]. Thus,
prion-inoculated animals harboring high infectivity levels, and even
abnormal PrP aggregates in their brains, may under certain circumstances remain asymptomatic carriers throughout their lifetime.
Notably, subclinical disease has been reported even after homologous
transmission, for instance, in studies using the oral route of infection and
low dosage inoculum [79,83,84].
3. Fish prion proteins
3.1. Duplicated PrPs in bony fish
PrP genes from land vertebrates have typically been cloned using
conventional procedures such as cDNA hybridization and PCR
screening. In contrast, fish PrP genes could not be isolated with
these methods due to their large DNA sequence divergence. The first
fish PrP homologue was identified in Fugu rubripes by searching
genome databases for ORFs encoding key structural features of PrPs
[64]. Subsequent cloning and sequencing of PrP genes in salmon, trout,
carp, stickleback, sea bream and zebrafish [67,68,85,86] revealed that
bony fish possess two PrP orthologs, PrP-1 and PrP-2, which probably
arose during a fish-specific genome duplication [87]. Northern and
Western Blot analyses indicated that the fish proteins are expressed at
particularly high levels in adult fish brains, as well as in muscle, skin,
heart and gills [67]. Although quite variable in size and sequence, PrP1 and PrP-2 present characteristic features of mammalian PrPs: an Nterminal signal peptide, conserved N-glycosylation sites, two cysteine
residues that form a disulphide bond, a central hydrophobic stretch,
and the C-terminal signal for the attachment of a GPI-anchor (Fig. 1).
An intriguing feature of fish PrPs is the presence of a highly conserved
motif of 13 amino acids in length between the repetitive region and
the hydrophobic stretch [67]; the structural or functional importance
of this motif is not evident from its sequence.
The repetitive domains of fish PrPs are easily recognizable but at the
same time clearly different from those of tetrapod PrPs. For example,
while a constant number of nearly identical octarepeats is typical for
mammals (or hexarepeats in birds), in fish the number and length of
degenerate repeats vary from species to species. Nevertheless, all PrP
repeats can be considered variations of two basic types of repeats (A and
B), which evolved differentially in every vertebrate class [67]. The
sequence of the globular domain also has diverged considerably
between fish and mammals, owing to high rates of amino acid
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substitutions, insertions and deletions. Notably, these changes predominantly affect residues outside key structural motifs, and are not
expected to affect the globular fold [67].
Apart from PrP-1 and PrP-2, other genes partially resembling PrP
have been reported in fish. Originally described as PrP-like and Shadoo
[88,89], they are also known as PrP-1-rel and PrP-2-rel because of their
genomic location, directly downstream of PrP-1 and PrP-2, respectively [67]. The encoded products are GPI-anchored polypeptides of
only 150–180 amino acids in length, expressed in brain tissues, and
containing the characteristic PrP hydrophobic stretch. Further
similarities to PrPs include a β-strand motif in PrP-rel-1 identical to
the first β-strand of PrP, and the presence of degenerated B- and Alike repeats in PrP-rel-2. This situation is reminiscent to that of Prnp
and its genomic neighbor Doppel. Thus, PrP-rels, like Doppel, may
have arisen from the tandem duplication of an ancestral PrP gene, and
then diverged as a result of high substitution rates and the differential
loss of motifs. Interestingly, although PrP-rels are predicted to be
unstructured proteins, their mammalian homologue Shadoo has
recently received attention because it appears to influence biological
and pathogenic activities of PrP in vivo [90,91].
3.2. Zebrafish PrP-1 and PrP-2
The characterization of PrP homologues in the zebrafish opened the
exciting possibility to study prion biology in a new model organism. Like
other bony fish, the zebrafish has two prion protein orthologs, PrP-1 and
PrP-2, mapped to chromosomes 10 and 25, respectively [67]. Genomic
studies revealed that the two genes originated from the duplication of a
large chromosomal block that corresponds to those containing the Prnp
gene in mammals. That both gene copies are fully functional has been
biochemically confirmed in fish and mammalian cells [92,93]. In these
studies, it was shown that the encoded polypeptides become properly
glycosylated and attached to the plasma membrane via a GPI-anchor. As
is often the case with duplicated genes [94], PrP-1 and PrP-2 are thought
to have retained complementary subsets of ancestral PrP functions.
Accordingly, zebrafish PrPs have evolved distinct spatiotemporal
patterns of embryonic expression: while PrP-1 is ubiquitously expressed
at high levels in very early stages of embryogenesis (blastula and
gastrula stages), PrP-2 is strongly upregulated at later stages in
developing neurons [92]. At later larval stages and into adulthood,
both genes continue to be expressed in the CNS, although PrP-2 at much
higher levels than PrP-1. The differences between the expression
patterns of PrP-1 and PrP-2 are due to the divergent evolution of DNA
transcriptional regulatory sequences upstream of each gene (Barth and
Málaga-Trillo, unpublished data). Interestingly, the expression of
zebrafish PrPs (particularly PrP-2) in the developing nervous system
bears striking similarities to that of PrP in the mouse embryo [32]. It
remains to be determined if, like PrP-1, mammalian PrP plays a role
during blastula and early gastrula stages.
3.3. PrP loss of function phenotypes in zebrafish embryos
One of the many experimental advantages of the zebrafish is the
possibility to knockdown the expression of any given gene using
morpholino antisense oligonucleotides. When microinjected into
early fish embryos, morpholinos rapidly bind to their target mRNAs
and block their translation [95]. Recently, this method was successfully applied to study PrP loss of function in the zebrafish. In contrast
to the absence of clear phenotypes in PrP knockout mice, knockdown
of zebrafish PrPs revealed that these genes play essential roles during
distinct phases of embryonic development. Specifically, PrP-1 knockdown causes lethal embryonic arrest during gastrulation, whereas
PrP-2 knockdown severely impairs brain development. Notably, the
PrP-1 phenotype can be rescued by PrP-1 or PrP-2 overexpression,
indicating that although the two proteins are expressed in different
developmental contexts, they share essentially the same biological
activity. Furthermore, the ability of mouse PrP to partially revert the
PrP-1 phenotype strongly suggests that this activity of PrP is
conserved between fish and mammals [92]. It is therefore surprising
that the loss of such an important function would not be evident in PrP
knockout mice, especially since PrP is expressed at high levels in the
mouse embryo. A plausible explanation for this discrepancy is that the
mouse knockout phenotype may become masked by genetic
compensation or developmental plasticity [96].
The relative simplicity of the zebrafish gastrula makes the PrP-1
phenotype an excellent choice to analyze conserved mechanisms of
PrP function at the molecular and cellular levels. Detailed characterization of PrP-1 knockdown embryos revealed that the developmental
arrest was caused by the loss of tissue cohesion and the consequent
failure to carry out epiboly, an early morphogenetic cell movement.
Because epiboly relies on the control of cell–cell adhesion, this finding
led to the discovery that PrP-1 regulates the stability of E-cadherin/ßcatenin adhesive complexes at the plasma membrane [92]. Ongoing
biochemical work indicates that the mechanism behind this effect is
the ability of PrP-1 to control the levels and membrane localization of
ß-catenin as well as the levels and phosphorylation state of Srcrelated kinases (Sempou and Málaga-Trillo, in preparation). Related
unpublished in vivo pharmacological data suggest that PrP-1 acts to
prevent the degradation of some of these proteins.
These results are in agreement with long proposed roles of PrP as a
signal transduction molecule [46]. Concretely, the experiments with
zebrafish gastrulae indicate that a complex signaling cascade is
initiated by the interaction between PrP-1 molecules across opposite
cell membranes. Accordingly, in vitro experiments show that fish,
amphibian, avian and mammalian PrPs share the intrinsic ability to
engage in such homophilic trans-interactions, promoting the formation of cell–cell contacts, and triggering intracellular signaling via Srcrelated kinases [92]. Of special interest within the scope of this review
is the finding of heterologous interactions between zebrafish and
mammalian PrPs [92], which raise the question of whether PrP
conversion and TSE transmission can take place across such distant
species.
Altogether, the combined work in zebrafish embryos and
mammalian cells indicate that PrPs play crucial roles in cell–cell
communication (Fig. 1). These data are in line with the notion that PrP
provides protective signals required for neuronal growth and/or
survival [97,98]. Understanding the precise nature of such signals will
be crucial to uncover the early cellular mechanisms of prion
pathogenesis in the mammalian brain.
4. Risk of prion disease in fish
4.1. TSEs and the food chain
The appearance of BSE in Europe has been causally linked to the
common practice of feeding cattle with meat and bone meal (MBM) of
bovine origin [99]. Approximately 200,000 BSE cases were reported
between 1985 and 2000, and it has been estimated that roughly three
million infected animals could have entered the human food chain
before developing a clinical disease [81]. Due to its low cost and high
nutritional value, MBM has become an important ingredient in the
diet of non-ruminant livestock and farmed fish. Hence, there is the
theoretical risk that farmed animals unintentionally fed with prioncontaminated MBM might develop TSEs or serve as asymptomatic
carriers of a disease [100]. Since BSE was initially reported, the
European Commission (EC) has implemented a comprehensive set of
TSE risk-reducing measures to protect humans from BSE, as well as to
control and eventually eradicate prion diseases in animals. Among
these, a total EU-wide feed ban on the use of rendered animal proteins
in the nutrition of all animals was imposed, with minor exceptions
(i.e. use of fish feeds in the diets of non-ruminants like pigs and
chicken).
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Experimental demonstration that TSEs can propagate in a new
host without typical pathognomonic brain lesions, clinical disease or
fatality, has led to the conclusion that prion-infected individuals could
go undetected within an exposed population [100]. This implies that
farmed animals may carry the disease but not develop it until they are
slaughtered. Human consumption of meat from these animals would,
in turn, give rise to repeated infection cycles long before clinical signs
can be recognized. Moreover, the relatively recent discovery of
unconventional TSE phenotypes, such as atypical scrapie in sheep
and goats [101,102], and bovine amyloidotic spongiform encephalopathy (BASE) in cattle [103], usually displaying decreased or altered
PrPSc resistance to proteolytic treatment and less pronounced clinical
signs, has further made it difficult to define prion diseases in absolute
terms and thus, to implement effective rules to eliminate them.
4.2. Risk of prion transmission to fish
According to the aforementioned arguments, fish, poultry and pigs
represent potential candidate hosts for prion infection. In order to assess
the risk of prion transmission to fish, five main parameters should be
considered: 1. the potential use of prion-contaminated MBM as a
nutritional supplement in aquaculture, 2. the consumption of infected
farmed fish by humans, 3. the use of feeds or other products (i.e. gelatin,
milk replacers) derived from TSE-affected fish for mammalian or piscine
nutrition, 4. the use of fishmeals cross-contaminated with MBM in
mammalian diets, and 5. the escape of infected fish, or the release of
infected waste from aquaculture facilities into the marine environment.
These scenarios are depicted in Fig. 2.
With fish mariculture turning into a very important and rich
protein source for humans, consumers may become concerned about
the possibility of farmed fish developing prion disease, or serving as
passive carriers of prion infectivity. All farmed fish receive commercial
feeds containing 40–55% protein, and since some of it is likely to be of
animal origin, the possibility of feed contamination with mammalian
prions cannot be excluded. Interestingly, in the late 1980s, total fish
feed production in the UK was in the order of 75,000 tons per year,
with the use of about 3750 tons of MBM in fish feed [104].
However, how realistic is the thesis that fish prion proteins may
misfold and form abnormal aggregates? The striking conservation of
structural motifs between mammalian and piscine PrP molecules
407
makes it rather difficult to exclude such a possibility. Regarding the
convertibility potential of fish PrPs, it has been suggested that the
extended type A-repeat domain in sea bream PrP-1 and other fish PrPs
could confer on these proteins the ability to self-polymerize and
aggregate [85]. In addition, it has been noted that motifs similar to
those observed in fish PrPs can also be identified in unrelated proteins
such as the piscine annexin 11 and the mammalian lectin L-29 [77].
Notably, both these proteins display self-aggregation properties, and
annexin 11 is, like PrP, a neuro-specific membrane protein.
4.3. Transmission studies in commercially important fish species
At present, only a few studies have explored the experimental
transmission of TSEs to teleost fish. The first attempts in this direction
involved the oral and parenteral inoculation of two commercially
important fish species, rainbow trout and turbot, with the 139A
mouse-adapted scrapie strain [105]. Although the fish displayed no
clinico-histopathological signs during the three month-experimental
period, a mouse bioassay revealed that they carried residual
infectivity. Specifically, mice intracranially inoculated with trout
intestinal extracts one day after oral challenge were positive for
brain PrPSc deposition approximately 200 days post inoculation (p.i.),
despite the absence of clinical symptoms. Similar results were
obtained when mice were intracerebrally (i.c.) inoculated with spleen
extracts from trouts and turbots, 15 days after parenteral inoculation
of the fish with scrapie material. Finally, brain tissue from parenterally
inoculated turbots 15 and 90 days after challenge was also able to
elicit PrPSc accumulation in the brains of recipient mice, without
causing clinical disease.
Recently, we reported data on the oral transmission of BSE and
scrapie to gilthead sea bream (Sparus aurata) [106]. Interestingly, at
two years p.i., a number of fish that had been force-fed BSE- or
scrapie-infected brain homogenates developed abnormal plaque-like
deposits in their brains. Specifically, the brains of two out of five fish
inoculated with scrapie developed signs of abnormal protein
aggregation at 24 months p.i. These aggregates were positively
stained with polyclonal antibodies raised against fish PrPs, but
showed no proteinase K (PK)-resistance or Congo red birefringence
(Fig. 3A). The brains of the BSE-challenged fish, however, displayed a
much more striking picture, having already developed the first signs
of abnormal deposition at eight months p.i. A general progression in
size, PK-resistance and morphological features was observed thereafter, resulting in an impressive number of aggregates in all the brain
regions examined at 24 months p.i. Three out of five fish sacrificed at
this time point showed 500–800 deposits per brain section each, 70–
85% of which were PK-resistant and had a mean diameter of 30 μm.
These aggregates were PAS-positive, congophilic and birefringent in
polarized light, indicating an amyloid or amyloid-like fibrillar
structure (Fig. 3B). In contrast to the TSE-challenged individuals, no
signs of abnormal aggregation or any other lesions were observed in
the brains of the control fish “challenged” with bovine or ovine brain
homogenates prepared from healthy animals. Altogether, the development of abnormal brain deposits in BSE-challenged sea bream
constitutes an unprecedented histopathology in fish.
4.4. Neuropathology in TSE-challenged sea bream
Fig. 2. Prion diseases and the food chain. Potential scenarios for the transmission of
prions between mammals and fish are illustrated. Arrows indicate the direction of
possible transmission events.
The lesions observed in the brains of the BSE-infected sea bream
share some remarkable similarities with those typically seen in prionaffected mammalian brains. For instance, the deposits in fish brains
were only detectable where neuronal parenchyma was present, a
feature greatly resembling the localization of prion amyloid plaques in
mammals [107]. The most extensively affected regions included the
cerebellum (which displayed a mammal-like deposition motif [108]),
the lateral nucleus of the ventral telencephalic area (a fish counterpart
to the basal nucleus of Meynert in mammals [109]), the lateral
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Fig. 3. Experimental transmission of prions between mammals and fish. Inoculation of fish with ovine (A), bovine (B) and mouse (C) prions and their various outcomes. The
photographs on the upper right corner illustrate the development of abnormal amyloid deposition in the optic tectum of BSE-challenged sea bream, using the periodic acid Schiff
(PAS) staining reaction. The right image (scale bar = 10 μm) is a zoom-in of the lesion indicated in the left image (scale bar = 100 μm). i.c., intracerebrally.
telencephalic pallium (homologue to the mammalian hippocampus
[110]), and finally the thalamus and diencephalon. Notably, no
spongiosis was observed in any of the brain regions examined. However,
cases of TSE subtypes that develop little or no vacuolation have also been
reported for human prion diseases [111]. Interestingly, the neurodegeneration observed in BSE-challenged sea bream affected only
neurites, as opposed to the “classically defined” degeneration of
neuronal somata. Based on the morphological progression of the
abnormal deposits and the level of neurite involvement, aggregates
were classified into two main categories. “Pre-mature” deposits,
observed mostly at earlier time points and consisting mainly of
dystrophic neurites that have lost their coherence, appear to mark
initial stages of neurodegeneration. “Mature” deposits, observed
24 months p.i., seem to result from the complete deconstruction of
fibers, leading to the development of a more homogenous and
flocculated extracellular material, with increased PrP-immunoreactivity, congophilia and birefringence in polarized light. The two types of
aggregates in the brains of BSE-challenged sea bream could be
interpreted as different stages of pathogenesis, with the former
temporally preceding the latter. For scrapie-challenged sea bream, the
distribution and morphology of the observed neuropathology could not
be evaluated and classified as mentioned earlier, due to the limited
number of fish developing abnormal deposits in this group.
4.5. Absence of “clinical symptoms” in fish: presymptomatic carrier
state?
Visual examination of challenged fish throughout the post
inoculation period revealed no signs of altered swimming behavior
or eating habits. However, previous studies have shown that species
once thought to be resistant to certain TSE strains can be life-long
carriers of the infection without ever developing a clinical disease. For
example, the absence of clinical symptoms has been reported in mice
following the primary transmission of sheep and hamster scrapie.
However, sequential passage of infectious material did result in the
development of clinical disease [82]. Interestingly, differential
susceptibility to specific pathogens is also known among piscine
species. For instance, closely related fish species may exhibit
differential resistance to viral infections, as illustrated by the
development of clinical disease in sea bass but not in sea bream
infected with nodavirus [112,113]. On the other hand, the absence of
clinical symptoms in BSE-challenged sea bream could be related to the
remarkable ability of teleost fish to continuously produce new
neurons throughout their lifetime. It is well known that adult fish
can regenerate destroyed retinal tissue, optic nerve axons, and
degenerated brain stem neurites, resulting in functional recovery of
these structures [114,115]. Given the fact that farmed fish remain in
aquaculture facilities up to 20 months before reaching the required
commercial weight, the lack of clinical disease in infected animals
may become a crucial issue of unanticipated relevance for public
health.
4.6. Differential susceptibility of fish species to prion infection
In a parallel study, we inoculated European sea bass (Dicentrarchus
labrax) with the same infectious material, namely BSE and scrapie.
Interestingly, in contrast to the sea bream, none of the challenged
individuals showed any clinical symptoms or histopathological
evidence of abnormal deposition at any point throughout the time
course of the study. Sea bass and sea bream are both Perciformes, the
largest class of teleosts. Although they belong to different families,
Sparidae (sea bream) and Moronidae (sea bass), the two species are
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quite closely related at the phylogenetic and anatomical levels. Since
the amino acid sequence of sea bass PrP is not available, it is not clear
how it might differ from that of sea bream PrP. However, the two
proteins display different reactivities against anti-fish PrP antibodies.
Conceivably, such differences could confer on the sea bass PrP
resistance to the PrPSc in the inoculum, and thus, reinforce the
“transmission barrier” in these experiments.
Another factor possibly involved in the different reaction of the
two species to prion inoculation might be the ease with which
exogenous PrPSc can cross the intestinal barrier and reach the CNS
[116]. In teleosts, like in other vertebrates, oral pathogens are initially
confronted by activated immune cells on the enteric wall. Macrophages, whose efficiency contributes to the resistance against
pathogens, play an important role in this process. While in sea bass,
enteric phagocytes seem to become extensively activated upon
microbial attack [117], it has been reported that the intestinal mucosa
of the sea bream may be relatively permissive to infection by certain
bacteria [118].
Although plausible, these hypotheses do not entirely explain the
differential sensitivity of the two species to prion infection, since cases
of pathogenesis in which the clinically affected species is the sea bass
and not the sea bream are also known [112]. It is noteworthy,
however, that the different susceptibility of the two species can be
regarded as an internal control in our transmission experiments.
Specifically, the fact that despite the close relationship between the
two species, only sea bream were affected, strengthens the hypothesis
that the observed neuropathology represents a novel piscine
amyloidosis triggered by mammalian prions. Thus, transmissibility
of prions to fish exhibits species specificity, a common feature of TSEs.
the GI tract may serve as a potential portal for exogenous PrPSc, due to
the high PrP mRNA levels detected in the posterior intestine of zebrafish
larvae [68]. In mammals, studies have shown that the pathogenic agent
may invade the CNS directly, by exploiting the extended autonomous
intestinal innervation [120]. In particular, it has been proposed that BSE
neuroinvasion may take place via the parasympathetic nerve fibers of
the vagus nerve into the brain stem of orally-challenged cattle [81]. Such
a route of dispersion requires the adequate expression of endogenous
PrPC within the peripheral nervous system [121]. In the sea bream,
immunohistochemical examination of intestinal tissue revealed three
main regions of intense PrP-immunopositivity, namely the serosa, the
myenteric plexus (corresponding to Auerbach's plexus in mammals)
and the submucous plexus (a homologue to mammalian Meissner's
plexus). Both the myenteric and the submucous plexus contain many
parasympathetic nerves and ganglia, one of which is a nucleus of the
vagus nerve running to the medulla oblongata in the brain stem of
mammals and teleosts. The hypothesis that the infectious agent might
use the enteric nervous system to invade the fish CNS is supported by
the progressive changes observed in the regional distribution of
abnormal deposits in sea bream brain [81]. Initially, the region affected
is the posterior part of the brain, with the brain stem displaying the most
severe lesion profile. Subsequently, anterior anatomical regions show
signs of abnormal aggregation as well, although posterior brain areas
continue to be affected. Interestingly, it has been proposed that direct
neuroinvasion of the infectious agent following per os challenge might
be related to the absence of clinical symptoms and the reduced attack
rate of the transmitted disease [122], an observation that is in agreement
with the asymptomatic amyloidosis developed by BSE-challenged sea
bream.
4.7. Possible routes of neuroinvasion: tissues harboring infectivity
5. Future directions
Immunohistochemical examination of peripheral tissues from TSEinoculated sea bream including spleen and intestine revealed no signs of
abnormal protein accumulation at any of the time points tested.
Therefore, it is likely that the transit of prions from the fish
gastrointestinal (GI) tract to the CNS might have occurred long before
the first time point of sacrifice, perhaps only a few hours or days after
oral challenge, leaving no trace of infectivity in the peripheral tissues
tested thereafter. Transmission studies in rainbow trout and turbot have
shown that specific binding of exogenous PrPSc (139A scrapie strain) can
be detected in the intestinal mucosa for up to seven days p.i., despite the
absence of active uptake by the epithelial cells [105,119].
Preliminary data from a more recent study demonstrate the
importance of residual infectivity, suggesting that after oral challenge
with the ME7 mouse-adapted scrapie, prions can cross the intestinal
wall of the sea bream and be delivered from the peritoneal cavity into
the spleen (Figueras et al., unpublished data). More specifically,
inoculated fish were sacrificed at regular time points, and various
tissues, including spleen and intestine, were used to intracranially
inoculate mice (Fig. 3C). The TSE-challenged fish never showed
histopathological or clinical symptoms throughout the 18 monthexperimental period. However, serial passage to mice of tissues from
fish sacrificed one week p.i. induced vacuolation in the brains of some
of the mice, 300 days after the intracranial inoculation. Concretely,
spongiosis without clinical disease was detected in five out of 19
recipient mice inoculated with scrapie-infected fish spleens, and in 12
out of 17 individuals challenged with scrapie-infected fish intestines.
These findings suggest, on one hand, that the TSE agent might have
been able to reach the fish brain in a remarkably short period of time,
either after peripheral propagation or even by direct neuroinvasion.
On the other hand, this study emphasizes the need for a thorough
examination of peripheral tissues in asymptomatic individuals
following prion inoculation.
There is no doubt that the intestine represents the main entry point
for oral pathogens. In zebrafish, for example, it has been proposed that
5.1. Monitoring for infectious neurodegenerative disease in farmed fish
The rapid worldwide expansion of fish farming calls for the
implementation of efficient measures to ensure animal health and
food safety. The fact that pathogens can spread considerably more easily
through flowing water than between land animals is one of the main
difficulties when dealing with aquatic diseases. Over the last years, the
aquaculture industry has been faced with an increasing number of
infectious diseases affecting both farmed and feral populations.
Surveillance and monitoring programs have therefore been developed
in many countries, in an attempt to prevent and control the emergence
of new fish pathologies [123]. The list of notifiable diseases varies
depending on the specific needs of each region. In Europe, all countries
are asked to comply with the Council Directive (2006/88/EC) on health
requirements for aquaculture animals. Nevertheless, it would be
sensible to enrich the list of large-scale spot checks in fish farms by
including the histological examination of fish brains, in order to detect
possible signs of feed-induced or even spontaneous PrPSc amyloidosis.
As described earlier, BSE-infected sea bream developed the first signs of
abnormal aggregation already at eight months p.i., a time period that lies
within the grow-out phase of fish breeding. Moreover, the fact that
these animals remained asymptomatic throughout the experimental
period raises the possibility that affected fish may generally go
undetected before ending up on our plates.
5.2. Further transmission studies in commercially important fish species
Gilthead sea bream and European sea bass are the two most
important farmed fish in the Mediterranean Sea, with the main
producers being Greece, Turkey, Spain, France and Italy. In 2004, the
annual production levels of both species reached 160,000 tons
worldwide. Therefore, assessing the risk of TSE transmission to these
fish is an issue of major importance for public health. On a global scale,
the practice of aquaculture is experiencing steady growth rates, driven
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by the increasing demand for aquatic food products. According to
projections by the Food and Agriculture Organization of the United
Nations (FAO), the global production of farmed fish will have to reach
80 million tons by 2050, in order to maintain the current level of fish
consumption per capita. Amazingly, not all countries have introduced
laws to regulate the use of rendered mammalian proteins for fish
nutrition and, in some cases, those that have, have incurred in minor and
major violations [124]. Hence, the likelihood that contaminated MBMs
might have been inadvertently fed to farmed fish is considerably high. In
this regard, the amyloidosis observed in the brains of BSE-fed sea bream
demonstrates that certain fish species can develop neuropathology
following ingestion of prion-contaminated meals. Therefore, further
transmission studies with other commercially important fish species,
including salmon (Salmo salar), trout (Oncorhynchus mykiss), and carp
(Cyprinus carpio) should be conducted in order to evaluate the
likelihood of acquiring TSEs upon consumption of infected feeds
(Fig. 4). In addition, primary TSE transmission studies in piscine species
that are considered future candidates for mariculture would be
advisable, especially those closely related to sea bream, such as the
common dentex (Dentex dentex, Sparidae) and the red porgy (Pagrus
pagrus, Sparidae).
5.3. Zebrafish as a model for prion pathobiology
Clearly, transmission experiments in commercially important
species are necessary to directly assess the risk that TSE-infected
fish would pose to humans. On the other hand, addressing the cellular
and molecular aspects of prion transmission to fish requires the use of
established model organisms. The zebrafish is best fitted for this
purpose, not only because of its well-characterized neuroanatomy
and neural development, but also because of the availability of useful
research tools, such as mutant strains, GFP-transgenic lines, specific
antibodies, cell lines and a fully sequenced genome. Additionally,
zebrafish larvae are optimally suited for the study of neurological
diseases because they display quantifiable neuropathological and
behavioral phenotypes similar to those observed in humans [125]. It
would be particularly interesting to know whether scrapie or CJD
prions can be efficiently inoculated to transgenic zebrafish expressing
mouse or human PrPs. Similarly, expression of pathogenic PrP
mutants in wildtype and transgenic fish could be of great value in
elucidating the genetic basis of prion disease. Engineering of these
animals, currently underway, will provide an ideal tool to systematically analyze prion replication in fish brains, as well as the
development of PrP aggregates, brain lesions, and possibly clinical
disease. Another fascinating perspective is the possibility to study the
effect of exogenously added prions on zebrafish embryos. Such
experiments could significantly facilitate the analysis of PrPC and PrPSc
behavior in vivo, thus contributing valuable insights into the
relationship between prion conversion and PrP dysfunction. Altogether, transmission experiments in zebrafish could help to effectively evaluate the susceptibility of fish to TSEs, as well as the mechanistic
link between PrP function and prion pathogenesis.
Fig. 4. Future scenarios for transmission studies. Examples of primary and secondary inoculation studies required for the evaluation of prion transmissibility between piscine and
mammalian species. Tg = transgenic, wt = wildtype, ZF = zebrafish, boPrP = bovine PrP, ovPrP = ovine PrP, huPrP = human PrP, moPrP = mouse PrP.
E. Málaga-Trillo et al. / Biochimica et Biophysica Acta 1812 (2011) 402–414
411
5.4. Assessing transmissibility and convertibility
5.5. Dissecting the physiological roles of PrP in the zebrafish
Issues of prion transmissibility between fish and mammals need to
be given full consideration because of the possibility that fish fed with
prion-contaminated MBM may further transmit TSEs through the
food chain. To evaluate this scenario, secondary transmission
experiments between BSE-affected fish brains and mice carrying the
bovine Prnp gene are currently underway. Further studies in
transgenic fish will also help assess the risk of potential transmission
of piscine amyloidoses to mammals or to other fish species (Fig. 4).
For instance, the use of transgenic zebrafish carrying the bovine Prnp,
or mice carrying the gene coding for fish PrP on a PrP knockout
background would answer a plethora of questions concerning not
only host-related pathogenic determinants but also the specific
conditions required for certain intra-species transmissions.
The discovery of residual prion infectivity in fish intestines and
spleens is also a matter of great concern, as it suggests that even fish
species resistant to prion infection might harbor infectivity shortly
after ingestion of contaminated meals, thus being able to transmit the
disease to consumers. Similar studies should be undertaken employing a variety of fish species and TSE strains, including atypical scrapie
and BSE, to assess residual infectivity levels not only in spleen and
intestine, but also in blood and other “high risk” tissues (Fig. 4).
Furthermore, bioassays should also include “humanized” mice in
order to directly assess the risk of transmission to humans.
Another basic question that requires further clarification is the
convertibility potential of fish PrPs. Ongoing experiments using the
sensitive protein misfolding cyclic amplification (PMCA) method
[126,127] may reveal whether piscine PrPs have the intrinsic ability to
misfold and aggregate. In addition, the use of TSE-infected cell lines
could become an important tool to investigate possible molecular
interactions between mammalian PrPSc and fish PrPC, leading to the
conversion of piscine PrPs. Aside from transmissibility and convertibility issues, the composition of the deposits found in the brains of the
BSE-challenged sea bream deserves further examination. Concretely,
the identification of other aggregated molecules in brain deposits
could help define the properties of these novel amyloid plaques, and
perhaps their mechanistic connection to neurodegeneration. For
instance, previous data on the composition of amyloid deposits
present in degenerating neuronal tissue have suggested that the
components of such aggregates may represent the outcome of the
degeneration process rather than the cause of neuronal cell death
[128]. Thus, the study of novel proteinaceous deposits may contribute
new leads to the debate about the causal link between neurotoxicity
and protein oligomerization.
The molecular basis of prion disease is closely associated with the
structural and functional properties of PrP. Therefore, deep understanding of prion pathogenesis in fish and mammals cannot be
reached without basic knowledge about the physiological roles of PrP.
The knockdown phenotypes described in zebrafish constitute the first
experimental evidence that the lack of PrP can cause dramatic
abnormalities in a whole organism [92]. In addition, the finding that
PrPs regulate cell–cell communication via known cellular pathways
provides an ideal opportunity to establish how PrP may exert its
proposed neuroprotective role (Fig. 5). It is noteworthy that some of
the molecules affected in PrP-1 knockdown embryos (Src-kinases, Ecadherin, β-catenin, etc.) are not only essential for early zebrafish
development but also play important roles in neuronal function and
during neurodegeneration [96]. Therefore, it will be crucial to further
characterize the signaling pathways under the control of PrP-1 using
biochemical, cell biological, proteomic and imaging tools. Such
experiments are currently being carried out in fish embryos and
embryonic stem cells. Ideally, this basic knowledge should be
integrated into the design and interpretation of studies addressing
the function of PrP in mammalian cells, as well the experimental
transmission of prion disease between fish and mammals.
Characterization of the PrP-2 knockdown phenotype is currently in
progress. Preliminary results show that PrP-2 is required for the
proliferation and differentiation of different groups of embryonic
neurons (Málaga-Trillo and Luncz, unpublished data). A similar
phenotype has been reported for PrP knockout mice [41], although
the defect does not impair neural development to the extent seen in
the zebrafish [92]. Because PrP-2 also shares the ability to elicit cell–
cell adhesion and intracellular signaling, it is likely to regulate neural
development via the same molecular pathways controlled by PrP-1 in
the early gastrula. Thus, morphological and molecular analyses of the
PrP-2 phenotype may provide valuable mechanistic insights into the
roles of PrP during neuronal differentiation, axon growth, and
synaptogenesis. Integrating the emerging zebrafish data within the
vast amount of information available from mammalian cells and
knockout mice promises to become a lengthy but rewarding task.
6. Concluding remarks
For many years, prion researchers have relied heavily on the use of
mammalian models as experimental systems. This strategy, and
particularly the development of genetically modified mice, has led to
outstanding discoveries in the field. At the same time, the
Fig. 5. Exploring the roles of PrP in health and disease. It has been proposed that neuronal death results from failure by PrP to provide an unknown neuroprotective function. It
remains to be clarified how the various cellular and molecular processes influenced by PrP in vivo may converge into such a neuroprotective role.
412
E. Málaga-Trillo et al. / Biochimica et Biophysica Acta 1812 (2011) 402–414
experimental difficulties associated with the study of TSEs have
exposed the need for new animal models in prion biology. Recently,
researchers have explored the utility of fish to address questions
concerning the physiological roles of PrP and the risk of TSEs in these
organisms. An interesting picture has emerged, suggesting that
transmission of prions could potentially take place between commercially important fish and mammals, although the implications of
these findings for human and animal health remain to be clarified.
Much remains to be done in order to confirm or rule out the existence
of bona fide prions in fish. Nevertheless, the use of fish models in TSE
research will likely uncover novel aspects of prion transmission across
a wide range of hosts, relevant to human health. In addition, the
finding that fish and mammalian PrPs share important roles in cell–
cell communication might help elucidate the molecular mechanisms
by which prions induce neuronal death, setting the stage for the
identification of new anti-prion therapeutic targets.
Acknowledgements
This work is supported by a DFG grant (MA 2525/2-1) and University
of Konstanz funds awarded to EMT. ES, TS and AF are funded by EU
grants (QLK5-2002-00866 for ES, TS, AF and FOOD-CT-2004-506579 for
ES, TS). ES is a scholar of the Greek States Scholarships Foundation (IKY),
and the sea bream and sea bass transmission experiments were part of
her doctoral thesis. AF would like to thank Raquel Aranguren and Beatriz
Novoa for contributing to the secondary transmission study of ME7infected sea bream tissues to mice.
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Contents lists available at ScienceDirect
Biochimica et Biophysica Acta
j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / b b a d i s
Review
Microdomain-forming proteins and the role of the reggies/flotillins during axon
regeneration in zebrafish☆
Claudia A.O. Stuermer ⁎
University of Konstanz, Department of Biology, 78465 Konstanz, Germany
a r t i c l e
i n f o
Article history:
Received 15 September 2010
Received in revised form 30 November 2010
Accepted 2 December 2010
Available online 13 December 2010
Keywords:
Axon regeneration
Microdomain
Reggie/flotillin
Recruitment
Targeted delivery
a b s t r a c t
The two proteins reggie-1 and reggie-2 (flotillins) were identified in axon-regenerating neurons in the central
nervous system and shown to be essential for neurite growth and regeneration in fish and mammals. Reggies/
flotillins are microdomain scaffolding proteins sharing biochemical properties with lipid raft molecules, form
clusters at the cytoplasmic face of the plasma membrane and interact with signaling molecules in a cell type
specific manner. In this review, reggie microdomains, lipid rafts, related scaffolding proteins and caveolin—
which, however, are responsible for their own microdomains and functions—are introduced. Moreover, the
function of the reggies in axon growth is demonstrated: neurons fail to extend axons after reggie knockdown.
Furthermore, our current concept of the molecular mechanism underlying reggie function is presented: the
association of glycosyl-phophatidyl inositol (GPJ)-anchored surface proteins with reggie microdomains elicits
signals which activate src tyrosine and mitogen-activated protein kinases, as well as small guanosine 5′triphosphate-hydrolyzing enzymes. This leads to the mobilization of intracellular vesicles and to the
recruitment of bulk membrane and specific cargo proteins, such as cadherin, to specific sites of the plasma
membrane such as the growth cone of elongating axons. Thus, reggies regulate the targeted delivery of cargo—
a process which is required for process extension and growth. This article is part of a Special Issue entitled
Zebrafish Models of Neurological Diseases.
© 2010 Elsevier B.V. All rights reserved.
1. Introduction
The zebrafish and goldfish visual pathway has served as a model
system for the exploration of rules governing over the development of
the retinotopic projection of retinal ganglion cell (RGC) axons onto
the optic tectum [1,2]. The restoration of the retinotopic map by
regenerating RGC axons after optic nerve lesion has also been
intensively analyzed in this system. Because of its remarkable
regeneration-supporting properties, the fish optic nerve is, moreover,
suited to search for factors underlying successful regeneration of
injured CNS fiber tracts per se. It is expected that insights into
conditions allowing successful regrowth in fish might provide
valuable clues for stimulation of axon regeneration in the mammalian
CNS where regeneration does not spontaneously occur to any
significant extent. Experiments along these lines have led to the
discovery of two proteins, reggie-1 and reggie-2 [3], which turned out
to be essential for growth and regeneration [4], hence their name reggie. Evidence for a role of the reggies in axon growth and regeneration,
together with our current understanding of the underlying molecular
☆ This article is part of a Special Issue entitled Zebrafish Models of Neurological
Diseases.
⁎ Tel.: +49 7531 882236.
E-mail address: [email protected].
0925-4439/$ – see front matter © 2010 Elsevier B.V. All rights reserved.
doi:10.1016/j.bbadis.2010.12.004
mechanisms, will be reviewed in this article. But before reggies are
considered in the context of axon growth, their properties as
constituents of “lipid rafts,” or better microdomains will be discussed
and compared to other microdomains and microdomain-forming
proteins in zebrafish. Prior to this, however, the reasons why axons in
the CNS of fish regenerate—as opposed to mammals which do not—
will be considered in brief.
2. Axon regeneration in the CNS and the identification of the
reggie proteins
One reason for successful regeneration in the CNS is the growthpermissive nature of the glial cell environment in the fish optic nerve
[5,6]. The mammalian CNS, by contrast, presents several growth
inhibiting molecules on glial cells and in the extracellular matrix
which block axon growth and prevent regeneration [7]. The second
reason for the success of axon regeneration is also dependent on the
special neuron-intrinsic properties of nerve cells in fish [8]. Retinal
ganglion cells (RGCs), for instance, promptly upregulate growthassociated proteins after optic nerve lesion which enables the neurons
to regrow their axons and to provide them with cytoskeletal proteins,
cell adhesion molecules, the necessary receptors for guidance cues
and, of course, bulk membrane material [4,8,9]. This ability to switch
on expression of genes which were active during development
but downregulated thereafter is by far less developed in mammals
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although in principle possible [10 and see below]. More than a dozen
growth-associated proteins in RGCs have been identified at the
molecular level, and these were recently complemented by a
proteomics approach in mammalian cortical neurons [11].
The two proteins discovered in this context, reggie-1 and reggie-2
[3,11], are 45% identical to one another and have a molecular weight
of 48 kD. Reggies were also identified in mammals [12,13] where they
occur in basically all cells examined so far. In the rat retina, they are
upregulated in exactly the 3% of RGCs that, as demonstrated by
Richardson et al. [10], can regenerate their axon in a growthpermissive environment.
However, it was not immediately clear how reggies regulate
regeneration. Unlike cell adhesion molecules which are also upregulated during axon regeneration [14,15] and mediate the interaction of
the elongating growth cone with the environment, the reggies were
found to reside at the cytoplasmic face of the plasma membrane. They
are anchored to the membrane by myristoyl and palmitoyl residues
[16,17] and through stretches of hydrophobic amino acids in their
N-terminal head domain (Fig. 1). It was also observed that the reggies do
not only occur at the plasma membrane but also in association with
many intracellular vesicles [9,18,19], including the Rab11 recycling
compartment. Consistent with their identity as microdomain proteins,
reggies were independently identified as components of the protein
fraction known as DRMs (detergent resistant membranes). The DRM
contains molecules that are insoluble in non-ionic detergents (such as
Triton X-100) and that are regarded as “lipid raft” components. Together
with other DRM proteins, the reggies “float” after sucrose density
centrifugation [12], and hence, they were named flotillins [12]. Reggie-1
corresponds to flotillin-2, and reggie-2 to flotillin-1. Reggies/flotillins
are often used as markers for the lipid raft fraction in biochemical work.
We prefer the term “reggie microdomain” (instead of lipid raft) because
reggies assemble by oligomerization into clusters of ≤100 nm [9,20] and
are, therefore, visible at the light microscopic level by immunofluorescence analysis [13,20,21] with specific antibodies. The microdomains
appear as dots or puncta at the plasma membrane of cells and along
axons, growth cones, filopodia and as densely packed rows of dots, for
instance, at cell–cell contacts [21,22] and in the T cell cap [20,23]. This
“pearl on a string-like” arrangement in T cell caps and at cell contacts
(Fig. 2) is formed by a reduction of the distance between individual
microdomains [22].
Fig. 1. The microdomain scaffolding proteins and their domains. Reggie-1 and reggie-2 (flotillin) possess an N-terminal head domain (light blue), with two hydrophobic regions
(yellowish) and acylation sites (red) for membrane attachment. The tail domain (gray) has strikingly many EA repeats for α-helical coiled-coil formation and oligomerization. The
other microdomain scaffolding proteins share some degree of sequence homology with reggie and among each other and also form oligomers.
C.A.O. Stuermer / Biochimica et Biophysica Acta 1812 (2011) 415–422
417
Fig. 2. Distribution and function of reggie microdomains in T cells and neurons. A) In T cells, reggie-1 (and -2)—visualized here by a reggie-1 specific antibody (red)—is clustered at
one pole of the cell, the preformed reggie cap which represents a platform for the association of specific GPI-anchored proteins, such as PrP and Thy-1 and signaling. B) Reggie
microdomains cluster at cell–cell contact sites (arrowheads) by reducing the distance between individual microdomains (red, immunofluorescence with anti-reggie-1 antibodies).
C) The growth cone from a zebrafish RGC axon shows the distribution of reggie microdomains (puncta) at its central region as well as in its filopodia. D) Individual zebrafish RGC
neuron and its axon immunostained with a reggie-1 specific antibody and showing the punctate microdomains along the axon. E) Hippocampal neurons differentiate in vitro
(E, control siRNA) and extend neurites whereas their counterparts fail to produce neurites after exposure to reggie-1 specific siRNAs (F). G) Hippocampal neurons often produce
club-shaped endings where a growth cone should have been, after perturbation of reggie-1 expression. Scale bars: 20 μm.
3. Microdomains and lipid rafts
Microdomains, caveolae and lipid rafts represent functional units
in cellular membranes whose existence depends on mainly saturated
sphingolipids and cholesterol in a liquid ordered phase and
segregated from unsaturated lipids in the liquid disordered phase
[24,25]. Rafts range in size from a few nanometer encompassing by
estimate six GPI-anchored proteins (nanoclusters) [26] to several
hundred nanometers (known as “clustered rafts”) [25] are dynamic
and generally short-lived. They can be stabilized by the actin
cytoskeleton [27] which influences their distribution, half life and
size. The existence of lipid rafts has been heavily disputed [28]
particularly since they often escape microscopic observation due to
their small size [26]. Recent results now demonstrate the phase
separation of sphingolipids and cholesterol in giant plasma membrane vesicles of vertebrate cells which support again the raft concept
[29].
Microdomains—in contrast to rafts—depend on specific scaffolding
proteins that have an affinity for the lipid raft environment and share
many raft properties. A family of microdomain proteins with a
characteristic membrane interaction domain at their N-terminal
“head” domain consists of stomatin, prohibitin, flotillin/reggie, podocin
and erlin [9,30–32]. The scaffolding proteins (Fig. 1) possess a region for
membrane interaction and stretches of amino acids (tail domain) which
promote oligomerization [33]. These protein oligomers, when labeled
by antibodies or tags, make microdomains visible at the light
microscopic level and amenable to direct analysis of distribution and
function.
Lipid rafts and microdomains confer inhomogeneities to the
plasma membrane and allow the formation of membrane compartments [25] which are used by specific membrane proteins for cluster
formation, protein–protein interactions and signaling [34] as well as
for the recruitment and targeted delivery of membrane proteins to
specific sites [9]. GPI-anchored proteins are typical cell-surface
residents of rafts and microdomains whereas proteins with acyl
residues (palmitoylation, myristoylation and others) typically reside
at the cytoplasmic face of the microdomain plasma membrane.
Certain transmembrane proteins also acquire a preference for the lipid
raft and microdomain environment, in particular certain growth
factor and transmitter receptors [25].
While being intensively discussed among cell biologist and experts
in lipidomics, rafts and microdomains do not play such a central role
in research on zebrafish development or disease. Few exceptions are
the microdomains scaffolded by the proteins caveolin (i.e. caveolae),
podocin and reggie which will be described next and whose function
in zebrafish embryos and/or adult fish will be discussed subsequently.
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4. Microdomains and caveolae
Among the most intensively studied microdomains are caveolae,
100 nm flask-shaped plasma membrane invaginations, whose formation depends on caveolin [35]. Each of the other scaffolding proteins,
reggie/flotillin, stomatin, podocin, prohibitin and erlin, forms its own
microdomain without producing membrane indentations or other
morphologically conspicuous structures.
There was a debate as to whether reggies represent additional
constituents of caveolae which would imply that they serve as a
second scaffold of the caveolar invaginations [12]. Reggie proteins,
however, are present in basically every cell analyzed so far, whereas
caveolin is not. Caveolin, not reggie, is absolutely required for
caveola formation. Moreover, caveolae are absent from neurons and
lymphocytes [13,20,21,36] which do have reggie microdomains.
Even in cells with caveolae, such as astrocytes, caveolae and reggie
proteins occupy non-overlapping regions of the plasma membrane
[21], so that reggies are clearly not constituents of caveolae. In fact,
we are not aware of any report on microdomains with mixed
protein scaffolds.
The microdomain scaffolding proteins are evolutionarily highly
conserved [32,37] suggesting important functions across kingdoms of
life. Accordingly, inspection of databases shows the presence of
homologs of caveolin, stomatin, prohibitin, reggie/flotillin, podocin
and erlin in zebrafish. Aside from their sequence, however, not much
is known on their function. We found no report on zebrafish stomatin,
prohibitin and erlin. But work was published on caveolin-1 and
caveolin-3 in the early embryo, on podocin during kidney formation,
and on reggie-1 and reggie-2 during axon regeneration. The reggies
will be considered in greater detail because they were discovered in
the fish CNS and functionally characterized in the context of axon
regeneration [3,4]. Moreover, results from fish together with insights
from various cell types and species has led to a new concept
explaining reggie functions [9] but before its presentation published
data on zebrafish caveolin-1, caveolin-3 and podocin will be
summarized.
5. Zebrafish caveolin and caveolae
In mammals, caveolin and caveolae have been implicated in many
functions such as endocytosis, transcytosis, signaling, trafficking of
cholesterol (for review see reference 38) so that it came as a surprise
that the caveolin-1 knockout mice had at first sight no apparent
phenotype. But mice turned out to suffer from pulmonary defects,
particularly under the challenge of exercise, and to lack the ability to
appropriately adapt their blood pressure [39].
5.1. Caveolin-1
In zebrafish, caveolin-1 and caveolae were shown to exist during
the development in neuromasts and in notochord cells (and other
structures) [40] which exhibited caveolae in abundance. Morpholino-mediated knockdown to assess the potential role of caveolin in
embryogenesis showed that the formation of neuromasts was
perturbed and the notochord disrupted [40]. Another publication
reports additional defects in the eye and in somites at 12 h post
fertilization (hpf) as well as disruption of the actin cytoskeleton
[41]. Later defects emerged in the vascular endothelium [41] and in
the smaller size of the embryos [40]. In addition, the lateral line
developed abnormally most likely because of the defects in
neuromasts and notochord [40,41]. The embryos might suffer from
other problems as, for instance, reduced cholesterol levels of the
plasma membrane since caveolin has been proposed to function as a
carrier for cholesterol [42] and cholesterol is needed for the
existence of lipid rafts and microdomains [25]. Whether morpholino-treated fish develop severe abnormalities due to imbalanced
cholesterol levels has not been reported nor is clear how caveolindependent receptor-ligand interaction and signal transduction may
change. The enzymes involved in cholesterol synthesis [43] are
expressed in zebrafish and in the embryo as maternal messages at
the one and two cell stage predicting that cholesterol biosynthesis is
crucial already in the early blastocytes. Altogether, it appears that
caveolin-1 and caveolae control the differentiation of specific cell
types in the embryo, yet the underlying mechanism is not well
understood.
5.2. Caveolin-3
Zebrafish also expresses a homolog of mammalian caveolin-3
(cav-3) which is a muscle-specific isoform in mammals and fish
[44,45]. Cav-3 in zebrafish was found in the first differentiating
muscle precursor cells and later in skeletal and cardiac muscle fibers
where expression correlated with the presence of caveolae. Cav-3,
however, also appeared in pectoral fin, facial muscle and notochord
[44]. Downregulation of cav-3 expression caused severe muscle
abnormalities and uncoordinated movement and increased the
number of muscle pioneer-like cells adjacent to the notochord. The
signaling pathways which depend on cav-3 and caveolae and steer
muscle development have not been described and are to this end
unknown.
6. Zebrafish podocin
Podocin is expressed by podocytes that are necessary for the
blood filtration barrier in the kidney glomerulus [46,47]. Podocytes
form the slit diaphragm, a specialized cell–cell adhesion complex at
the podocyte foot processes surrounding glomerular blood vessels.
Slit diaphragm formation depends on the cell adhesion proteins (of
the immunoglobulin superfamily) nephrin and Neph as well as
podocin [48]. Much as in higher vertebrates, zebrafish nephrin and
podocin are specifically expressed in pronephric podocytes and
required for the development of pronephric podocyte cell structures [49]. Morpholino-mediated knockdown of podocin (or nephrin)
resulted in loss of a slit diaphragm at 72 and 96 hpf and failure to form
normal podocyte foot processes. Absence of normal podocin (or
nephrin) expression resulted in defects in glomerular filtration and
aberrant passage of high molecular filtrate [49,50]. The exact cellular
and molecular mechanism of podocin function during slit diaphragm
formation is not known. However, human podocin mutations Cterminal to the membrane association (head) domain resulted in loss
of podocin at the cell membrane and aberrant accumulation in ER
secretory pathway vesicles [51]. Podocin mutants also resulted in failed
delivery of nephrin to the slit diaphragm thus indicating a role of
podocin in nephrin trafficking [48].
7. Reggie microdomains
Reggie microdomains are defined by hetero-oligomeric clusters of
reggie-1 and reggie-2. To this end, it has not been explicitly examined
whether reggie-1 and reggie-2 can perform separate functions.
However, it has become clear that reggie-2 undergoes proteasomal
degradation when reggie-1 is downregulated (53), for example after
siRNA and morpholino-mediated interference with reggie-1 expression levels in neurons, epithelial cells and zebrafish embryo. It is
unclear why reggie-2 is so tightly regulated in dependence of reggie-1
nor has it been thoroughly analyzed whether reggie-1 and reggie-2
can subserve separate functions in other cell types. Therefore, we do
not distinguish between reggie-1 and -2 in the text when they were
not addressed individually.
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7.1. Association of the GPI-anchored prion protein and Thy-1 with reggie
microdomains
Reggie-1 and -2 were isolated from larval goldfish by coimmunoprecipitation with an antibody against goldfish Thy-1 [3].
This is in line with the fact that Thy-1, as a GPI-anchored protein,
associates preferentially with reggie microdomains [20,21,52,53] not
only in fish but also in all vertebrate cells studied so far. Moreover,
Thy-1 is upregulated in fish RGCs after nerve lesion like reggie [53]
and co-localizes with reggie in microdomains in growth cones, along
axons, at cell–cell contact sites and in the T cell cap [9]. In the
uninjured fish visual system, Thy-1 as well as reggie, is colocalized in
the few axons from new RGCs that are constantly added to the fish
retina [3]. Thus, Thy-1 seems to cocluster preferentially with reggie
microdomains in vitro and in vivo. This applies also to the GPIanchored cellular prion protein [PrPc; 20] and the zebrafish homologs
PrP-1 and PrP-2 [54]. That this association of PrP and Thy-1 with
reggie-1 and -2 is functionally important was demonstrated first in T
cells as will be described below. Further insights into the role of the
reggies came from studies on insulin stimulation of adipocytes (fat
cells) [55,56] and on cell–cell contact formation in various types of
cells [9].
7.2. Reggie and PrP signaling in T cells
In T cells, reggie microdomains are pre-clustered at one pole of the
cell, representing ‘the preformed reggie cap’ [23] where the T cell
receptor complex and the signaling molecules associated with T cell
activation coalesce upon stimulation [20]. The preformed reggie cap is
important for T cell activation. A dominant-negative reggie construct
disrupts the cap and interferes with the formation of the immunological synapse [19]. In T cells, the activation of GPI-anchored proteins
by antibody cross-linking is sufficient for TCR complex assembly in the
cap [34]. This applies, for instance, to Thy-1 and PrP, whose activation
leads to their selective association with the reggie cap [20,21].
When clustered in the reggie cap, PrP elicits the phosphorylation
of the MAP (mitogen-activated protein) kinase ERK1/2 and evokes a
distinct Ca2+ signal leading to the recruitment of CD3 [20,57], the
major T cell receptor component, to the membrane in the cap. Yet,
CD3 recruitment is not sufficient for the full activation of the T cell
receptor complex [20], which would require cross-linking of CD3 or
stimulation by antigen. T cell receptor recruitment involves the
communication with actin dynamics through src tyrosine kinases
(fyn, lck, src), Rho-GTPases and the signaling molecule Vav [19]. The
conclusion from these results is that the microdomains consisting of
reggie oligomers serve as platforms for PrP cluster formation,
signaling, actin rearrangement and the recruitment of CD3 (from
internal stores) to the cap [9]. The involvement of reggie in the
recruitment of a membrane protein (Glut4) to specific sites of the
plasma membrane has previously been observed in adipocytes [55,56]
as is discussed next.
419
It is known that the small GTPases regulate actin dynamics and vesicle
trafficking [4,60–62]. Vesicles carrying reggie were observed to shuttle
between the plasma membrane and more distant intracellular sites [61]
including the recycling compartment. This indicates that reggie
microdomains at vesicle membranes participate in the signaling
required for vesicle mobilization, trafficking and targeting. These events
require the activity of small GTPases that, in turn, have a preference for
rafts [9,58,63].
The reggie/flotillin-binding protein CAP/ponsin and its relatives
ArgBP2 and vinexin were implied in the interaction with the actin
cytoskeleton [56] in which reggie/flotillin is also involved [64] and
with substrate and cell adhesion proteins, such as integrins and
cadherins. Together, these findings suggest that reggie activates a
pathway for polarized delivery of cargo [9]. The type of cargo depends
on the cell type and includes, for example, transporters (such as
Glut4) [55], cell adhesion molecules (such as cadherins, integrins)
[54,56], receptors, ion channels and many other proteins that need to
be targeted to specific sites of cells [9]. The sites are “marked” by the
clustering of reggie/flotillin microdomains and co-clusters of GPIanchored proteins, which provides external signals and specificity
(Fig. 3). Reggies/flotillins at membranes of intracellular vesicles
[18,61] might represent similar signaling centers for clustering of
signaling molecules to promote vesicle transport, sorting and targeted
delivery of cargo such as cadherin (Fig. 3) [9].
7.4. Reggies, PrP and cadherin recruitment
Reggies are clustered at contact sites between cells [20,21] and so
is PrP. PrP–PrP trans-interactions in contacts between mammalian
7.3. Reggie-associated signaling and the recruitment of Glut4 in
adipocytes
In adipocytes (fat cells) flotillin-1/reggie-2 interact with the c-cblassociated adaptor protein (CAP) and via CAP with a signaling complex
that activates the small guanosine 5′-triphosphate-hydrolyzing enzyme
(GTPase) TC10 [55] in lipid rafts (reggie/flotillin microdomains). This
results in the translocation of the glucose transporter Glut4 from a
specific vesicular storage compartment to the plasma membrane
[55,56]. This step requires the exocyst and the (Ras-related) GTPase
RalA. TC10 and RalA associate with vesicles, such as the recycling
compartment and (transport) vesicles en route to the plasma
membrane [58,59] which also carry reggie—in agreement with the
role of the reggie proteins in activation of TC10 and other small GTPases.
Fig. 3. Recruitment and targeted delivery of membrane and membrane proteins
regulated by reggie. PrP is typically associated with reggie microdomains and clustered
at contact sites between cells (cell 1 with cell 2). PrP trans-interaction in reggie
microdomains leads to signal transduction including the CAP/reggie-associated
signaling complex which activates the small GTPase TC10. This leads to the mobilization
of cargo (cadherin)-loaded vesicles which also have reggie microdomains, through
TC10 and RalA. Vesicles are transported (along cytoskeletal elements) to the plasma
membrane and fuse resulting in membrane growth and cadherin delivery at cell contact
sites.
420
C.A.O. Stuermer / Biochimica et Biophysica Acta 1812 (2011) 415–422
epithelial and neuronal cells or zebrafish embryonic blastocytes cause
PrP clustering and co-clustering with reggies [9,54]. PrP clusters in
reggie microdomains apparently regulate cell contact formation in
zebrafish embryos as well as in mammalian cells (Fig. 3) as well as
downstream actin rearrangement [54]. In zebrafish embryos, PrP-1
accumulates selectively at contact sites between cells at the blastula
and gastrula stage. PrP clustering results in signaling. This signal is
necessary for the PrP-dependent recruitment of epithelial (E)cadherin from the internal vesicle pool to the contacts between cells
in zebrafish as well as in various mammalian cells [9,54]. In zebrafish,
PrP downregulation by morpholinos has dramatic effects: the cells
lose contact and the embryo dies from its failure to proceed through
gastrulation [54]. Search for the underlying mechanism showed that
E-cadherin was retained in intracellular recycling vesicles instead of
emerging at the cell-surface where E-cadherin should be engaged in
homophilic adhesion.
How GPI-anchored proteins such as PrP communicate with reggie
for the recruitment of cadherins and other membrane proteins
remains to be solved. How proteins with lipid anchors confined to
one leaflet of the lipid bilayer are capable of signal transduction
without associated transmembrane proteins is also unsolved and
debated in many reports and reviews dealing with lipid rafts and their
function [25]. Recent results from a biophysical simulation approach,
however, suggest that cluster formation of GPI-anchored proteins
with lipid anchors in the outer leaflet of the plasma membrane is
crucial and efficient in influencing the adjacent cytoplasmic leaflet,
which offers another cluster of proteins (such as the reggie
microdomain) with their own lipid anchors—the myristoyl and
palmitoyl residues in reggie (Matthias Weiss, pers. commun.).
Moreover, sphingolipids with lipid chains longer than the width of
one leaflet are enriched in rafts and microdomains. It is thus
conceivable that co-clusters of proteins in the outer and inner leaflet
of the plasma membrane can transduce signals into the cell, activating
src tyrosine kinases that are also lipid-anchored and able to interact
with reggie (such as fyn) [65].
Thus, reggies appear to function as platforms necessary for the
assembly of specific cell-surface proteins, for signal transduction and
activation of small GTPases to regulate actin dynamics. Specificity and
the trigger for signaling appear to come from the cell-surface associates
of reggie/flotillin microdomains, which are cell type dependent (insulin
receptor in adipocytes, PrP (Thy-1) in T cells and PrP (Thy-1) at cell
contact sites in the embryo and cells in vitro) and domain specific (the
cap in T cells, contact sites). The cell-surface-derived signals often (or
always) target GTPases and actin dynamics, conceivably to activate and
recruit specific vesicles from the intracellular pool such as Glut4 and
E-cadherin.
Thus, consistent with finding reggie-1 and -2 in basically all cell
types and species as distant as flies, fish and mammals [53], their
function seems to be of general importance for the communication
between cells and for membrane protein sorting, trafficking and
delivery. How does this relate to axon growth?
8. Reggies regulate axon regeneration
To examine whether and how reggies might regulate axon
regeneration and growth, we applied morpholinos in a piece of
gelfoam to the transected right nerve of adult zebrafish and a control
morpholino to the left transected nerve [4] following a procedure by
Becker et al. [66]. The morpholinos are being taken up by the severed
axons and transported retrogradely to the RGCs of origin. The
morpholinos are labeled by a fluorescent tag (lissamine) thus
allowing identification of morpholino-laden RGCs in retina whole
mounts. Miniexplants of retinae of treated fish send out axons. The
quantification of axons extending from the explants showed a 45%
decrease from morpholino-treated RGCs compared to control. A
second experiment gave even more striking effects: a fluorescent dye
was applied to the regenerating axons behind the first transection and
morpholino application site in the optic nerve which allows to
quantify the regenerating axons by the second label in their parent
RGCs. This approach showed a 70% reduction of RGCs with regenerating
axons in the optic nerve showing that reggie morpholinos block axon
regeneration and, in other words, suggest that reggies are necessary for
axon growth and regeneration.
This conclusion was supported by individual neurons from the
mouse hippocampus in culture in which reggie expression levels were
downregulated by reggie-specific siRNAs [4]. These neurons failed to
differentiate and did not form axons or failed to elongate them. Axons
often showed immobile club-shaped endings where a growth cone
should have been and did not grow (Fig. 2). Other neurons had shorter
or no processes (Fig. 2), were abnormally large and malformed and
produced conspicuous bulges instead of axons and dendrites. The
elongation process was apparently blocked because, as we believe,
membrane proteins or membrane and proteins from the internal
vesicle pool were not appropriately supplied to the prospectively
growing tips [9]. Thus, the mechanism of membrane and protein
recruitment which is necessary for process growth is controlled by
reggies. Therefore, reggies clearly regulate regeneration and axon
growth.
Such phenotypes indicate a disturbance in the communication
with the cytoskeleton, which was examined in N2a neuroblastoma
cells. Reggie mis- and downregulation perturbed the activation levels
of the Rho-GTPases Rac, Rho and cdc42 which changed the activation
of the downstream effectors WASP, Arp2/3, cortactin and cofilin and
of focal adhesion kinase (FAK), p38 and ras [4,62]. This is compatible
with the view that the reggie proteins can affect and modify actin
dynamics [64], in connection with the recruitment of membrane and
proteins (or simply membrane building blocks) from the constitutive
secretory pathway and recycling compartment to the growing tips [9].
9. An explanation of reggie functions
Taken together, it seems that reggies are crucial for the
recruitment of membrane and specific membrane proteins (or
membrane building blocks) to specific regions or domains of the
cell (Fig. 3). Neurons with elongating and regenerating axons, where
reggies are enriched at growing tips, seem to suffer badly when
reggies are missing since blockage of reggie function impairs the
delivery of material for growth. Membrane proteins are recruited
from vesicular pools (recycling compartment, transport vesicles,
Golgi-associated vesicles) and moved along cytoskeletal elements,
and this requires the small GTPases of the Rho- and Ras-family and
their effectors. Reggie-associated signal transduction and membrane
protein recruitment appear to be activated by surface molecules with
specific affinities for the reggie microdomain environment such as
GPI-anchored proteins and certain receptor types. The surface
proteins can be activated by ligands, binding partners (PrP–PrP
trans-interaction) or cross-linking antibodies mimicking ligands
which activate signaling molecules located in reggie microdomains:
src tyrosine kinases, small GTPases, cbl and interacting adaptor
complexes. Signaling provokes the recruitment of vesicles and
thereby the targeted delivery of membrane building blocks and
proteins for axon growth, adhesion, navigation as well as for contact
formation between cells and for the establishment of specific
membrane domains such as the leading edge, growth cone, its
lamellipodia and filopodia. This implies a role of the reggies in the
recruitment of specific vesicles and certain membrane proteins from
vesicles and stores (transporters, receptors, ion channels and
adhesion molecules) [9].
This role of the reggies in the mediation of the recruitment of
proteins/membrane from intracellular pools requires their existence in
all cells of vertebrates and invertebrates and explains why they are so
important for axon growth. Upregulation of reggie expression is a
C.A.O. Stuermer / Biochimica et Biophysica Acta 1812 (2011) 415–422
prerequisite for axon growth/regeneration not only in fish but also in
the mammalian CNS [4,13]. In fish, all RGCs express reggie at high levels
and regenerate their axons. In the mammalian retina, only 3% of the
neurons regenerate their axons and express reggie. When new methods
are applied to stimulate the remaining neurons to upregulate reggie,
axon regeneration might be successful to an extent that would allow
recovery of function in mammals. First results in this direction are
encouraging (unpublished results: Jan Koch, Paul Lingor, Matthias Bähr
[Göttingen] in collaboration with Gonzalo Solis and C.A.O. Stuermer
[Konstanz]). In light of such results it is tempting to speculate that
upregulation of reggie in injured neurons after spinal cord lesion might
promote axon regrowth and improve the fatal conditions after lesion.
Upregulation of reggie and the ensuing growth response might provide
neurons with the necessary “power” to resist and counteract effects
from neurodegenerative diseases.
This unexpected role of the reggies predicts on the one hand
related functions of other microdomain proteins and requires on the
other hand identification of all players in the reggie-dependent
signaling pathway. Moreover, whether reggies act as molecular
shuttles between plasma membrane and intracellular vesicle pools
needs to be addressed experimentally. The most crucial issue in the
context of axonal regeneration is the identification of the factors that
control reggie upregulation in neurons during axon outgrowth and
regeneration in fish and in mammals so to stimulate neurons to
produce new processes upon injury and resist degeneration.
Acknowledgment
Our work is supported by the Deutsche Forschungsgemeinschaft
and Fonds der Chemischen Industrie. I want to thank Ulrike Binkle,
Marianne Wiechers and Vsevolod Bodrikov for the figures and all
co-workers for the experiments and results in our reggie research.
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