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1From the Department of Biology, Swiss Federal Institute of Technology (ETH) Zurich, and the 2Brain Research Institute, University of Zurich, Zurich, Switzerland.
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METHODS. Mutant larvae were observed for defects in visual behavior by testing their optokinetic response (OKR). In addition, electroretinograms (ERG) were measured and retinal morphology was examined by standard histology, immunocytochemistry, TUNEL assay, and electron microscopy.
RESULTS. Both, gup and sly showed no OKR at any light intensity tested, whereas bal embryos showed some remaining OKR behavior at more than 40% of contrast. Consistent with the OKR result, gup and sly did not show an ERG response at any light intensity tested, whereas bal mutants exhibited small a- and b-waves at high light intensities. All three laminin mutants showed altered ganglion cell layers, optic nerve fasciculations, and lens defects. Again, bal showed the least severe morphologic phenotype with no additional defects. In contrast, both, gup and sly, showed severe photoreceptor outer segment shortening and synapse alteration (floating ribbons) as well as increased cell death.
CONCLUSIONS. Lamb1 and lamc1 chains play an important role in the morphogenesis of photoreceptors and their synapses. In contrast, lama1 is not involved in outer retina development.
, ß, and 
chains. So far, 15 laminin isoforms have been identified in different tissues and at different developmental stages.1 2 They can bind to integrins, dystroglycan, syndecans, and other cell surface molecules that act as their cellular receptors. Laminin proteins are crucial elements of the extracellular matrix and are known to be involved in central nervous system (CNS) development, in particular in forming and maintaining neuronal contacts, both during development and in the adult. This is also the case in the retina, where laminins are present in many different compartments—namely, Bruch’s membrane, the interphotoreceptor matrix, the external limiting membrane (ELM), the outer plexiform layer (OPL), inner plexiform layer (IPL), and inner limiting membrane (ILM).3 Because of the presence in these retinal compartments, laminins are thought to play a central role in development and maintenance of photoreceptor structure and synaptic function.3 In addition to their effects on the neural retina, it has been shown recently that laminins are crucial for lens development.4 5 6 In humans, mutations in laminin-encoding genes lead to severe diseases such as junctional epidermolysis bullosa,7 congenital muscular dystrophy,8 and Pierson syndrome,9 10 a disease causing a severe nephrotic syndrome followed by early-onset end-stage renal disease accompanied by ocular impairments such as extreme nonreactive narrowing of the pupils and complex maldevelopment of the eyes, including lens abnormalities, atrophy of the ciliary muscle, corneal changes, and retinal alterations.
In the zebrafish, three laminin mutants, named bashful (bal; lama1), grumpy (gup; lamb1), and sleepy (sly; lamc1) have been identified due to their reduced body length and defects in notocord differentiation.11 12 13 Anterior–posterior axon trajectory defects in the retinotectal projection are apparent in gup and sly mutant larvae after DiI and DiO labeling.14 These two mutants were also identified in a screen for defects in visual behavior with defective optokinetic response (OKR), ERG, and retinal morphology.15
We describe the morphologic, behavioral, and electrophysiological alterations in the retinas of these mutants.
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Fish Maintenance and Breeding
Fish were maintained and bred as described elsewhere.16 We used the zebrafish strains Tübingen (TÜ) and Tup Long Fin (TL).17 grumpy (guptg210), sleepy (slyti263a), and bashful (baltp82) larvae were obtained by mating of identified heterozygous carriers, and the embryos were sorted according to their small body and eye size. A comparison of the strength of these alleles with the published data5 12 13 14 18 19 suggests that the alleles used are functional null alleles. Embryos were raised at 28°C in E3 medium (5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl2, and 0.33 mM MgSO4) and staged according to development in days postfertilization (dpf).
Retinal Histology
Larvae were anesthetized at 4°C on ice before fixation. For light microscopy, they were then immediately fixed in 4% paraformaldehyde in 0.2 M phosphate buffer (PB; pH 7.4) for 1 hour (4°C). Larvae for electron microscopy (EM) were fixed in 1% paraformaldehyde and 2.5% glutaraldehyde in 0.1 M PB overnight. For standard histology, fixed larvae were dehydrated in a graded series of ethanol-water mixtures and embedded in resin (Technovit 7100; Kulzer, Wehrheim, Germany). Microtome sections (3 µm) were prepared and mounted on slides (SuperFrost Plus; Menzel-Gläser, Braunschweig, Germany). They were then air dried at 60°C, stained with Richardson solution (1% azure, 1% methylene blue, and 1% borax in deionized water), and coverslipped with mounting medium (Entellan; Merck, Darmstadt, Germany).
Quantifications
Photoreceptor Layer: Thickness Quantification.
Toluidine blue-stained resin-embedded sections with a thickness of 5 µm were evaluated by using bright-field optics. In each larva, we measured the photoreceptor layer thickness as the distance, from the inner end of the photoreceptor soma to Bruch’s membrane. In each laminin mutant, three sections from two to three different fish were quantified. In each section, 10 loci in proximity to the optic nerve were analyzed. Thus, 20 to 30 loci were analyzed per replicate. The bar graphs were designed and the statistic tests performed in commercial software (Prism 4.03; GraphPad, San Diego, CA). Significance between bal, gup, and sly retinas was determined by one-way ANOVA and the Tukey post hoc test (Prism; GraphPad, San Diego, CA).
Lens Phenotype Quantification in the bal Mutant.
Toluidine blue–stained, resin-embedded sections and unstained cryostat sections visualized by differential interference contrast (DIC) optics were screened for lens phenotypes in 35 eyes of 19 different bal larvae.
Electron Microscopy
The EM-fixed larvae were washed in 0.1 M PB for 2 hours and postfixed in 1% osmium tetroxide for 1 hour, 20 minutes. After they were rinsed in 0.1 M PB, the specimens were dehydrated in a graded series of ethanol-water mixtures up to 100% ethanol. After preinfiltration in 1:1 100% ethanol/embedding resin (Fluka, Buchs, Switzerland), larvae were infiltrated in pure embedding resin overnight. Larvae were then positioned in embedding capsules (Beem caps; Canemco & Marivac, Canton de Gore, Quebec, Canada) with fresh resin and polymerized at 60°C for approximately 16 hours. Ultrathin (60 nm) transverse sections were prepared, contrasted with lead citrate, examined, and photographed with a transmission electron microscope (model EM 900; Carl Zeiss Meditec, Oberkochen, Germany).
Cell Death Detection
Fixed larvae were cryoprotected in 30% sucrose for at least 4 hours. Entire larvae were then embedded in tissue-freezing medium (Cryomatrix; Reichert-Jung, Vienna, Austria) and rapidly frozen in liquid N2. Cryosections (25 µm thick) were cut at –20°C, mounted on slides (Super Frost Plus; Menzel-Gläser; Braunschweig, Germany), air dried at 37°C for at least 2 hours, and stored at –20°C until further use. For cell death detection, the slides were thawed, and apoptosis was detected by using the TUNEL (terminal deoxynucleotidyl transferase [TdT]mediated deoxyuridinetriphosphate [dUTP] nick end-labeling) method,20 as previously described.21
Immunohistochemistry
Fixed larvae were cryoprotected in 30% sucrose for at least 4 hours. Entire larvae were then embedded in tissue-freezing medium (Cryomatrix; Reichert-Jung) and rapidly frozen in liquid N2. Cryosections (15 µm thick) were cut at –20°C, mounted on slides (Super Frost Plus; Menzel-Gläser), air dried at 37°C for at least 2 hours, and stored at –20°C until further use. For immunohistochemistry, the slides were thawed, washed three times in PBS (50 mM, pH 7.4), and incubated in blocking solution (BS; 10% NGS and 1% BSA in 0.3% PBS/Triton X-100) for 1 hour. Sections were then incubated overnight in primary antibody in BS at 4°C. The immunolabeling was visualized by using Alexa488-conjugated anti-mouse IgG or anti-rabbit IgG (1:1000; Invitrogen-Molecular Probes, Basel, Switzerland) as a secondary antibody. The following primary antibodies were used for immunostaining: monoclonal mouse anti-zpr1 (1:20; University of Oregon Stock Center), monoclonal mouse anti-glutamine synthetase (GS, 1:700; Chemicon, Harrow, UK), and polyclonal rabbit anti-syntaxin3 (Synt3, 1:400; Alamone Laboratories, Jerusalem, Israel).
After the immunostaining, the slides were coverslipped and analyzed under a fluorescence microscope (Axioscope; Carl Zeiss Meditec, with AxioVision 4 as the imaging software).
OKR Measurements
Visual performance was assessed by measuring the OKR as previously described.22 To measure eye velocity, single larvae were placed dorsal-side-up in the center of a Petri dish (35 mm diameter) containing 3% prewarmed (28°C) methylcellulose. Moving sine-wave gratings were projected by a linearized HP vp6111 projector onto a screen within the visual field of the larva. The apparent distance from the larva’s right eye to the screen was 4.65 cm, and the projection size on the screen was 8 x 6 cm, subtending a visual angle of 65.6° horizontally and 53.1° vertically. Eye movements were triggered by the visual stimulation and recorded by an infrared-sensitive CCD camera. Custom-written software (based on LabView IMAQ ver. 5.1; National Instruments, Austin, TX) was used to control the stimulation and the camera and to analyze the resulting images. Contrast sensitivity functions for wild-type (wt) and laminin mutant larvae were measured as eye velocity as a function of the spatial frequency of the moving grating with alternating movement direction (0.33 Hz). The averaged eye velocity for each spatial frequency was calculated by integration of eye-velocity traces. Graphs were designed and the statistical tests performed with commercial software (Prism 4.03; GraphPad). Significant differences between bal (n = 5), gup (n = 5), sly (n = 5), and their respective siblings (n = 5) were determined by a two-way ANOVA and Bonferroni post hoc test.
Electroretinographic Recordings
Electroretinograms (ERGs) were recorded from 5 dpf larvae as previously described.23 All specimens were dark-adapted for 30 minutes before positioning them in the recording chamber. Each larva was placed on the surface of a moist sponge with E3 medium and paralyzed by directly applying a droplet of the muscle relaxant (Esmeron; 0.8 mg/mL in larval medium; Organon Teknika, Eppelheim, Germany). Light flashes of 200 ms duration were separated by 7-second intervals. Unattenuated irradiation at the position of the subject as measured by a photometer with photopic sensitivity profile was 5.7 mW/cm2.
A virtual instrument (VI) (NI LabVIEW ver. 5.1; National Instruments) was used to control the stimulation and to record ERG traces on computer. Sampling was performed in buffered acquisition mode with a sampling rate of 1000 Hz, and responses were averaged between five acquisitions.
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1, ß1, and 1 are important both for notochord differentiation and for proper intersegmental blood vessel formation.11 12 13 18 In addition, defects in vision have been reported for ß1, and 1 laminin mutant larvae.15 To assess the general retinal morphology of these laminin deficient fish, we analyzed laminin mutant and wt retinas with standard histologic techniques (Figs. 1A 1B 1C 1D) at 5 dpf. All three laminin mutants showed similar retinal alterations: They possessed either no lens (Figs. 1B 1C) or a small lens (Fig. 1 D). In addition, the ganglion cell layer (GCL) was affected in all three mutants (Figs. 1B 1C 1D) . The classic morphology at 5 dpf, with approximately three rows of ganglion cells (GCs) and the optic nerve entering in one big bundle of nerve fibers was broken up. GCs clearly formed more than three rows of cell bodies and were arranged in a less orderly fashion (Figs. 1B 1C 1D) , probably because of the available space left by the reduced or lacking lens. In the wt, fasciculation of GC axons became apparent first at the exit point of the GC layer, where they formed the optic nerve growing toward the optic nerve head in a tight, fasciculated bundle (Fig. 1A) . In contrast, several smaller fascicles originating from GCs of the mutant GCL projected toward the normally fasciculated optic nerve.
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1 mutants showed remarkable shortening of photoreceptor (PhR) outer segments (Figs. 1C 1D) . Quantification of the PhR layer thickness revealed significantly reduced photoreceptors in the gup and sly mutants (Fig. 1E) . In comparison to the thickness measured in wt siblings (41.5 ± 0.40 µm), the PhR layer thickness in gup was significantly reduced to 27.5 ± 2.0 µm and in sly to 18.4 ± 1.0 µm. In contrast, bal mutants showed wt-like PhR layer thickness of 41.0 ± 0.6 µm. To determine the extent to which the photoreceptors and their synapses are affected by the respective laminin mutations we used immunohistochemistry to label double cones (zpr1-labeling) and ribbon synapses (Syntaxin 3;24 ) specifically in the OPL at 5 dpf. All three mutants showed zpr1-labeling. However, whereas the labeling in bal mutants (Fig. 2B) was almost identical with that in the wt (Fig. 2A) , gup (Fig. 2C) and sly (Fig. 2D) larvae expressed zpr1-labeling only in double cone inner segments and somata, thus suggesting outer segment degeneration. As it is known that laminins are highly expressed in the synaptic layers of the retina,3 we wanted to know whether the different laminin mutations would affect the expression of specific synaptic markers of the OPL. Therefore, we labeled ribbon synapses by using a Syntaxin 3 antibody. In this staining, ribbon synapses were labeled to the same extent in wt and all three laminin mutants (Fig. 2E 2F 2G 2H) , thus indicating that the remaining synapses between photoreceptors and second order neurons still possess normal ribbon synapses.
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Retinal Cell Death in Laminin Mutants
We next asked whether the retinal defects in these mutants are associated with an increase in apoptotic cell death by applying the TUNEL assay.20 The number of TUNEL-positive cells detected in 5 dpf wt (Fig. 4A) and bal (Fig. 4B) mutant larvae was very low with no more than one to three labeled cells per section and thus corresponded closely to the rate reported from zebrafish normal development.21 In contrast, the amount of cell death observed in gup (Fig. 4C) and sly (Fig. 4D) was very high, showing numerous dying cells all over the retina. In the sly mutant, most of the apoptotic cells were labeled in the outer nuclear layer (ONL), thus suggesting severe photoreceptor degeneration (Fig. 4D) .
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Contrast sensitivity of all three laminin mutant larvae was severely reduced compared with that in respective wt siblings (Fig. 6 ; bootstrap resampling test, P < 0.05). However, the bal mutant (Fig. 6A) showed some residual OKR at light intensities of 50%, 70%, and 100%, thus suggesting that bal mutants are still able to see at higher light intensities.
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According to our results, a mutation in the lama1 gene encoding for the 1 laminin chain (bal) leads to defective laminin111 (formerly known as laminin 1; see Ref. 26 ) and defective laminin121 but leaves all other 13 known laminins unaffected. In contrast, mutations in lamb1 and lamc1 lead to malformation of six different laminins in gup and 10 in sly, respectively.
Studies of the role of laminins in the zebrafish eye4 5 have shown that a defect in laminin expression leads to alterations in lens morphology. Semina et al.4 recently showed that lama1 mutant zebrafish show disrupted lens development with subsequent lens degeneration. The same was shown for lama1 knockdowns generated by using morpholino antisense oligonucleotides.5 In addition to theses findings, Lee and Gross6 showed that retroviral insertional mutations in lamb1 and lamc1 also lead to severe lens defects. In the present study we showed that lama1, lamb1, and lamc1 mutants have severe lens defects, varying from small lenses to no lenses. This deficiency is likely to be due to a compromise in lens capsule integrity.6 However, because we still found lenses in some of the mutant eyes we suggest that the lense capsule defect leads to defective lens formation during early development, and later on, to a complete clearing of the lens from the laminin mutant eye.
In the OKR and ERG experiments, bal showed wt-like ERG but OKR responses only at high light intensities. We think that this difference in behavioral/functional performance in bal is caused by the altered lens morphology and the resultant low image quality on the mutant retina. In contrast, gup and sly did not show any measurable signals in any of the two functional tests due to the pathologically altered morphology of their photoreceptors that show shortened and degenerating outer segments, altered synapse ultrastructure, and thus impaired synaptic transmission. Similar phenotypes were observed in lamb2 chain–deficient mice.27 However, in contrast to gup and sly, lamb2 chain–deficient mice still show a measurable a-wave, as well as other zebrafish mutants with alterations in photoreceptors and their synapses such as lazy eyes28 and no OKR.29 Therefore, we suggest that the photoreceptor alterations in lamb1 and lamc1 mutant zebrafish lead to a more severe deterioration of photoreceptor function and signal transmission than that observed in beta2 chain–deficient mice and other zebrafish mutants. This hypothesis is strengthened by the proven localization of different laminin chains, respectively special laminin trimers at different parts of the retinal tissue3 (e.g., laminin332 in the interphotoreceptor matrix and OPL of the developing rat retina or lama1, lamb1, and lamc1 in the ILM and in Bruch’s membrane) where they fulfill tissue specific functions. Thus, we suggest that lamb1 and lamc1 play a crucial role for the development and maintenance of the photoreceptors and their synapses, whereas lama1 does not or is functionally compensated by lama5, which is likely to be expressed in this retinal region as well.3
We found severe disorganization of the GCL and optic nerve in all three laminin mutants examined in the present study. Similar phenotypes have been described in other publications on laminin mutant4 6 or morphant5 zebrafish. There are two possible reasons for the GCL defect: One is that the malformation of the GCL may simply be associated with the fact of the reduced or lacking lens in the laminin mutants. Semina et al.4 tested this hypothesis by comparing lama1 mutants to embryos in which the lens vesicle had been surgically removed. Their results suggests that many of the anterior and posterior ocular defects in the lama1 mutant are independent of the lens degeneration and, therefore, we suggest that this also holds true for the lama1, lamb1, and lamc1 mutants of our present study. The second hypothesis to explain the GCL defect is that the lack of any of the -, ß-, or -chains needed to build up the heterotrimeric laminin111 protein affects the structural integrity of the ILM. This membrane is known to contain laminin111.3 Because of the interdependency between the laminins and the Müller cells in building up the ILM,25 this change in structural integrity influences the termination locus of Müller cell end feet and in consequence the GCL lamination. This hypothesis is supported by reports of similar GCL alterations in other zebrafish laminin mutants4 6 and morphants.5 Therefore, a malformed ILM probably leads to a deteriorated GCL.
In conclusion, we found that lama1, lamb1, and lamc1 mutations lead to lens defects and morphologic alterations of the GCL and the optic nerve, whereas only lamb1 and lamc1 mutations lead to severe photoreceptor dysfunction.
The affected retinal tissues are located in the retinal compartments known to contain laminin proteins in rodents, such as Bruch’s membrane, the interphotoreceptor matrix, and the ELM, OPL, IPL, and ILM.3 The lack or mutation of one chain of a trimeric laminin protein leads to developmental malformation and/or degeneration in the respective retinal compartments such as the photoreceptors, the OPL, or the GCL. As the mutation in the lama1 chain (bal) affects only the lens and the GCL and leaves other layers normal, we favor the idea that lama1 chains are not involved in outer retina development and maintenance or may be replaced by lama5 or other lama chains to compensate for the absent lama1 chain. The recent study by Semina et al.4 furthers this hypothesis by showing that other alleles of lama1 mutation show similar phenotypes, affecting the GCL and lens, such as baltp82. Thus, all three laminin chains examined in this study are crucial in the formation of a proper ILM and a normal GCL, but only lamb1 and lamc1 are vital for photoreceptor development and maintenance. Finally, the outcome of this hypothesis is that the location of the respective laminin chains throughout the zebrafish retina correlates nicely with the laminin chain distribution suggested by Libby et al.27 in the rat retina.
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Acknowledgements |
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Supported by the Swiss National Science Foundation (SCFN), the Velux Foundation (OB), and the EMBO Young Investigator Program (SCFN).
Submitted for publication October 9, 2006; revised February 26, 2007; accepted April 20, 2007.
Disclosure: O. Biehlmaier, None; Y. Makhankov, None; S.C.F. Neuhauss, None
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked "advertisement" in accordance with 18 U.S.C. 
1734 solely to indicate this fact.
* Each of the following is a corresponding author: Oliver Biehlmaier, Swiss Federal Institute of Technology (ETH) Zurich, Department of Biology, and the Brain Research Institute, University of Zurich, Winterthurerstrasse 190, CH 8057 Zurich, Switzerland; biehlmaier{at}hifo.unizh.ch. Stephan C. F. Neuhauss, University of Zurich, Institute of Zoology, Winterthurerstrasse 190, CH 8057 Zurich, Switzerland; stephan.neuhauss{at}zool.unizh.ch.
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1 containing laminins are essential for basement membrane integrity in the zebrafish eye. Invest Ophthalmol Vis Sci. 2007;48:2483–2490.This article has been cited by other articles:
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  P. Thornhill, D. Bassett, H. Lochmuller, K. Bushby, and V. Straub Developmental defects in a zebrafish model for muscular dystrophies associated with the loss of fukutin-related protein (FKRP) Brain, June 1, 2008; 131(6): 1551 - 1561. [Abstract] [Full Text] [PDF]
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 J. Lee and J. M. Gross Laminin {beta}1 and {gamma}1 Containing Laminins Are Essential for Basement Membrane Integrity in the Zebrafish Eye Invest. Ophthalmol. Vis. Sci., June 1, 2007; 48(6): 2483 - 2490. [Abstract] [Full Text] [PDF]
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