DDMOD-408; No of Pages 9
Drug Discovery Today: Disease Models
DRUG DISCOVERY
TODAY
DISEASE
MODELS
Vol. xxx, No. xx 2014
Editors-in-Chief
Jan Tornell – AstraZeneca, Sweden
Andrew McCulloch – University of California, SanDiego, USA
Bone biology in animal models, including testing of bone biomaterials
Fish: a suitable system to model human
bone disorders and discover drugs with
osteogenic or osteotoxic activities
Vincent Laizé1,3, Paulo J. Gavaia1,3, M. Leonor Cancela1,2,3,*
1
2
Centre of Marine Sciences, University of Algarve, 8005-139 Faro, Portugal
Department of Biomedical Sciences and Medicine, University of Algarve, 8005-139 Faro, Portugal
This review discusses the suitability and advantages of
teleost fish for studying underlying mechanisms of
normal and pathological development and mineralization of vertebrate skeleton, presents a selection of
zebrafish mutants and transgenic lines modeling hu-
Section editors:
Oskar Hoffmann – Pharmacology and Toxicology, UZA II
Pharmacy Center, Vienna, Austria.
Wilhelm Hofstetter – Clinical Research Department,
University of Bern, Bern, Switzerland.
man skeletal diseases and highlights currently available
fish systems for identifying and characterizing novel
osteogenic and osteotoxic molecules.
Introduction
The last decade has seen an exponential interest in the use of
model organisms capable of providing suitable alternatives to
mammalian models and allowing quicker and less expensive
approaches, leading to significant improvements in our
knowledge on the molecular basis of human pathologies.
In this respect, the study of skeletal diseases has not been
an exception but requires the use of vertebrates for obvious
reasons. Fish, in particular zebrafish and medaka, have been
used with a growing success due to their many similarities
with mammals in molecular pathways and mechanisms involved in the onset of patterning and development of skeletal
structures. Many reports have confirmed the suitability of fish
systems to model many of the pathological defects affecting
human skeletal formation, osteoclastic bone resorption, osteoblastic bone mineralization and extracellular matrix
maintenance, but also to visualize the onset of normal and
*Corresponding author.: M.L. Cancela ([email protected], [email protected])
3
All authors contributed equally.
1740-6757/$ ß 2014 Elsevier Ltd. All rights reserved.
abnormal skeletal structures, and the possibility of rapidly
detecting drug-induced osteogenic or osteotoxic effects in
phenotype-based assays.
Teleost fish and human skeletons are remarkably
similar
Despite some small differences that can be attributed to evolutionary distance (their last common ancestor existed approximately 420 million years ago), anatomic and developmental
features of teleost fish and mammalian skeletons are remarkably similar, with much of the skull, axial and appendicular
skeleton formed by identical bones, and with a high conservation of the developmental events and underlying mechanisms
of skeletogenesis, including the early formation of a cartilaginous anlage followed by bone formation through endochondral and dermal ossification [1,2]. Main differences and
similarities between fish and mammalian bone are summarized in Table 1 (see [3] for a more comprehensive comparison).
The most striking difference is probably the occurrence of
acellular bone (devoid of osteocytes) and mononucleated
osteoclasts in most teleost fish species, while mammals have
exclusively cellular bone and multinucleated osteoclasts. Interestingly, osteocytic bone and multinucleated osteoclast
http://dx.doi.org/10.1016/j.ddmod.2014.08.001
e1
Please cite this article in press as: Laizé V, et al. Fish: a suitable system to model human bone disorders and discover drugs with osteogenic or osteotoxic activities, Drug Discov
DDMOD-408; No of Pages 9
Drug Discovery Today: Disease Models | Bone biology in animal models, including testing of bone biomaterials
Vol. xxx, No. xx 2014
Table 1. Features of fish and human bone: similarities and differences
Fish skeleton
Human skeleton
Endochondral and membranous ossification
Hydroxyapatite-like calcium-phosphate crystals
Bone formation by osteoblasts positive for alkaline phosphatase
Bone resorption by osteoclasts positive for tartrate-resistance acid phosphatase
Mesenchymal origin of osteoblasts; hematopoietic origin of osteoclasts
Osteocytic and anosteocytic bone
Osteocytic bone
Dendritic processes in a well-developed lacunocanalicular system
Dendritic processes mostly absent and no lacunocanalicular system
Mono- and multinucleated osteoclasts
Multinucleated osteoclasts
Mechanosensing by osteoblasts and lining cells
Mechanosensing by osteocytes
Collagen I and II in bone
Collagen I in bone
Perichordal ossification of the vertebral column
Endochondral ossification of the vertebral column
16 types of cartilage
3 types of cartilage
occur in zebrafish [4,5]. Also noteworthy is the fact that most
teleost fish ossify the vertebrae directly over the notochord,
while mammals have a cartilaginous precursor [6,7]. Fish also
have a much higher diversity of cartilage types and intermediate tissues than what is found in adult mammals [4,8,9].
Despite those differences, the availability of fish mutants
exhibiting features resembling human pathologies has contributed to validate fish as a model organism to study mechanisms underlying skeleton development and skeletal
disorders.
Among teleost fishes, zebrafish Danio rerio (Hamilton,
1822) has been the target of a growing interest from the
scientific community. Zebrafish is a small freshwater fish
from tropical regions of South and Southeast Asia for which
numerous models of human diseases have been identified or
developed during the last decade, leading to its recognition as
a suitable model for biomedical research (reviewed in [10]).
Zebrafish embryos develop externally and are transparent,
allowing for direct observation of embryonic development
and organ growth. Additional features such as small size (3–
4 cm long), high fecundity (mature females can lay hundreds
of non-adhesive eggs every week), short generation time
(adulthood attained in 3–4 months) and rapid development
(most body structures are visible 48 h after fertilization),
robustness (they are easy to manipulate, adapt to a wide
range of environments and can be kept in large shoals)
and availability of genetic techniques, have reinforced the
attractiveness of the zebrafish as a laboratory model. Finally
and importantly, the zebrafish genome has been almost
entirely sequenced and annotated, with most human genes
having orthologs in zebrafish [11]. In addition, it is estimated
that approximately 70% of human disease genes have functional homologs in zebrafish [12]. The key regulators of bone
formation have been highly conserved, and corresponding
zebrafish orthologs share significant sequence similarities
and overlapping of expression patterns [13]. For phenotype-based assays related to skeletogenesis, the transparency
and external/rapid development of fish embryos is a clear
e2
References
[1,59]
[1]
[5]
[5]
[59]
[5]
[60–62]
[63]
[59,61,64]
[1,8]
[6]
[8]
advantage. It is possible to monitor each stage of skeleton
development and determine at high resolution the role of
single cells in bone and cartilage formation/mineralization.
However, zebrafish is not a mammalian species and the lack
of some organs (e.g. lung or mammary gland) represents a
limitation to the modeling of some human diseases. In
addition, phenotypic characteristics of diseases caused by
orthologous genes can differ between fish and human. Because it suffered the teleost-specific whole genome duplication event, zebrafish genome contains numerous paralogous
genes that resulted in gene sub-functionalization and/or
neofunctionalization, a situation that may limit, in some
cases, the use of zebrafish as a disease model. However, this
is also true in studies using the mouse model [11,14]. Still,
zebrafish is a relevant model organism to study vertebrate
development and human diseases, and is rapidly acquiring
standard prominence worldwide as an alternative and complement to rodent models, which remain the standard system
in pre-human drug tests.
Fish models of human skeletal disorders
In the last decade, there has been an increasing effort in
producing mutant and transgenic fish lines with the objective
of modeling human skeletal disorders and improving the
visualization of the skeleton. These achievements have decisively contribute to facilitate studies towards uncovering
novel drugs with the potential to treat the many skeletal
pathologies that affect millions of people worldwide and
which incidence is growing due to the increase in human
life span. Human diseases such as osteogenesis imperfecta,
bone loss (osteoporosis and osteopenia), craniosynostosis,
craniofacial defects and spinal deformities (scoliosis, holospondyly) are examples, among many others, of pathologies
for which fish mutants are available and being used to unveil
the corresponding changes in normal molecular pathways,
thus contributing to discover therapeutic molecules capable
of rescuing these pathologies (Table 2). Although medaka and
guppy have also been used to model human bone disorders,
www.drugdiscoverytoday.com
Please cite this article in press as: Laizé V, et al. Fish: a suitable system to model human bone disorders and discover drugs with osteogenic or osteotoxic activities, Drug Discov
DDMOD-408; No of Pages 9
Vol. xxx, No. xx 2014
Drug Discovery Today: Disease Models | Bone biology in animal models, including testing of bone biomaterials
Table 2. Example of fish models of human bone and skeletal disorders (see also [90])
Human bone/skeletal disorders (bone/skeletal phenotype)
Fish model systems
Affected gene(s)
References
Osteogenesis imperfecta (OI)
(reduced bone density, bone fragility, skeletal deformities)
Osteoporosis
(reduced bone mineral density)
Glucocorticoid-induced osteoporosis (GIOP)
(reduced bone mineral density upon use of steroids)
Iron-induced osteoporosis
(reduced bone mineral density upon iron overload)
Raine syndrome (RNS)
(increased ossification)
Multiple osteochondromas (MO)
(cartilaginous bone tumors leading to skeletal deformities)
Zebrafish chihuahua (chi) mutant
Zebrafish frilly fins (frf) mutant
rankl-induced medaka
col1a1
bmp1
rankl
[65,66]
Mucolipidosis II (ML-II)
(skeletal, craniofacial and joint abnormalities)
Craniosynostosis
(premature fusion of cranial sutures)
Holospondyly
(fusion of vertebral centra)
Fibrodysplasia ossificans progressiva (FOP)
(heterotopic endochondral ossification)
Idiopathic scoliosis
(spinal deformity)
Arterial calcification of infancy
(ectopic mineralization)
Holoprosencephaly (HPE)
(craniofacial defects)
Campomelic dysplasia
(craniofacial defects)
Ehlers-Danlos syndrome (EDS)
(craniofacial defects)
DiGeorge syndrome (DGS)
(craniofacial defects)
Cranio-lenticulo-sutural dysplasia (CLSD)
(craniofacial defects and short stature)
Osteopathy related to mineral homeostasis
(failure to form mineralized bone)
Osteopathy related to abnormal ECM deposition
(craniofacial defects)
Osteopathy related to delayed mineralization
(delayed vertebrae calcification)
Hyperossification
(hyperossification and skeletal overgrowth)
[67]
Prednisolone-treated zebrafish
[56,68]
Iron-overloaded zebrafish
[28]
Zebrafish fam2b mutant
fam20b
[69]
ext2
extl3
papst1
gnptab
[70,71]
[72]
Zebrafish dolphin (dol) and stocksteif (sst) mutants
cyp26b1
[73]
Zebrafish stocksteif (sst) mutant
Retinoic acid-treated zebrafish
Zebrafish lost-a-fin (laf) mutant
cyp26b1
[74]
acvr1/alk8
[75]
Guppy curveback mutant
Not determined
[76]
Zebrafish dragonfin (dgf) mutant
enpp1
[16]
Zebrafish sonic-you (syu) mutant
shh
[77]
Zebrafish jellyfish (jef) mutant
sox9a
[78]
Zebrafish b4galt7 mutant
b4galt7
[79]
Zebrafish van-gogh (vgo) mutant
tbx1
[80]
Zebrafish crusher (cru) mutant
sec23a
[81]
Zebrafish no bone (nob) mutant
entpd5
[16]
Zebrafish
Zebrafish
Zebrafish
Zebrafish
creb3l2
uxs1
sec24d
Not determined
[82–84]
[85]
rpz
[86]
Zebrafish
Zebrafish
Zebrafish
Zebrafish
dackel (dak) mutant
boxer (box) mutant
pinscher (pic) mutant
gnptab morphant
feelgood (fel) mutant
man o’war (mow) mutant
bulldog (bul) mutant
bone calcification slow (bcs) mutant
Zebrafish rapunzel (rpz) mutant
most diseases models have been developed from zebrafish
mutants. Among the first mutants developed and characterized, the zebrafish chihuahua, which lacks a functional type 1
collagen gene, exhibits a skeletal dysplasia resembling human
osteogenis imperfecta characterized by extreme bone fragility. Zebrafish mutants sonic you – lacking a functional sonic
hedgehog gene, which is a key regulator of vertebrate organogenesis – and jellyfish – lacking a functional sox9a gene, which
is a transcription factor involved in chondrocyte differentiation – exhibit skeletal dysplasia resembling human craniofacial syndromes. Guppy is also a model and its mutant
curveback exhibits a skeletal dysplasia resembling idiopatic
scoliosis. Following these first mutants, many more have
been developed and characterized (Table 2) and its numbers
are continuously growing. A simple search in bibliography
using as key words ‘zebrafish’ and ‘human diseases’ can
undoubtedly show the exponential interest in zebrafish in
the last decade. Recently, zebrafish has also been proposed as
a model of excellence to study the molecular and cellular
events underlying the development of ectopic mineralizations, which are observed in a number of human pathologies
such as vascular calcification, skin calcifications, renal
www.drugdiscoverytoday.com
e3
Please cite this article in press as: Laizé V, et al. Fish: a suitable system to model human bone disorders and discover drugs with osteogenic or osteotoxic activities, Drug Discov
DDMOD-408; No of Pages 9
Drug Discovery Today: Disease Models | Bone biology in animal models, including testing of bone biomaterials
calcium stones, among others [15]. For example, the dragonfish mutant shows a broad range of ectopic calcifications
around perichondral bone in the craniofacial skeleton as well
as in the brain, the neural tube and the heart. On the
contrary, the no bone mutant fails to produce mineralized
bone [16].
Furthermore, the growing number of transgenic lines
expressing fluorescent proteins under the control of promoters of bone-related genes such as osx (sp7), oc2, runx2, sox9,
sox10, barx1, col2 and col10 among others [17–21], provide a
plethora of in vivo tools which allow studies with a level of
morphological detail and at a temporal scale never achieved
before. Thus it can be anticipated that these improvements,
both technical and in availability of in vivo models with
improved capabilities will lead to new discoveries at a much
higher pace than before. Information on the availability of
mutant and transgenic zebrafish lines can be found at the
European Zebrafish Resource Center (EZRC; http://
www.ezrc.kit.edu) or at the Zebrafish International Resource
Center (ZIRC; http://zebrafish.org).
Fish systems to discover osteogenic drugs
Given the important advances in technology, public in general as well as public and private decision makers expect that
these should accelerate the discovery of novel treatments,
pressing those involved in biomedical research as well as
pharma and biotech companies, to bring new drugs into
the market. Thus, economical as well as political pressures
require more and more the use of high throughput methods
to achieve the expected results. Given the advantages
highlighted above, zebrafish and medaka stand up among
other fish as models of choice to be used in biomedical
research, in particular for this quest for novel drugs suitable
to be used in bone therapeutics. Its importance can be also
measured by the growing number of articles focusing on this
issue in the last decade [10,22–26]. Here we describe available
systems used in screenings for osteogenic and mineralogenic
drugs. The success of these approaches confirms that fish, in
particular the cellular boned zebrafish, is an excellent model
for discovering therapeutic molecules for human skeletal
disorders (Fig. 1). Accordingly, Table 3 presents a number
of relevant studies which used zebrafish as an in vivo model to
uncover, following screening of various panels of drugs,
novel osteogenic and/or mineralogenic drugs, or drugs capable of reverting pathological conditions, thus proving once
more the importance of this model organism for discovering
therapeutic molecules of interest for human skeletal disorders. For example, the recent screening of 320 compounds in
the zebrafish mutant Snca1 led to the identification of clemizole as a potential treatment for Dravet syndrome [27]; in
another study using zebrafish, deferoxamine was found to
effectively counteract iron-overload-induced inhibition of
osteogenesis [28]. Similarly, zebrafish has also been used as
e4
Vol. xxx, No. xx 2014
a choice model to unveil the effects of pollutants and other
toxic molecules on skeletogenesis, with high relevance for
environmental monitoring and ecotoxicology.
Skeletogenesis in fish larvae
Looking at the effect of a molecule in the whole-organism is a
preferable approach since it not only allows the identification
of potential pitfalls but also the determination of therapeutic
activity, range of action and general toxicity. Fish, in particular zebrafish, embryos/larvae are small, transparent and
available in large quantities, and thus can be easily accommodated in 96-well plates for high-throughput screening of
molecule libraries. In zebrafish, the onset of bone formation
and mineralization occurs as early as 2 day post-fertilization
(dpf) and is first evident at 3 dpf in the cleithrum then in the
branchial and cranial skeletons [29]. Furthermore, it can be
assessed easily through whole-mount bone-specific staining
due to the transparency of the larvae at these early stages. The
fast development of the fish skeleton allows for short duration assays (few days), something difficult in traditional
mammalian models. Larvae are initially grown in petri dishes
then arrayed in 96-well plates at a density up to five fish per
well. Molecules are added directly to the liquid medium in
which the embryos/larvae develop. In wild-type strains, ossification is detected through alizarin red S staining in 6–11 dpf
zebrafish larvae [30] and quantified from the area and intensity of staining determined from color analysis of ventral
pictures of the head skeleton [31] (Fig. 1b). Zebrafish transgenic lines using fluorescent reporters highlighting particular
structures in the skeleton can also be used to observe specific
bone elements/cells and quantify their area/number without
the need for euthanasia. For example, Tg(oc2:GFP; osx:mCherry) transgenic zebrafish line (Fig. 1c), where GFP and
mCherry production is under the control of osteocalcin 2
and osterix promoters, respectively, can be used to determine
the size of the operculum or the number of pre- versus mature
osteoblasts. Similarly, col10a1:nlGFP transgenic zebrafish
line, where GFP production is under the control of collagen
10 promoter, a marker of early osteoblastic precursors, can be
used to assess events preceding mineralization in cranial and
axial skeleton [21]. In addition to its common use as a
colorant in bone research, Alizarin red S is also used as a
fluorochrome label of mineralized tissues, often as a complement to fluorescent reporters in transgenic fish ([32]; Bensimon-Brito et al., in preparation). In all cases, osteogenic/
osteotoxic effects are determined from the comparative analysis of treated versus control fish.
Osteogenesis in regenerating teleost fish fin
The caudal fin of teleost fish is remarkably simple, accessible
and can be restored upon amputation or damage. Because of
these characteristics, it has logically become an excellent
system for investigating underlying mechanisms of epimorphic
www.drugdiscoverytoday.com
Please cite this article in press as: Laizé V, et al. Fish: a suitable system to model human bone disorders and discover drugs with osteogenic or osteotoxic activities, Drug Discov
DDMOD-408; No of Pages 9
Vol. xxx, No. xx 2014
Drug Discovery Today: Disease Models | Bone biology in animal models, including testing of bone biomaterials
(a)
Skeletal deformities
Regenerating
fin rays
Developing
operculum
Mineralizing
scales
Mineralogenic vertebraderived cell lines
11 dpf
(b)
Cl
Tg(oc2:GFP; osx:mCherry)
16 dpf
(c)
Op
Operculum
Nc
PT
Ps
Cb5
(d)
(e)
Lepido
-trichia
(f)
AR-S
stained
nodules
5 dpa
Drug Discovery Today: Disease Models
Figure 1. (a) Fish systems used to study/screen molecules for osteogenic effects. (b) Ventral view of whole-mount alizarin red S (AR-S) – alcian blue (AB)
stained zebrafish larvae at 11 days post-fertilization (dpf). Calcified structures appear in red; area and intensity of staining is determined for the notochord
(Nc), operculum (Op), parasphenoid (Ps) cleithrum (Cl), ceratobranchial 5 (Cb5) and pharyngeal teeth (PT). (c) Lateral view of Tg(oc2:GFP; osx:mCherry)
transgenic zebrafish at 16 dpf. Dotted white line indicates operculum area. (d) Lateral view of AR-S stained regenerating caudal fin of a juvenile zebrafish. Black
triangle indicates the amputation plan; Dotted black line indicates regenerated area; Solid black line indicates area of new bone formation. (e) AR-S-stained
elasmoid scale from the dorsal region of a juvenile gilthead seabream (Sparus aurata L.). (f) AR-S stained mineral nodules deposited within the extracellular
matrix of gilthead seabream VSa16 cell line (osteoblast-like cells).
www.drugdiscoverytoday.com
e5
Please cite this article in press as: Laizé V, et al. Fish: a suitable system to model human bone disorders and discover drugs with osteogenic or osteotoxic activities, Drug Discov
DDMOD-408; No of Pages 9
Drug Discovery Today: Disease Models | Bone biology in animal models, including testing of bone biomaterials
Vol. xxx, No. xx 2014
Table 3. Examples of fish systems to discover molecules affecting skeleton and bone formation
Molecules
Fish systems
Skeletal and bone effects
References
2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD)
17b-Estradiol
3-Methylcholanthrene
Developing medaka
Cultured goldfish scales
Developing zebrafish
Regenerating zebrafish
Cultured gilthead seabream bone cells
Cultured goldfish scales
Cultured gilthead seabream bone cells
Cultured goldfish scales
Developing zebrafish
Impaired chondro- and osteogenesis
Increased osteoclastic activity
Increased rate of skeletal deformities
Reduced de novo bone formation
Reduced ECM mineralization
Suppressed osteoclastic activity
Reduced ECM mineralization
Suppressed osteoclastic and osteoblastic activites
Craniofacial defects
[87]
[58]
[36]
Developing zebrafish
Iron-overload zebrafish
Developing zebrafish
Developing zebrafish
Increased bone formation
Rescued iron-induced osteoporosis
Bone loss
Reduced bone mineralization
[31]
[28]
[31]
[89]
Calcitonin
Vanadium
Bisphenol A
Ethyl tert butyl ether (ETBE)
Tertiary amyl methyl ether (TAME)
Vitamin D3 analogs
Deferoxamine
Parathyroid hormone (PTH)
Dorsomorphin
regeneration, that is the complete reformation of missing tissues
[33–35]. When caudal fin tissues are amputated, a thick wound
epidermis is formed at the amputation plane, then a mass of
undifferentiated mesenchymal cells, called blastema, proliferate
beneath the wound epidermis and will later differentiate into
the various cell types necessary to the faithful restoration of
missing fin structures. Full regeneration is usually achieved after
10–15 days depending on the fish species, the level of amputation and culture conditions. Although zebrafish are raised at
28 8C, assays of fin regeneration are typically performed at
33 8C to speed up regeneration process, which is well advanced
after only 5 days. Fin regeneration and de novo bone mineralization is determined after alizarin red staining, imaging and
morphometric analysis of regenerated areas (Fig. 1d; see brief
protocol in [36])
Mineralogenesis in cell and scale cultures
In addition and as a complement to in vivo studies, mineralogenic cell lines developed from several fish species [37–39]
can be used to study more in depth mechanisms affecting
bone and cartilage cell function and metabolism and the
molecular pathways involved in various processes such as
extracellular matrix mineralization [40–46] or the mineralogenic effect of various molecules, for example retinoic acid
[47,48], polyunsaturated fatty acids [49], vanadate [46,50–52]
and polycyclic aromatic hydrocarbon [36]. In vitro mineralization is easily detected and quantified through the alizarin
red S staining of hydroxyapatite-like crystals deposited within
the extracellular matrix of fish bone-derived cell lines upon
exposure to a mineralogenic cocktail ([37]; Fig. 1f).
While cell cultures have been used to study mechanisms of
extracellular matrix mineralization, they have limited value
in the study of cell–cell and cell–matrix interactions. Elasmoid scales of teleost fish (Fig. 1e) are dermal bone elements
that develop late (30 dpf in zebrafish) and serve as a reservoir
of calcium [53,54]. They are small, easily accessible, abundant
e6
[58]
[52]
[57]
[88]
(hundreds of highly similar scales per fish), transparent and
have the ability to quickly regenerate (4 weeks in zebrafish),
when lost or removed [25,55]. As for regenerating fin rays,
scales have been recently used in biomedical studies aiming
at the better understanding of mechanism of bone regeneration [25,55] but also as an in vivo disease model for osteoporosis studies [56]. The possibility of culturing fish scales in
vitro as a bone unit, where osteoclasts and osteoblasts cohabit
on both sides of the mineralized matrix and interact in a way
resembling in vivo conditions, has allowed their use to assess
effects of anti/pro-osteogenic molecules [57,58] and as a
valuable ex vivo system to discover new drugs affecting bone
formation and/or resorption.
Conclusions
Through the availability of mutant and transgenic lines, fish
has become an attractive model system to study bone disorders. Furthermore, fish provide unique insights into underlying molecular mechanisms that cannot be gained in
traditional mammalian systems due to shortcomings, morphological constrains and technical limitations. Fish are also
cost-effective models with the potential to identify, in a
shorter time, novel drugs to treat bone disorders as well as
screening for osteotoxic molecules. Finding new treatments
for human diseases is challenging and future works should
aim at developing novel fish systems capable of modeling
more bone disorders and at automating screening processes.
Conflict of interest
The authors have no conflict of interest to declare.
Acknowledgements
This work was supported by the Portuguese Funding Agency
for Science and Technology (FCT) through AQUATOX
(PTDC/MAR/112992/2009) research project, by the Atlantic
Area Transnational Cooperation Programme of the European
www.drugdiscoverytoday.com
Please cite this article in press as: Laizé V, et al. Fish: a suitable system to model human bone disorders and discover drugs with osteogenic or osteotoxic activities, Drug Discov
DDMOD-408; No of Pages 9
Vol. xxx, No. xx 2014
Drug Discovery Today: Disease Models | Bone biology in animal models, including testing of bone biomaterials
Community through MARMED (2011-1/164) project, and by
the National Strategic Reference Framework (QREN I&DT)
through ZEBRAFEEDS (QREN 23000) project.
[22]
[23]
References
[1] Hall BK. Bones and cartilage: developmental and evolutionary skeletal
biology. Academic Press; 2005.
[2] Javidan Y, Schilling TF. Development of cartilage and bone. Methods Cell
Biol 2004;76:415–36.
[3] Apschner A, Schulte-Merker S, Witten PE. Not all bones are created equal –
using zebrafish and other teleost species in osteogenesis research. Methods
Cell Biol 2011;105:239–55.
[4] Hall BK, Witten PE. Plasticity of and transitions between skeletal tissues in
vertebrate evolution and development. In: Anderson JS, Sues H-DD.,
editors. Major transitions in vertebrate evolution. Indiana University
Press; 2007. p. 13–56.
[5] Witten PE, Huysseune A. A comparative view on mechanisms and
functions of skeletal remodelling in teleost fish, with special emphasis on
osteoclasts and their function. Biol Rev Camb Philos Soc 2009;84:
315–46.
[6] Arratia G, Schultze H-P. Reevaluation of the caudal skeleton of certain
actinopterygian fishes: III. Salmonidae. Homologization of caudal skeletal
structures. J Morphol 1992;249:187–249.
[7] Nordvik K, Kryvi H, Totland GK, Grotmol S. The salmon vertebral body
develops through mineralization of two preformed tissues that are
encompassed by two layers of bone. J Anat 2005;206:103–14.
[8] Benjamin M. The cranial cartilages of teleosts and their classification. J
Anat 1990;169:153–72.
[9] Witten PE, Huysseune A, Hall BK. A practical approach for the
identification of the many cartilaginous tissues in teleost fish. J Appl
Ichthyol 2010;26:257–62.
[10] Lieschke GJ, Currie PD. Animal models of human disease: zebrafish swim
into view. Nat Rev Genet 2007;8:353–67.
[11] Howe K, Clark MD, Torroja CF, Torrance J, Berthelot C, Muffato M, et al.
The zebrafish reference genome sequence and its relationship to the
human genome. Nature 2013;496:498–503.
[12] Langheinrich U. Zebrafish: a new model on the pharmaceutical catwalk.
Bioessays 2003;25:904–12.
[13] Spoorendonk KM, Hammond CL, Huitema LFA, Vanoevelen J, SchulteMerker S. Zebrafish as a unique model system in bone research: the power
of genetics and in vivo imaging. J Appl Ichthyol 2010;26:219–24.
[14] Santoriello C, Zon LI. Hooked! Modeling human disease in zebrafish. J
Clin Invest 2012;122:2337–43.
[15] Hosen MJ, Vanakker OM, Willaert A, Huysseune A, Coucke P, De Paepe A.
Zebrafish models for ectopic mineralization disorders: practical issues
from morpholino design to post-injection observations. Front Genet
2013;4:74.
[16] Huitema LFA, Apschner A, Logister I, Spoorendonk KM, Bussmann J,
Hammond CL, et al. Entpd5 is essential for skeletal mineralization and
regulates phosphate homeostasis in zebrafish. Proc Natl Acad Sci U S A
2012;109:21372–77.
[17] DeLaurier A, Nakamura Y, Braasch I, Khanna V, Kato H, Wakitani S, et al.
Histone deacetylase-4 is required during early cranial neural crest
development for generation of the zebrafish palatal skeleton. BMC Dev
Biol 2012;12:16.
[18] Hammond CL, Moro E. Using transgenic reporters to visualize bone and
cartilage signaling during development in vivo. Front Endocrinol
(Lausanne) 2012;3:91.
[19] Knopf F, Hammond C, Chekuru A, Kurth T, Hans S, Weber CW, et al. Bone
regenerates via dedifferentiation of osteoblasts in the zebrafish fin. Dev
Cell 2011;20:713–24.
[20] Nichols JT, Pan L, Moens CB, Kimmel CB. Barx1 represses joints and
promotes cartilage in the craniofacial skeleton. Development
2013;140:2765–75.
[21] Renn J, Büttner A, To TT, Chan SJH, Winkler C. A col10a1:nlGFP
transgenic line displays putative osteoblast precursors at the
[24]
[25]
[26]
[27]
[28]
[29]
[30]
[31]
[32]
[33]
[34]
[35]
[36]
[37]
[38]
[39]
[40]
[41]
[42]
medaka notochordal sheath prior to mineralization. Dev Biol
2013;381:134–43.
Brittijn SA, Duivesteijn SJ, Belmamoune M, Bertens LFM, Bitter W, de
Bruijn JD, et al. Zebrafish development and regeneration: new tools for
biomedical research. Int J Dev Biol 2009;53:835–50.
Fleming A. Zebrafish as an alternative model organism for disease
modelling and drug discovery: implications for the 3Rs [WWW
Document]; 2007, http://www.nc3rs.org.uk/news.asp?id=421.
Löhr H, Hammerschmidt M. Zebrafish in endocrine systems: recent
advances and implications for human disease. Annu Rev Physiol
2011;73:183–211.
Metz JR, de Vrieze E, Lock E-J, Schulten IE, Flik G. Elasmoid scales of
fishes as model in biomedical bone research. J Appl Ichthyol
2012;28:382–7.
Renn J, Winkler C, Schartl M, Fischer R, Goerlich R. Zebrafish and medaka
as models for bone research including implications regarding spacerelated issues. Protoplasma 2006;229:209–14.
Baraban SC, Dinday MT, Hortopan GA. Drug screening in Scn1a zebrafish
mutant identifies clemizole as a potential Dravet syndrome treatment.
Nat Commun 2013;4:2410.
Chen B, Yan Y-L, Liu C, Bo L, Li G-F, Wang H, et al. Therapeutic effect of
deferoxamine on iron overload-induced inhibition of osteogenesis in a
zebrafish model. Calcif Tissue Int 2014;94:353–60.
Gavaia PJ, Simes DC, Ortiz-Delgado JB, Viegas CSB, Pinto JP, Kelsh RN,
et al. Osteocalcin and matrix Gla protein in zebrafish (Danio rerio) and
Senegal sole (Solea senegalensis): comparative gene and protein expression
during larval development through adulthood. Gene Expr Patterns
2006;6:637–52.
Walker MB, Kimmel CB. A two-color acid-free cartilage and bone stain for
zebrafish larvae. Biotech Histochem 2007;82:23–8.
Fleming A, Sato M, Goldsmith P. High-throughput in vivo screening for
bone anabolic compounds with zebrafish. J Biomol Screen 2005;10:823–
31.
Mackay EW, Apschner A, Schulte-Merker S. A bone to pick with zebrafish.
Bonekey Rep 2013;2:445.
Akimenko M-A, Smith A. Paired fin repair and regeneration. In: Hall BK,
editor. Fins into limbs: evolution, development, and transformation. The
University of Chicago Press; 2007. p. 152–62.
Nakatani Y, Kawakami A, Kudo A. Cellular and molecular processes of
regeneration, with special emphasis on fish fins. Dev Growth Differ
2007;49:145–54.
Yoshinari N, Kawakami A. Mature and juvenile tissue models of
regeneration in small fish species. Biol Bull 2011;221:62–78.
Laizé V, Gavaia PJ, Viegas MN, Caria J, Luis N. Osteotoxicity of 3methylcholanthrene in fish. Aquat Procedia 2014 [in press].
Pombinho AR, Laizé V, Molha DM, Marques SMP, Cancela ML.
Development of two bone-derived cell lines from the marine teleost Sparus
aurata; evidence for extracellular matrix mineralization and cell-typespecific expression of matrix Gla protein and osteocalcin. Cell Tissue Res
2004;315:393–406.
Rafael MS, Marques CL, Parameswaran V, Cancela ML, Laizé V. Fish bonederived cell lines: an alternative in vitro cell system to study bone biology.
J Appl Ichthyol 2010;26:230–4.
Vijayakumar P, Laizé V, Cardeira J, Trindade M, Cancela ML.
Development of an in vitro cell system from zebrafish suitable to study
bone cell differentiation and extracellular matrix mineralization.
Zebrafish 2013;10:500–9.
Fonseca VG, Rosa J, Laizé V, Gavaia PJ, Cancela ML. Identification of a new
cartilage-specific S100-like protein up-regulated during endo/
perichondral mineralization in gilthead seabream. Gene Expr Patterns
2011;11:448–55.
Fonseca VG, Laizé V, Valente MS, Cancela ML. Identification of an
osteopontin-like protein in fish associated with mineral formation. FEBS J
2007;274:4428–39.
Laizé V, Pombinho AR, Cancela ML. Characterization of Sparus aurata
osteonectin cDNA and in silico analysis of protein conserved features:
evidence for more than one osteonectin in Salmonidae. Biochimie
2005;87:411–20.
www.drugdiscoverytoday.com
e7
Please cite this article in press as: Laizé V, et al. Fish: a suitable system to model human bone disorders and discover drugs with osteogenic or osteotoxic activities, Drug Discov
DDMOD-408; No of Pages 9
Drug Discovery Today: Disease Models | Bone biology in animal models, including testing of bone biomaterials
[43] Rafael MS, Laizé V, Florindo C, Ferraresso S, Bargelloni L, Cancela ML.
Overexpression of four and a half LIM domains protein 2 promotes
epithelial-mesenchymal transition-like phenotype in fish pre-osteoblasts.
Biochimie 2012;94:1128–34.
[44] Rafael MS, Laizé V, Cancela ML. Identification of Sparus aurata bone
morphogenetic protein 2: molecular cloning, gene expression and in
silico analysis of protein conserved features in vertebrates. Bone
2006;39:1373–81.
[45] Tiago DM, Marques CL, Roberto VP, Cancela ML, Laizé V. Mir-20a
regulates in vitro mineralization and BMP signaling pathway by targeting
BMP-2 transcript in fish. Arch Biochem Biophys 2014;543:23–30.
[46] Tiago DM, Laizé V, Bargelloni L, Ferraresso S, Romualdi C, Cancela ML.
Global analysis of gene expression in mineralizing fish vertebra-derived
cell lines: new insights into anti-mineralogenic effect of vanadate. BMC
Genomics 2011;12:310.
[47] Conceição N, Laizé V, Simões B, Pombinho AR, Cancela ML. Retinoic acid
is a negative regulator of matrix Gla protein gene expression in teleost fish
Sparus aurata. Biochim Biophys Acta 2008;1779:28–39.
[48] Fernández I, Tiago DM, Laizé V, Cancela ML, Gisbert E. Retinoic acid
differentially affects in vitro proliferation, differentiation and
mineralization of two fish bone-derived cell lines: different gene
expression of nuclear receptors and ECM proteins. J Steroid Biochem Mol
Biol 2014;140:34–43.
[49] Viegas MN, Dias J, Cancela ML, Laizé V. Polyunsaturated fatty acids
regulate cell proliferation, extracellular matrix mineralization and gene
expression in a gilthead seabream skeletal cell line. J Appl Ichthyol
2012;28:427–32.
[50] Tiago DM, Cancela ML, Laizé V. Proliferative and mineralogenic effects of
insulin, IGF-1, and vanadate in fish osteoblast-like cells. J Bone Miner
Metab 2011;29:377–82.
[51] Tiago DM, Cancela ML, Aureliano M, Laizé V. Vanadate proliferative and
anti-mineralogenic effects are mediated by MAPK and PI-3K/Ras/Erk
pathways in a fish chondrocyte cell line. FEBS Lett 2008;582:
1381–5.
[52] Tiago DM, Laizé V, Cancela ML, Aureliano M. Impairment of
mineralization by metavanadate and decavanadate solutions in a fish
bone-derived cell line. Cell Biol Toxicol 2008;24:253–63.
[53] Carragher JF, Sumpter JP. The mobilization of calcium from calcified
tissues of rainbow trout (Oncorhynchus mykiss) induced to synthesize
vitellogenin. Comp Biochem Physiol Part A Physiol 1991;99:
169–72.
[54] Mugiya Y, Watabe N. Studies on fish scale formation and resorption II:
effect of estradiol on calcium homeostasis and skeletal tissue resorption in
the goldfish, Carassius auratus, and the killifish, Fundulus heteroclitus. Comp
Biochem Physiol Part A Physiol 1977;57:197–202.
[55] De Vrieze E, Sharif F, Metz JR, Flik G, Richardson MK. Matrix
metalloproteinases in osteoclasts of ontogenetic and regenerating
zebrafish scales. Bone 2011;48:704–12.
[56] De Vrieze E, van Kessel MAHJ, Peters HM, Spanings FAT, Flik G, Metz JR.
Prednisolone induces osteoporosis-like phenotype in regenerating
zebrafish scales. Osteoporos Int 2014;25:567–78.
[57] Suzuki N, Hattori A. Bisphenol A suppresses osteoclastic and
osteoblastic activities in the cultured scales of goldfish. Life Sci
2003;73:2237–47.
[58] Suzuki N, Suzuki T, Kurokawa T. Suppression of osteoclastic activities by
calcitonin in the scales of goldfish (freshwater teleost) and nibbler fish
(seawater teleost). Peptides 2000;21:115–24.
[59] Shahar R, Dean MN. The enigmas of bone without osteocytes. Bonekey
Rep 2013;2:343.
[60] Cao L, Moriishi T, Miyazaki T, Iimura T, Hamagaki M, Nakane A, et al.
Comparative morphology of the osteocyte lacunocanalicular system in
various vertebrates. J Bone Miner Metab 2011;29:662–70.
[61] Totland GK, Fjelldal PG, Kryvi H, Løkka G, Wargelius A, Sagstad A, et al.
Sustained swimming increases the mineral content and osteocyte density
of salmon vertebral bone. J Anat 2011;219:490–501.
[62] Turner CH, Warden SJ, Bellido T, Plotkin LI, Kumar N, Jasiuk I, et al.
Mechanobiology of the skeleton. Sci Signal 2009;2:pt3.
e8
Vol. xxx, No. xx 2014
[63] Witten PE, Hansen A, Hall BK. Features of mono- and multinucleated
bone resorbing cells of the zebrafish Danio rerio and their contribution to
skeletal development, remodeling, and growth. J Morphol 2001;250:197–
207.
[64] You L, Temiyasathit S, Lee P, Kim CH, Tummala P, Yao W, et al. Osteocytes
as mechanosensors in the inhibition of bone resorption due to
mechanical loading. Bone 2008;42:172–9.
[65] Fisher S, Jagadeeswaran P, Halpern ME. Radiographic analysis of zebrafish
skeletal defects. Dev Biol 2003;264:64–76.
[66] Asharani PV, Keupp K, Semler O, Wang W, Li Y, Thiele H, et al. Attenuated
BMP1 function compromises osteogenesis, leading to bone fragility in
humans and zebrafish. Am J Hum Genet 2012;90:661–74.
[67] To TT, Witten PE, Renn J, Bhattacharya D, Huysseune A, Winkler C. Ranklinduced osteoclastogenesis leads to loss of mineralization in a medaka
osteoporosis model. Development 2012;139:141–50.
[68] Barrett R, Chappell C, Quick M, Fleming A. A rapid, high content, in vivo
model of glucocorticoid-induced osteoporosis. Biotechnol J 2006;1:
651–5.
[69] Eames BF, Yan Y-L, Swartz ME, Levic DS, Knapik EW, Postlethwait JH, et al.
Mutations in fam20b and xylt1 reveal that cartilage matrix controls
timing of endochondral ossification by inhibiting chondrocyte
maturation. PLoS Genet 2011;7:e1002246.
[70] Clément A, Wiweger M, von der Hardt S, Rusch MA, Selleck SB, Chien C-B,
et al. Regulation of zebrafish skeletogenesis by ext2/dackel and papst1/
pinscher. PLoS Genet 2008;4:e1000136.
[71] Wiweger MI, Zhao Z, van Merkesteyn RJP, Roehl HH, Hogendoorn PCW.
HSPG-deficient zebrafish uncovers dental aspect of multiple
osteochondromas. PLoS One 2012;7:e29734.
[72] Flanagan-Steet H, Sias C, Steet R. Altered chondrocyte differentiation and
extracellular matrix homeostasis in a zebrafish model for mucolipidosis II.
Am J Pathol 2009;175:2063–75.
[73] Laue K, Pogoda H-M, Daniel PB, van Haeringen A, Alanay Y, von Ameln S,
et al. Craniosynostosis and multiple skeletal anomalies in humans and
zebrafish result from a defect in the localized degradation of retinoic acid.
Am J Hum Genet 2011;89:595–606.
[74] Spoorendonk KM, Peterson-Maduro J, Renn J, Trowe T, Kranenbarg S,
Winkler C, et al. Retinoic acid and Cyp26b1 are critical regulators
of osteogenesis in the axial skeleton. Development 2008;135:
3765–74.
[75] Bauer H, Lele Z, Rauch GJ, Geisler R, Hammerschmidt M. The type I serine/
threonine kinase receptor Alk8/Lost-a-fin is required for Bmp2b/7 signal
transduction during dorsoventral patterning of the zebrafish embryo.
Development 2001;128:849–58.
[76] Gorman KF, Tredwell SJ, Breden F. The mutant guppy syndrome
curveback as a model for human heritable spinal curvature. Spine (Phila
Pa 1976) 2007;32:735–41.
[77] Schauerte HE, van Eeden FJM, Fricke C, Odenthal J, Strähle U, Haffter P.
Sonic hedgehog is not required for the induction of medial floor plate cells
in the zebrafish. Development 1998;125:2983–93.
[78] Yan Y, Miller CT, Nissen RM, Singer A, Liu D, Kirn A, et al. A zebrafish sox9
gene required for cartilage morphogenesis. Development
2002;129:5065–79.
[79] Nissen RM, Amsterdam A, Hopkins N. A zebrafish screen for craniofacial
mutants identifies wdr68 as a highly conserved gene required for
endothelin-1 expression. BMC Dev Biol 2006;6:28.
[80] Piotrowski T, Ahn D, Schilling TF, Nair S, Ruvinsky I, Geisler R, et al.
The zebrafish van Gogh mutation disrupts tbx1, which is involved in
the DiGeorge deletion syndrome in humans. Development
2003;130:5043–52.
[81] Lang MR, Lapierre LA, Frotscher M, Goldenring JR, Knapik EW. Secretory
COPII coat component Sec23a is essential for craniofacial chondrocyte
maturation. Nat Genet 2006;38:1198–203.
[82] Melville DB, Montero-Balaguer M, Levic DS, Bradley K, Smith JR,
Hatzopoulos AK, et al. The feelgood mutation in zebrafish
dysregulates COPII-dependent secretion of select extracellular
matrix proteins in skeletal morphogenesis. Dis Model Mech
2011;4:763–76.
www.drugdiscoverytoday.com
Please cite this article in press as: Laizé V, et al. Fish: a suitable system to model human bone disorders and discover drugs with osteogenic or osteotoxic activities, Drug Discov
DDMOD-408; No of Pages 9
Vol. xxx, No. xx 2014
Drug Discovery Today: Disease Models | Bone biology in animal models, including testing of bone biomaterials
[83] Eames BF, Singer A, Smith GA, Wood ZA, Yan Y-L, He X, et al. UDP xylose
synthase 1 is required for morphogenesis and histogenesis of the
craniofacial skeleton. Dev Biol 2010;341:400–15.
[84] Sarmah S, Barrallo-Gimeno A, Melville DB, Topczewski J, Solnica-Krezel L,
Knapik EW. Sec24D-dependent transport of extracellular matrix proteins
is required for zebrafish skeletal morphogenesis. PLoS ONE
2010;5:e10367.
[85] Xi Y, Chen D, Sun L, Li Y, Li L. Characterization of zebrafish mutants with
defects in bone calcification during development. Biochem Biophys Res
Commun 2013;440:132–6.
[86] Green J, Taylor JJ, Hindes A, Johnson SL, Goldsmith MI. A gain of function
mutation causing skeletal overgrowth in the rapunzel mutant. Dev Biol
2009;334:224–34.
[87] Dong W, Hinton DE, Kullman SW. TCDD disrupts hypural
skeletogenesis during medaka embryonic development. Toxicol Sci
2012;125:91–104.
[88] Bonventre JA, White LA, Cooper KR. Craniofacial abnormalities and
altered wnt and mmp mRNA expression in zebrafish embryos exposed
to gasoline oxygenates ETBE and TAME. Aquat Toxicol 2012;120–
121:45–53.
[89] Yu PB, Hong CC, Sachidanandan C, Babitt JL, Deng DY, Hoyng SA, et al.
Dorsomorphin inhibits BMP signals required for embryogenesis and iron
metabolism. Nat Chem Biol 2008;4:33–41.
[90] Neuhauss SCF, Solnica-Krezel L, Schier AF, Zwartkruis F, Stemple DL,
Malicki J, et al. Mutations affecting craniofacial development in zebrafish.
Development 1996;123:357–67.
www.drugdiscoverytoday.com
e9
Please cite this article in press as: Laizé V, et al. Fish: a suitable system to model human bone disorders and discover drugs with osteogenic or osteotoxic activities, Drug Discov