European Journal of Neuroscience, Vol. 18, pp. 1377±1386, 2003
ß Federation of European Neuroscience Societies
Retinal function and morphology in two zebra®sh models
of oculo-renal syndromes
Ronja Bahadori,1, Matthias Huber,2,3, Oliver Rinner,1 Mathias W. Seeliger,4 Silke Geiger-Rudolph,5 Robert Geisler5
and Stephan C.F. Neuhauss1
1
Swiss Federal Institute of Technology (ETH) Zurich, Department of Biology, and the Brain Research Institute, University of Zurich,
Winterthurerstrasse 190, CH-8057 Zurich, Switzerland
2
Max-Planck Institute for Developmental Biology, Department of Physical Biology,
3
Department I, Tubingen University Eye Hospital
4
Retinal Electrodiagnostics Research Group, Department II, Tubingen University Eye Hospital
5
Max-Planck Institute for Developmental Biology, Department of Genetics, Tubingen, Germany
Keywords: electroretinogram, genetic mapping, mutation, optokinetic response, photoreceptor, retinal dystrophy, Senior±Loken
syndrome
Abstract
We characterized visual system defects in two recessive zebra®sh mutants oval and elipsa. These mutants share the syndromic
phenotype of outer retinal dystrophy in conjunction with cystic renal disorder. We tested the function of the larval visual system in a
behavioural assay, eliciting optokinetic eye movements by high-contrast motion stimulation while recording eye movements in parallel.
Visual stimulation did not elicit eye movements in mutant larvae, while spontaneous eye movements could be observed. The retina
proved to be unresponsive to light using electroretinography, indicative of a defect in the outer retina. Histological analysis of mutant
retinas revealed progressive degeneration of photoreceptors, initiated in central retinal locations and spreading to more peripheral
regions with increasing age. The inner retina remains unaffected by the mutation. Photoreceptors display cell type-speci®c
immunoreactivity prior to apoptotic cell death, arguing for a dystrophic defect. Genomic mapping employing simple sequence-length
polymorphisms located both mutations on different regions of zebra®sh linkage group 9. These mutants may serve as accessible
animal models of human outer retinal dystrophies, including oculo-renal diseases, and show the general usefulness of a behavioural
genetic approach to study visual system development in the model vertebrate zebra®sh.
Introduction
The zebra®sh (Danio rerio) has become an attractive model organism to study the vertebrate visual system, including its diseases
(Brockerhoff, 2001; Goldsmith, 2001). Zebra®sh in general are amenable to large-scale genetic screens, because they can be maintained and
bred easily and embryonic development is extraordinarily rapid (Driever
et al., 1996; Haffter et al., 1996). This is particularly true for the visual
system. Photoreceptor outer segments appear at about 55 h post-fertilization (Schmitt & Dowling, 1999), supporting rudimentary visual
function by 3 days post-fertilization (dpf) (Easter & Nicola, 1996). By
5 dpf, the visual system is functional as shown by retinal morphology,
electroretinography and visually mediated behavioural responses
(reviewed in Bilotta & Saszik, 2001; Neuhauss, 2003).
These advantageous features have been exploited in forward genetic
approaches to study the vertebrate visual system by screening chemically mutagenized strains for visual defects.
In such screens, a number of visually impaired mutant strains have
been isolated, using simple re¯exive behaviours depending on
vision, such as the optokinetic response and the optomotor response
Correspondence: Dr S. C. F. Neuhauss, as above.
E-mail: [email protected]
R.B. and M.H. contributed equally to this work.
Received 13 March 2003, revised 24 June 2003, accepted 2 July 2003
doi:10.1046/j.1460-9568.2003.02863.x
(Brockerhoff et al., 1995, 1997, 2003; Neuhauss et al., 1999; Allwardt
et al., 2001). Similar to many human diseases, the heritable blindness
in most isolated mutants is part of a syndrome. For instance, heritable
loss of vision is often accompanied by defects in body pigmentation
(Neuhauss et al., 1999).
Two mutant strains [elipsa (eli) and oval (ovl)] were identi®ed by
virtue of absence of optokinetic behaviour following motion stimulation. Heritable blindness in these mutants is accompanied by a bend
body shape, oval eye shape and pronephric cysts, the original criteria
for isolation of these strains (Brand et al., 1996; Malicki et al., 1996;
Drummond et al., 1998). Alleles of both mutants were shown to lack
photoreceptors at late larval stages (Malicki et al., 1996; Neuhauss
et al., 1999; Doerre & Malicki, 2002).
Here we present a histological and physiological evaluation of the
visual system in these mutants. Both mutants were indistinguishable in
all our phenotypic assays, hence only data for the elipsa mutant are
shown. Behavioural and electroretinographic analysis revealed a complete lack of response to light. Cell death of photoreceptors is
progressive and spares the inner retina. Cell death is executed via
apoptosis, as revealed by a DNA fragmentation assay. Photoreceptors
in both mutants were found to be affected following expression of cell
type-speci®c markers, with cells in the central retina being more and
earlier affected than cells in the periphery. In a genome scan with
simple sequence-length polymorphism markers, we identi®ed the
genomic location of both loci.
1378 R. Bahadori et al.
Materials and methods
All experiments were in compliance with the ARVO Statement for the
use of Animals in Ophthalmic and Vision Research.
Fish maintenance and breeding
Fish were raised and crossed as previously described (Mullins et al.,
1994). Outcrossed sibling pairs were set up to identify heterozygous
carriers. Clutches of these identi®cation crosses were used for phenotypic analysis. Embryos were raised at 28 8C in E3 medium (in mM:
NaCl, 5; KCl, 0.17; CaCl2, 0.33; MgSO4, 0.33) and staged according to
development in dpf. Complementation was tested by crossing heterozygous parents of oval and elipsa and assessing their offspring. The
two mutants were found to be non-allelic by complementation assay
and unlinked by genomic mapping, consistent with Doerre & Malicki
(2002). Alleles used in this study were ovaltz288 and elipsatp49d.
Optokinetic assay
Single 5-dpf-old larvae were placed dorsal side up in the centre of a
Petri dish (diameter 3.5 cm) containing 3% methylcellulose in E3
medium to suppress whole-body movement. The dish was placed
inside a rotating drum (5 cm diameter) ®tted with black and white
stripes (25 black stripes, 11.58 width) moving with a speed of 228/s.
The drum was illuminated by white light from below using a Schott KL
1500 lamp. Right eye movements were recorded on a personal
computer using a CCD b/w video-camera at 5 frames per second.
The recorded movies were analysed using the NIH Image application
(public domain: http://rsb.info.nih.gov/nih-image/) and custom-made
analysis software programmed in Labview. The behaviour of at least
10 single larvae was measured for all genotypes.
Electroretinographic (ERG) recordings
For ERG recordings, dark-adapted (>2 h) larvae were anaesthetized
and paralysed in 0.02% buffered MESAB (3-aminobenzoic acid
methyl ester; Sigma) and 0.8 mg/mL Esmeron (Organon Teknika,
Eppelheim, Germany) solution. Sedated larvae (n ˆ 8 for eli, n ˆ 5
for ovl, n > 8 for each sibling group) were placed on a wet paper
towel sitting on a platinum wire serving as reference electrode under
dim red light. A glass microelectrode (approximate tip diameter of
20 mm) ®lled with E3 medium was placed on the centre of the cornea
by means of a micromanipulator (M330R, World Precision Instruments, Sarasota, USA). The microelectrode holder was directly
connected to a voltage follower (VF2, World Precision Instruments),
which in turn was interfaced with an input channel of the ERG
ampli®er. Larvae were placed inside a Ganzfeld bowl of a commercially available ERG set-up (Toennies Multiliner Vision, Jaeger/
Toennies, HoÈchberg, Germany). Recordings followed the protocol
described earlier (Seeliger et al., 2002), based on the human ERG
standard by the International Society for Clinical Electrophysiology
of Vision (Marmor et al., 2003). Shown here are the scotopic single
¯ash recordings at light intensities increasing from 1 mcds/m2 to
25 cds/m2. Ten responses per intensity were averaged, with an
interstimulus interval of 5 s (1, 3, 10, 30, 100 mds/m2) or 17 s
(0.3, 1, 3, 10, 25 cds/m2). In some cases, an additional bright ¯ash
of 100 cds/m2 was used.
Histology
Paraformaldehyde-®xed larvae (4% paraformaldehyde in 0.2 M phosphate buffer, pH 7.4 for 1 h at room temperature) were dehydrated in a
graded series of ethanol±water mixtures, then incubated in 1 : 1 and
1 : 3 ethanol and Technovit 7100 basic solution (Kulzer, Germany) for
1 h, respectively. After overnight in®ltration in Technovit 7100 basic
solution, larvae were positioned in Technovit 7100 polymerization
medium overnight at room temperature.
Microtome sections (3 mm) were prepared and mounted on SuperFrost Plus slides (Menzel-GlaÈser), air-dried at 60 8C, stained with
toluidine blue solution (0.1% in aqua dest.), overlaid with Entellan
(Merck, Darmstadt, Germany) and coverslipped.
Some larvae used for histology were raised in 0.2 mM phenylthiourea (Sigma) to inhibit pigment formation. Sections of at least four
eyes, typically more than 10 eyes, were sectioned at each developmental stage for each genotype.
Immunohistochemistry
Fixed larvae were cryoprotected in 30% sucrose for at least 2 h. Whole
larvae were embedded in Cryomatrix (Jung-Leica; Tissue Freezing
Medium), rapidly frozen in liquid N2; 10-mm-thick sections were cut at
20 8C, mounted on SuperFrost Plus slides and ®xed in ice-cold
acetone for 1 min before storing at 20 8C. Before further use, slides
were thawed, hot-air-dried and washed three times in phosphatebuffered saline (50 mM), pH 7.4, and treated with 20% normal goat
serum, 2% bovine serum albumin in phosphate-buffered saline/0.3%
Triton X-100 (PBST) for 1 h. Sections were then incubated for 2 h in
primary antibodies in PBST at 4 8C. Fret43 (1 : 100; University of
Oregon Stock Center), anti-rhodopsin (1 : 500; Biodesign International, Saco, USA), mouse anti-glutamine synthetase (1 : 700; Chemicon International, Temecula, USA), zn 8 (1 : 500; University of
Oregon Stockcentre) and rabbit anti-synaptosomal-associated protein
of 25 kDa (1 : 300, SNAP-25, StressGen, San Diego, CA, USA) were
used as primary antibody. After washing three times in PBST, sections
were incubated in anti mouse Alexa433 coupled antibody (1 : 500;
Jackson Laboratory) for 1 h, washed three times in PBST, mounted in
glycerol and analysed using a Zeiss Axioscope ¯uorescence microscope.
Cell death detection
The TUNEL (terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labelling) method (Gavrieli et al.,
1992) was used to identify cells that were undergoing apoptosis as
previously described (Biehlmaier et al., 2001). Cryosections were cut
40 mm thick and mounted on SuperFrost Plus slides.
Younger stage (36 h post-fertilization) embryos were stained as
whole mounts with acridine orange (Sigma). Embryos were manually
dechorinated and incubated for 30 min (5 mg/mL E3 medium) and
viewed on a Zeiss Axioscope ¯uorescence microscope after thorough
rinsing.
More than six individual retinas were labelled for each genotype at
the developmental stages analysed (3, 4, 5, 7 dpf). Quanti®cation was
performed by counting apoptotic cells in one section per eye with the
maximal number of apoptotic cells. Signi®cance levels were calculated using a two-tailed paired t-test.
Genomic mapping
Both mutations were induced in the genetic background of the
TuÈbingen strain. In order to obtain informative genetic polymorphism,
heterozygous carriers were outcrossed to the WIK strain and pooled
DNA from F2 homozygous mutants and siblings were analysed using
PCR in a pooled-segregant analysis. Mapping of the two loci was
carried out by genome scanning with 192 SSLP markers as described
(Geisler, 2002). Segregation analysis on single embryos was used to
con®rm linkage group assignation and to obtain a more precise map
position by using additional polymorphic single-length polymorphisms. Map position was calculated using the Kosambi equation, as
described in Geisler (2002).
ß 2003 Federation of European Neuroscience Societies, European Journal of Neuroscience, 18, 1377±1386
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Results
Behavioural analysis
Evoking an optokinetic nystagmus tested the visual response of
zebra®sh larvae. Larvae were placed in a rotating drum ®tted with
black and white stripes. Rotation of the drum in both directions was
found to consistently evoke eye movements in larvae older than 5 dpf
(Clark, 1981; Brockerhoff et al., 1995; Easter & Nicola, 1996, 1997;
Neuhauss et al., 1999). These movements consist of a smooth pursuit
phase in the direction of the rotating drum and a fast saccade to reset
eye position in wild-type larvae. In both mutants no response could be
evoked at a variety of different illumination conditions (Fig. 1). Nevertheless, spontaneous eye movements uncorrelated to visual stimulation
can be recorded, indicating that the neural circuits necessary for eye
movements are unaffected in the mutants. This argues for a defect in
vision rather than in the motor components needed for execution of
optokinetic eye movements.
Furthermore, both mutant larvae have lost the ability to adjust their
body pigmentation to bright light. In wild-type larvae, melanin-containing granules (melanosomes) are rearranged in pigment cells (melanophores) depending on ambient light conditions. When placed on a
dark background, melanosomes are widely distributed throughout the
cell, while in bright light they are concentrated on one spot. In most
blind larvae melanosomes are widely distributed even in bright light,
presumably due to lack of light perception. In eli and ovl homozygous
larvae, melanophores are always in the dark-adapted state, independent of ambient light conditions, consistent with them being unable to
sense light.
Ganzfeld ERG analysis
Because our behavioural assays indicated an inability of the mutant
larvae to respond to visual cues of the environment, we directly
assessed visual function on the retinal level by Ganzfeld electroretinography. The light-evoked ERG response intensity series recorded in
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dark-adapted larvae is shown in Fig. 2. Wild-type larvae between 5 and
7 dpf respond at low light intensities with a small negative de¯ection.
At brighter light levels, ®rst a small, negative a-wave is visible,
followed by a positive b-wave that grows in amplitude with increasing
¯ash intensity (Fig. 2A). Both eli and ovl homozygous larvae do not
show any response to light (Fig. 2B). This observation was con®rmed
by ERG recordings under photopic conditions and with ¯icker stimulation (data not shown). Therefore we conclude that the retinas of these
mutants are not able to perceive light, consistent with a defect in the
outer retina.
Retinal morphology
In order to assess the morphology of the retina, we prepared retina
sections at different stages of retinal maturation. Layering of the
zebra®sh retina is ®rst apparent at about 3 dpf. At this stage, retinas
of mutant larvae cannot be readily distinguished from retinas of their
wild-type siblings (Fig. 3). Starting at 4 dpf, small gaps become
apparent in the outer nuclear layer. By 5 dpf, photoreceptor outer
segments are severely shortened or absent, most pronounced in the
central regions of the retina. Photoreceptor nuclei in the central retina
assume a ¯attened, disorganized appearance without any visible outer
segments. Photoreceptor nuclei in the periphery are arranged normally
and small outer segments can be detected. At 6 dpf, degeneration has
spread. Photoreceptor nuclei are now only visible in the peripheral
retina, while the central outer retina is completely devoid of nuclei.
The inner retina appears to be unaffected. Horizontal cells as characterized by their ¯attened appearance beneath the outer plexiform
layer (OPL), as well as bipolar, amacrine and ganglion cells can be
detected on sections of mutant retinas in similar numbers to wild-types.
In order to ®nd additional indication for an intact inner retina, we used
immunohistochemical stainings against glutamine synthethase, revealing the morphology of Muller glia cells (Peterson et al., 2001). The
Muller cell morphology with elongated cell bodies and processes
spanning the whole inner retina was preserved in the mutant retinas,
Fig. 1. eli mutants do not respond to high-contrast motion stimulation. Eye traces of wild-type (upper trace) and eli mutant (lower trace). Angle of eye gaze is plotted
as a function of time. Spontaneous eye movements are recorded in both mutant and wild-type. After onset of drum rotation (arrow), stimulus-correlated optokinetic
eye movements are apparent in wild-type larvae (upper trace) but absent in mutant larvae, indicating loss of vision in the mutant.
ß 2003 Federation of European Neuroscience Societies, European Journal of Neuroscience, 18, 1377±1386
1380 R. Bahadori et al.
Fig. 2. ERG intensity series of wild-type (A) and eli mutant larvae (B) at 5 dpf recorded under scotopic conditions. Recordings from wild-type larvae show a typical
ERG composed of a-wave and b-wave, with b-wave amplitude increasing with stimulus intensity. (B) No electrical response can be evoked from the eli retina at all
stimulus intensities.
indicative of a largely intact inner retina. Consistent with these
®ndings, immunohistochemical stainings against the ganglion cell
epitope zn8 (recognizing the neurolin protein) and the presynaptic
SNAP-25 protein (synaptosomal-associated protein of 25 kDa)
revealed no defects in the mutant inner retina (data not shown).
The retinas of both mutant eyes develop similarly, whereas small
variations in the timing of disease progression are apparent among
individual larvae homozygous for both mutated genes.
Apoptosis
Standard histology indicates a loss of photoreceptor cells in the mutant
retinas. In order to distinguish the mode of cell death, we probed the
retina for apoptotic cell death. Apoptotic cell death, in contrast to
necrotic cell death, is characterized by a series of well-orchestrated
biochemical steps eventually leading to degradation of nuclear material. This mode of cell death is involved in cell removal during normal
development as well as cell death triggered by genetic diseases,
including human outer retinal dystrophies. The characteristic multiple
DNA strand breaks observed during apoptosis can be used to visualize
apoptotic cells in situ by the TUNEL method (Gavrieli et al., 1992).
During zebra®sh wild-type development, only few cells in the retina
undergo apoptosis (Fig. 4; Biehlmaier et al., 2001). In mutant retinas, a
massive and sharp increase of apoptotic cell death was detected in the
outer nuclear layer during the fourth day of development (homozygous
eli larvae: 13.5 1.9; siblings: 0.7 0.8; homozygous ovl larvae:
10.2 2.2; siblings 0.7 0.3; P < 0.001, n ˆ 6). Consistent with retinal histology, dying cells were mainly con®ned to the outer nuclear
layer. A few cells also die in other retinal layers, presumably as part of
Fig. 3. Retinal histology at different developmental stages. Transverse sections through the retina in wild-type (WT, left row, A, C, E and G) and eli (right row, B, D, F
and H) larvae. (A and B) WT and eli retinas display a laminated retina at 3 days post-fertilization (dpf). (C) At 4 dpf, photoreceptor outer segments are visible in the
WT (arrow in inset). (D) In eli retinas, no long outer segments are apparent and pyknotic nuclei (arrow in inset) and gaps appear in the outer retina. (E and F) At 5 dpf,
the mutant central retina gets more affected, while at 6 dpf (G and H), the central mutant retina is completely devoid of photoreceptors. WT photoreceptors have
elongated nuclei and marked outer segments (inset G), while mutant photoreceptor cells in the transition zone take on a ¯attened stubby appearance with no apparent
outer segments (inset H). Cells in the inner nuclear layer appear to be unaffected in the mutant. Scale bars, 25 mm (A±D); 40 mm (E±H).
ß 2003 Federation of European Neuroscience Societies, European Journal of Neuroscience, 18, 1377±1386
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ß 2003 Federation of European Neuroscience Societies, European Journal of Neuroscience, 18, 1377±1386
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1382 R. Bahadori et al.
Fig. 4. Apoptotic cell death is increased in eli retina. Transverse sections through wild-type (WT, A, C, E and G) and eli (B, D, F and H) retinas stained with the
TUNEL protocol for DNA fragmentation, indicative of apoptosis. A dramatic increase of apoptotic cells in the outer retina of mutant retinas became apparent at 4 days
post-fertilization (dpf) (D), while at later stages only a few cells are left to die. Note also the auto¯uorescence of photoreceptor outer segments in the WT and its
absence in mutant retinas (E±H). Scale bars, 25 mm (A±D); 40 mm (E±H).
ß 2003 Federation of European Neuroscience Societies, European Journal of Neuroscience, 18, 1377±1386
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Fig. 5. Double cones-speci®c labelling reveals vanishing of photoreceptors in the outer retina of eli mutants. Transverse section through wild-type (WT, A, C and E)
and eli (B, D and F) retinas labelled with the Fret43 antibody. Initially double cones are present in eli retinas (B), but vanish over time (D). At 7 days post-fertilization
(dpf), cones are only present in the peripheral ciliary margin (F). Scale bar, 50 mm.
ß 2003 Federation of European Neuroscience Societies, European Journal of Neuroscience, 18, 1377±1386
1384 R. Bahadori et al.
normal development. At earlier stages no signi®cant difference in the
incidence of apoptosis between wild-type and mutant was observed,
both by the TUNEL method and acridine orange staining, another label
for cells undergoing apoptosis. No increase of dying cells was
observed outside of the outer retina. At later stages only a few cells
are observed to undergo apoptosis in the outer retina, as few cells are
left in this retinal layer. Hence, mutations in both the ovl and eli genes
lead to a massive increase in apoptosis in the outer nuclear layer of the
retina, while sparing the inner retina.
Cell differentiation
Having shown that photoreceptor cells undergo apoptosis, we asked if
these cells were differentiated as photoreceptors before undergoing
apoptosis. In order to label cone photoreceptors, we used the monoclonal antibody Fret43, an antibody speci®c for differentiated zebra®sh
double cones (middle and long wavelength cones) (Larison &
Bremiller, 1990). These red and green light-sensitive double cones
are the most prevalent photoreceptor subtypes in early larval retinas
(Larison & Bremiller, 1990). Initially at 3 dpf, a similar number of
cones label in both wild-type and mutant retinas (Fig. 3A and B).
Starting at 4 dpf and continuing to 5 dpf, fewer cells with smaller and
disarrayed outer segments label in the mutant retinas (Fig. 5D). Consistent with histological ®ndings, double cones are only detectable in
the peripheral mutant retina, while the central retina is devoid of any
cone-speci®c staining at 7 dpf.
We saw a similar progressive loss of rod photoreceptors by labelling
these cells with a rod-speci®c antibody directed against rhodopsin
(data not shown).
The progressive loss of photoreceptors in the two mutants affects
differentiated cells by virtue of cell type-speci®c antigenicity, arguing
that death is dystrophic and does at least initially not affect differentiation of these cells.
Genomic localization of ovl and eli
As an entry point into the molecular genetic analysis of the two
mutants, we determined their respective map positions on the zebra®sh
genetic linkage map. Initial mapping employing a pooled segregant
analysis of simple polymorphic length polymorphisms resulted in
linking both mutations to zebra®sh linkage group (LG) 9. Segregation
analysis on single embryos con®rmed this LG assignation and was
used for more precise mapping. Genetic mapping by analysing up to
200 individual meiotic events determined the map position for ovl at
50.2 cM from top of LG 9, between markers z31224 and z6430 on the
MGH zebra®sh map (http://zebra®sh.mgh.harvard.edu/). The position
of eli was determined to be 67.4 cm from the top between markers
z20031 and z4577 on the same LG. Linkage was highly signi®cant
with LOD scores for all tested markers higher than 8, for most markers
higher than 20. Both mutants are curiously located on the same LG but
at distinct locations. Genetic complementation crosses between carriers of both mutations con®rmed the distinct genetic nature of these
two mutants.
Discussion
We have characterized the visual system of two zebra®sh mutants that
affect both photoreceptor and kidney development. Homozygous
larvae for both mutants failed to respond to visual stimuli both in
behavioural assays, as well as in ERG recordings. Histological analysis
revealed a progressive loss of photoreceptors in the outer retina. The
outer retina appears to be unaffected at 3 dpf, while showing a
progressive loss of photoreceptors at subsequent developmental stages,
leading to a complete lack of photoreceptors in the central retina with
only few remaining cells in the periphery. The mode of cell death is
apoptotic as shown by in situ DNA fragmentation assay. Before
undergoing apoptosis, photoreceptor cells express cell type-speci®c
antigens, indicating a loss of differentiated cells. Both mutants were
indistinguishable in their retinal defect. Curiously, both mutants map
to the same genetic LG, but are clearly distinct loci, both by genetic
mapping and classical genetic complementation assay.
In this study we show that eli and ovl share a number of characteristics of heritable outer retinal dystrophies. We interpret the loss of
photoreceptors observed in the mutant as dystrophic cell loss in
contrast to an aplasic defect. This conclusion is based on the expression
of cell type-speci®c markers, such as rhodopsin (rod photoreceptors)
and Fret43 (red/green double cones). Similar results were obtained in
another study using opsin-speci®c antisera, albeit these immunolabellings additionally revealed a different distribution of visual pigments,
likely due to a lack of proper outer segment formation (data not shown;
Doerre & Malicki, 2002). The expression of these markers and also the
initial presence of small outer segments indicate that these cells have
adopted a photoreceptor fate. Nevertheless, the mutant retinas never
show function by virtue of the ERG and visually mediated behaviour,
and clearly do not develop proper photoreceptor outer segments.
Hence, the data are also consistent with a developmental defect,
maybe associated with outer segment formation, rather than a degenerative defect in maintenance (Doerre & Malicki, 2002). Because the
outer nuclear layer and photoreceptor inner segments clearly form, we
deem it likely that the degenerative process is concomitant with outer
segment formation, and not causally linked to cell fate determination.
In this scenario, the extraordinary rapid development of the zebra®sh
retina leads to a temporal overlap of outer segment formation and
photoreceptor degeneration in the mutants. Hence, these two processes
would be unrelated but overlapping in time.
Cell death of photoreceptors can be caused by a number of alterations in photoreceptor biology, both cell autonomous such as defects in
the phototransduction cascade or structural properties of photoreceptor
outer segments, as well as cell non-autonomous, for instance in
retinoid metabolism (Gregory-Evans & Bhattacharya, 1998). The
occurrence of blindness as a syndrome with kidney cysts argues against
photoreceptor biology-speci®c malfunctions. A possible non-autonomous defect would be a lack of epithelial polarization, crucial for both
kidney and visual pathway function. However, such a defect is unlikely
because normal polarization of both kidney and pigment epithelium
was found (data not shown). This conclusion is supported by transplantation experiments by Doerre & Malicki (2002) that indicate that
the genetic defect in both ovl and eli is photoreceptor cell autonomous.
The occurrence of blindness as a syndrome with kidney cysts is
therefore probably due to a separate cell-autonomous defect in kidney
cells.
The two described mutants display a number of properties shared by
human congenital blindness. They are progressive and affect, at least
initially, exclusively the outer retina. In human patients with outer
retinal dystrophy, as well as in animal models of congenital blindness,
the mode of cell death is apoptotic and leaves the inner retina largely
intact. The spatial and temporal progression of apoptosis in these
mutants is reminiscent of human disorders, such as the central to
peripheral wave of photoreceptor destruction observed in macula
dystrophies. Nevertheless this might be more simply explained by
the way the zebra®sh retina proliferates. In both larval and adult teleost
retinas including zebra®sh, cells are continuously proliferating at
the periphery (the ciliary margin), generating all cell types, so that
cells of the central retina are more mature than those in the periphery
(Marcus et al., 1999). Therefore, in the zebra®sh retina an age gradient
exists with developmentally younger cells being more peripherally
ß 2003 Federation of European Neuroscience Societies, European Journal of Neuroscience, 18, 1377±1386
Retinal dystrophy and cystic renal disorder
positioned than older cells located more centrally. An earlier onset of
degeneration in the central retina may therefore be explained as an agerelated phenomenon rather than a position-speci®c effect.
The syndromic occurrence of retinal dystrophies and kidney defects
is also seen in a number of genetic human disorders, such as Bardet±
Biedl and Senior±Loken syndrome. Bardet±Biedl syndrome (BBS) is
characterized by retinal dystrophy and renal malformations, as well as
obesity, polydactyly, learning disorders and hypogenitalism (Katsanis
et al., 2001). Apart from retinal dystrophy and renal malformations, as
found in ovl and eli, it is obviously a formidable task to ®nd other
parallels of the phenotype in ®sh larvae. There are defects in six
genetic loci described leading to BBS in humans, with four of them
identi®ed at the molecular level. BBS 1 (Mykytyn et al., 2002), BBS 2
(Nishimura et al., 2001) and BBS 4 (Mykytyn et al., 2001) are caused
by mutations in novel genes with unknown function. BBS 6 as well as
McKusick±Kaufmann syndrome are caused by a mutation in a putative
chaperonin, crucial for proper folding of nascent proteins (Katsanis
et al., 2000; Slavotinek et al., 2000). Because these genes are widely
expressed in the body, the rather restricted phenotypes of ovl and eli
make them unlikely candidates as genetic models for BBS in humans.
More parallels can be established with Senior±Loken syndrome, an
autosomal recessive disorder, characterized by progressive loss of
vision and nephronophthisis with frequent medullary cystic disease
(Schuman et al., 1985). In these human syndromes, defects are likely
restricted to the eye and the kidney, similar to eli and ovl. Four loci
associated with nephronophthisis and retinitis pigmentosa have been
identi®ed in humans (Hildebrandt et al., 1997; Saunier et al., 1997;
Haider et al., 1998; Omran et al., 2002; Otto et al., 2002). The
nephrocystin 1 and 4 genes have been identi®ed and code for novel
proteins (Hildebrandt et al., 1997; Saunier et al., 1997; Mollet et al.,
2002; Otto et al., 2002). Both genes interact and are probably partners
in a nephrocystin multimolecular signalling complex involved in cell±
cell and cell±matrix adhesion (Mollet et al., 2002). These genes are
good candidates for the eli and ovl loci. We found a zebra®sh expressed
sequence tag coding for nephrocystin 4, which maps to LG 17 and can
therefore be excluded as a candiate gene (data not shown). Future
studies, including the elucidation of the molecular nature of these
syndromes in both humans and zebra®sh, will yield more insight into
similarities and possible differences of these syndromes.
Acknowledgements
The authors want to thank David Belet for technical assistance with histological
analysis. We also like to thank Oliver Biehlmaier for helpful criticism and
discussions as well as Jarema Malicki for sharing data prior to publication and
helpful discussions.
This work was supported by the Swiss National Science Foundation, ETH
internal grants, and the Velux Foundation, Glarus. O.R. is supported by a ZNZ
(Neuroscience Center Zurich) student fellowship, M.W.S. by the Deutsche
Forschungsgemeinschaft (SFB 430 C2) and R.G. by the German Human
Genome Project (DHGP Grant 01 KW 9919).
Abbreviations
BBS, Bardet±Biedl syndrome; dpf, days post-fertilization; cM, centi Morgan
(genetic distance); ERG, electroretinographic; LG, linkage group; PBST, phosphate-buffered saline/0.3% Triton X-100; TUNEL method, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labelling.
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