Retinal defects in the zebrafish bleached mutant

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Histological analysis of mutant retinae showed a severely affected outer retina ... epithelium and a disorganized outer nuclear layer containing few or no intact ...
Documenta Ophthalmologica 107: 71–78, 2003. © 2003 Kluwer Academic Publishers. Printed in the Netherlands.

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Retinal defects in the zebrafish bleached mutant Stephan C.F. Neuhauss1,2 , Mathias W. Seeliger3 , Carsten P. Schepp2 & Oliver Biehlmaier4 1 Brain

Research Institute, University of Zurich and Department of Biology, Swiss Federal Institute of Technology Zurich, Winterthurerstr. 190, CH-8057 Zurich, Switzerland; 2 Max-Planck Institute for Developmental Biology, Spemannstr. 35/I, 3 Retinal Electrodiagnostics Research Group, University Eye Hospital, Department II, Schleichstr. 12–16; 4 Experimental Ophthalmology, Röntgenweg 11, D-72076 Tuebingen, Germany Accepted 13 May 2002

Key words: apoptosis, electroretinography, retinal degeneration, zebrafish

Abstract The recessive zebrafish mutant bleached has, apart from its defects in pigmentation, a heritable defect leading to larval blindness. Here, we analyze the retina of homozygous bleached larvae, employing morphological and electrophysiological methods. Electroretinography revealed a complete lack of electrical signals in response to light. Histological analysis of mutant retinae showed a severely affected outer retina with a hypopigmented pigment epithelium and a disorganized outer nuclear layer containing few or no intact photoreceptors. Using the TUNEL assay for cell death detection, we noticed a strong increase of apoptotic cells in all retinal cell layers, starting in young larvae even before retinal support of visual function. At later stages cell death is most pronounced at the marginal zone, where new cells are constantly added to the retina. At early stages increased apoptosis is mainly confined to the retina, while at later stages elevated cell death is also apparent in extra-retinal tissues, particularly in the brain. Hence, the lack of visual responses in homozygous bleached larvae can be attributed to a severe defect of the outer retina, preceded by increased levels of apoptotic cell death in all retinal cell layers.

Introduction The zebrafish is an increasingly popular model vertebrate for studying the genetic basis of vertebrate development and degenerative diseases, including the visual system. The visual system is particularly well suited for a genetic analysis, since it develops extraordinary rapidly in the zebrafish and is functional early on in larval development. Only 5 days post fertilization (dpf), the retina is well layered with all cell types present, retinal ganglion cell axons have arborized on their main target region in the brain, the optic tectum, and the maturity of the visual system is evidenced by a number of visually mediated behaviors [1–4]. Additionally, sum field potentials of the retina in response to light can readily be recorded by electroretinography [2, 5–7]. These favorable features have been used to screen for mutant strains with defects in the morphology of the visual system [8–10]. In such screens, mutants

with defects in various aspects of retina development have been isolated (reviewed in Ref. [11]). Furthermore, functional properties of the visual system can be assessed by a variety of visually mediated behaviors, allowing screens for mutants abnormal in visual system function. For instance, the stereotypical eye movements of the optokinetic response are robustly evoked by moving stimuli in 5-day-old larvae, permitting screens for larvae defective in visually mediated behavior. In such a screen, the recessive mutant bleached (blc) was identified as being unresponsive to moving visual stimuli, albeit being able to display spontaneous eye movements [12]. The mutant was initially isolated in one of the large-scale genetic screens and named after its abnormal body and eye pigmentation phenotype. All three pigment types of larval zebrafish are affected in the mutant, with the melanin-containing melanophores degenerating, and yellow xanthophores and silvery iridiophores assuming a pale or dull ap-

72 pearance [13]. Similar to the reduced body pigmentation, the eyes are progressively hypopigmented and at later stages slightly reduced in size. Here we further analyze the visual system defect of homozygous blc larvae by electrophysiological and histological means. We show that the retina at 5 dpf is completely unresponsive to light as judged by electroretinography. Histological sections of blc mutant retinae revealed a damaged retina. Particularly, the outer retina and the pigment epithelium are severely affected with no intact photoreceptors left. We furthermore used an assay for apoptotic cell death (TUNEL, terminal deoxynucleotidyl transferase (TdT)-mediated deoxyuridinetriphosphate (dUTP) nick end-labeling) to assay for apoptotic cells in situ. An increase in apoptotic cell death was already apparent in all retinal cell layers of the young mutant larva before the retina is mature enough to perceive light. At later stages, increased cell death is also seen outside of the retina. Materials and methods Fish maintenance and breeding Fish were maintained and bred as previously described [14]. Homozygous bleached larvae were obtained by pairwise matings of identified carrier fish. All experiments in this study were performed using the th204b allele. As wild-type controls we either used larvae from the Tübingen (Tü) strains or unaffected siblings of the mutant crosses. We detected no difference between sibling and wild-type larvae. 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 post fertilization (dpf). Electroretinography (ERG) Five-dpf-old larvae were dark-adapted for a minimum of 4 h prior to the measurements and subsequently handled under dim red illumination. They were anesthetized with 0.02% buffered 3-aminobenzoic acid methyl ester (MESAB; Sigma, St. Louis, USA) and paralyzed with 0.8 mg/ml Esmeron (Organon Teknika, Eppelheim, Germany) by adding these solutions to the water. Recordings were performed as previously described [7]. Briefly, anesthetized larvae were placed in a lateral position on a wet paper towel sitting on a platinum wire utilized as reference electrode. Under visual control via a standard microscope

equipped with red illumination, a glass microelectrode with an opening of approximately 20 µm at the tip was placed on the center of the cornea. Stimulation and data acquisition were performed with a commercially available ERG setup (Toennies Multiliner Vision, Jaeger/Toennies, Höchberg, Germany) featuring a Ganzfeld bowl, a DC amplifier, and a personal computer for stimulus generation and data management. Bandpass filter cutoff frequencies were 1 and 300 Hz for all measurements. Single flash stimuli increasing from 1 mcds/m2 to 3 cds/m2 were used under scotopic conditions. Ten responses per intensity level were averaged, with an inter-stimulus interval (ISI) of 5 (1, 10, 30, 100 mcds/m2 ) or 17 s (1 and 3 cds/m2 ). Histology Larvae were anesthetized on ice at 4 ◦ C and then immediately fixed in 4% paraformaldehyde in 0.2 M phosphate buffer, pH 7.4, for 1 h (4 ◦ C). Fixed larvae were dehydrated in a graded series of ethanol–water mixtures, then incubated in 1:1 ethanol 99.9% and Technovit 7100 (Kulzer) basic solution for 2 h. After overnight infiltration in Technovit 7100 basic solution, larvae were positioned in Technovit 7100 (Kulzer) polymerisation medium for 2 h (37 ◦ C). Microtome sections (3 µm) were mounted on polyL -lysine coated slides, air dried at 60 ◦ C, stained with Richardson solution (1% azur, 1% methyleneblue and 1% Borax in deionized water), overlaid with DPX (Merck), and coverslipped. For each stage more than ten separate eyes were analyzed. Cell death detection The TUNEL (terminal deoxynucleotidyl transferase (TdT)-mediated deoxyuridine- triphosphate (dUTP) nick end-labeling) method [15] was used in a modified form to identify cells that were undergoing apoptosis. Fixed larvae (4% paraformaldehyde over night at room temperature) were cryoprotected in 30% sucrose for at least 4 h. Whole larvae were embedded in Tissue Tek (Sakura, Torrance, USA) and stored at −20 ◦ C. Sections, 25 µm thick were cut on a cryostat at −20 ◦ C, mounted on gelatin-coated slides, air dried at 37 ◦ C for at least 2 h, and then stored at −20 ◦ C. For the TUNEL assay, cryostat sections were thawed and dried at 37 ◦ C for 1 h, then postfixed in 4% paraformaldehyde for 20 min at RT and washed twice in 50 mM PBS, pH 7.4. After proteinase K treatment

73 (2 µg/ml in 10 mM Tris, pH 7.6) at 37 ◦ C for 5 min, slides were washed three times in 10 mM Tris, pH 7.6. Sections were then incubated with 70% ethanol / 30% acetic acid for 5 min at −20 ◦ C, washed twice for 5 min in 50 mM PBS, pH 7.4, then treated again with proteinase K (2 µg/ml in 50 mM Tris, pH 7.6) at 37 ◦ C for 5 min. Following three washes in Tris (50 mM, pH 7.6), all sections were incubated in blocking solution (10% NGS; 1% BSA; 1% fishgelatine; 0.3% Triton X100 in PBS) for 30 min at room temperature. After the blocking, slides were incubated with TUNEL reaction mix (In Situ Cell Death Detection Kit, Fluorescein; Roche Diagnostics) for 1 h at 37 ◦ C. Finally, samples were rinsed three times with PBS (pH 7.4), overlaid with glycerin and coverslipped. For all analyzed stages at least 10 separate eyes were used.

Results ERG analysis Previous results indicated a complete loss of visually mediated behavior in bleached (blc) larvae at 5 dpf. In order to test if the loss of vision is associated with a loss of retinal function, we evaluated electrical responses of the retina to light by recording electroretinograms (ERG). We recorded from 5-dpfold larvae, since robust behavioral and ERG responses have been reported at this stage [1, 6, 7, 16]. We dark-adapted both mutant larvae and non-affected siblings to measure an intensity series under scotopic conditions. In unaffected sibling larvae, we recorded responses typical for vertebrate ERGs (Figure 1A). Positive components (b-wave) first became apparent at about 30 mcds/m2 , and further grew in amplitude with increasing stimulus intensity, while the initial negative a-wave was either small or absent. Recordings from unaffected siblings were indistinguishable from those of wild-type larvae [7]. In homozygous blc larvae we were unable to record any response to light, even at maximum stimulus brightness (Figure 1b), indicating a complete loss of photoreceptor cell function. Hence our ERG recordings show that the cause of blindness in blc homozygous larvae is due to defects in the outer retina. Retina histology The macroscopic observation that the eye is hypopigmented and slightly reduced in size in combination

with the electrophysiological finding of the outer retina’s unresponsiveness to light, suggests a morphological defect in the retina. We therefore analyzed the retina of 5 dpf blc homozygous larvae and unaffected siblings on histological sections. At this stage, the wild-type retina is well layered and all cell types are formed (Figure 2a). The retina of unaffected siblings is indistinguishable from the wild-type, indicating that blc is a fully recessive mutation. The outer segments of the photoreceptors are clearly visible, the three nuclear layers and the two plexiform layers are well formed and separated. The retinal pigment epithelium (RPE) appears black and is filled with melanin containing granules. In contrast, histological sections of the blc mutant eye show a severely disorganized outer retina. The RPE contains no discernible melanin granules. The outer nuclear cell layer (ONL) and the outer plexiform (OPL) cell layer are absent and no photoreceptors with outer segments are present. A number of pyknotic nuclei and numerous vacuoles, presumably left by deceased cells, are visible. The inner retina is affected to a much lesser degree and still shows a clear separation of the inner plexiform and the ganglion cell layer. Therefore the histological data is consistent with the ERG results, proving a morphologically defected outer retina. Apoptotic cell death The disorganized outer retina and the presence of vacuoles and pyknotic in the blc mutant retina suggests a highly elevated rate of cell death caused by the mutation. In order to test if cell death is indeed elevated and if it occurs via apoptosis, we employed an in situ assay for DNA fragmentation. Apoptosis, in some cases referred to as programmed cell death, is characterized by a well-orchestrated sequence of biochemical steps, leading to the fragmentation of nuclear material. DNA fragmentation can be assayed by the TUNEL method [15] on histological sections. In the wild-type retina we observed little apoptotic cell death (Figure 4A,C), consistent with published results [17]. Apoptosis was strongly enhanced in blc retinae at all stages surveyed. Already at 60 h post fertilization, we saw an increased number of apoptotic nuclei in all retinal cell layers, while the ONL was still separated from the inner retina by the OPL (Figure 3). By 3 dpf numerous cells in all retinal cell layers undergo apoptotic cell death (Figure 4B). At this stage, dying cells are mostly found in the eye, while at 5 dpf also extraretinal tissues display

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Figure 1. ERG intensity series of wild-type (A) and blc mutant larvae (B) at 5 dpf recorded under scotopic conditions. Representative recordings from wild-type larvae show the typical response growth with increasing light stimulus intensity, while no electrical responses can be evoked from the blc retina at all intensities.

Figure 2. Transversal sections through wild-type (wt) and bleached (blc) retinae. (A) Histological section of wt larva at 5 dpf showing normal development across the retinal layers. The outer nuclear layer (ONL), photoreceptor outer segments (OS), outer plexiform layer (OPL), the retinal pigment epithelium (RPE) as well as the marginal zone (MZ) are clearly visible. (B) The histological section of the blc mutant eye completely lacks the OPL, and ONL. The RPE is void of melanin granules and appears ‘bleached’. Many pyknotic nuclei can be identified (arrowheads) and already deceased cells leave numerous vacuoles (arrows) in the ONL. Additionally, the marginal growth zone (MZ) is hypotrophic and shows vacuolization as well. The inner retinal layers appear to be little affected at this stage. Scale bar: A,B = 20 µm.

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Figure 3. TUNEL labeling of apoptotic cells in a blc retina section. Apoptotic cells are mainly confined to the retina and affect all cell layers. The outer nuclear layer (ONL) is well formed (arrow). Scale bar: 50 µm.

an increased rate of apoptosis (Figure 4D). Clearly all retinal cell layers are affected by apoptosis, but apoptosis is most prominent in the marginal zone, where new cells are added to the growing retina, indicating that cells also die at around the time of their last mitotic division.

Discussion The recessive zebrafish bleached (blc) mutation was induced in a large-scale chemical mutagenesis screen and most likely represents a single gene defect [18]. Mutant animals were initially identified by a marked loss of pigmentation, affecting body and eye pigmentation [13]. In a subsequent behavioral screen for visually impaired larvae, homozygous blc larvae were found to be defective in visually mediated behaviors, such as the optokinetic nystagmus and the optomotor response [12]. In order to uncover the cause of blindness in this mutant, we first determined by electroretinography if the retina is able to respond to light. We were unable to record any activity of the mutant retina, even in response to maximally bright stimuli.

This argues for a defect in the outer retina. Subsequent histological analysis of the retina confirmed morphological defects in the outer retina. In 5 dpf blc retinae no separated ONL or OPL was visible. No evidence for differentiated photoreceptors or outer segments was apparent (Figure 2). In younger larvae, the ONL is formed (Figure 3), indicating that degeneration is progressive in this mutant. Vacuoles and pyknotic nuclei in the standard histological sections pointed towards massive cell death in the retina. We used an in situ DNA fragmentation assay (TUNEL assay) to determine that cells in the mutant retina die via an apoptotic pathway, similar to human retinal dystrophies and their animal models [19–21]. Elevated apoptosis is apparent in the blc retina before 3 dpf prior to retinal function and is initially most apparent in the eyes. At later stages (5 dpf), elevated levels of apoptosis spread throughout the body. Apoptotic cells are then found in all cell layers, but are most frequent in the marginal growth zone of the eye. This proliferative peripheral region is composed of a group of specialized cells, which constantly add new cells to all retinal cell layers. The high concentration of

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Figure 4. TUNEL labeling of apoptotic cells in retinal section of wild-type (A,C) and bleached (B,D) larvae. At 3 dpf, there are few apoptotic cells in the wild-type (A), while blc (B) retinae show a large number of cells in all retinal cell layers undergoing apoptosis. At 5 dpf wild type larvae (C) only sporadic apoptotic cells are detected in the retina in contrast to blc larvae (D) with massive amount of dying cells, also apparent in non-retinal tissue (data not shown). Scale bar: 25 µm.

apoptotic cells in this region indicates that some cells in the blc retina undergo apoptosis right around the time of their final mitotic division. This contrasts with other zebrafish mutants where outer retinal cell death is most pronounced in central regions, arguing for an age-related phenomenon where older, and therefore more centrally located cells are most likely to die ([22]

and data not shown). Hence, the defect in blc is likely a developmental rather than a dystrophic defect, since it affects also new born and presumable undifferentiated cells. However, the initial formation of the retinal cell layers is not affected in the mutant, since the three classical nuclear layers are well formed in 3-dpf-old

77 larvae, even in view of an already increased rate of apoptotically dying cells. The location of the primary defect which leads to massive apoptosis in the blc mutant retina is still unclear. Our data is most consistent with a primary defect affecting all retinal cells, since elevated levels of apoptosis are observed in all retinal cell layers, even at early stages. An alternative explanation would be that the primary defect affects the RPE, which consequently fails to support retinal cells. We deem it unlikely that retinal cell death is secondary to RPE degeneration, since not only ONL cells which are direct dependent from the RPE, but also cells of the inner retina die early on. Although the increase in cell death is initially mainly apparent in retinal cells and in melanophores, the mutation affects other body tissues at later stages as well. It is presently unclear if this phase of apoptosis, ultimately leading to the death of the larva at around 8 dpf, is directly related to a lack of blc function in these cells or if it is an indirect consequence of other cells being affected. In blc zebrafish, both body pigmentation and the visual system are affected. Such a syndrome of body pigmentation and visual system abnormalities is quite common in vertebrates. In humans, a number of pigmentation defects are concurrent with visual defects. Most prominent is heritable oculocutaneous albinism affecting body pigmentation and visual system development. Nevertheless, the blc mutant is different in that cells die in all retinal cell layers, while the primary defect in albinism is in melanin formation leading to secondary defects in visual system development [23]. The blc mutant, particularly once its molecular nature is revealed, will shed light on the biological link between body pigmentation and development of the visual system. This and other zebrafish mutations affecting the visual system will continue to contribute to our understanding of vertebrate visual system development and maintenance, and particularly its genetic basis.

References 1. 2.

3. 4. 5.

6. 7. 8.

9.

10.

11.

12.

13.

14.

15.

16.

17.

Acknowledgements The authors want to thank Konrad Kohler for discussions, Friedrich Bonhoeffer (CPS, SCFN), Velux Stiftung Glarus (SCFN) and the Deutsche Forschungsgemeinschaft (Graduiertenkolleg Neurobiologie Tübingen (OB), SFB 430 C2 (MWS)) for support.

18.

19.

Easter SS Jr, Nicola GN. The development of vision in the zebrafish (Danio rerio). Dev Biol 1996; 180: 646–63. Brockerhoff SE, Hurley JB, Janssen-Bienhold U, Neuhauss SCF, Driever W, Dowling JE. A behavioral screen for isolating zebrafish mutants with visual system defects. Proc Natl Acad Sci U S A 1995; 92: 10545–9. Li L. Zebrafish mutants: behavioral genetic studies of visual system defects. Dev Dyn 2001; 221: 365–72. Bilotta J, Saszik S. The zebrafish as a model visual system. Int J Dev Neurosci 2001; 19: 621–9. Branchek T. The development of photoreceptors in the zebrafish, Brachydanio rerio. II. Function. J Comp Neurol 1984; 224: 116–22. Saszik S, Bilotta J, Givin CM. ERG assessment of zebrafish retinal development. Vis Neurosci 1999; 16: 881–8. Seeliger MW, Rilk A, Neuhauss SCF. Ganzfeld ERG in zebrafish larvae. Doc Ophthalmol 2002; 104: 57–68. Malicki J, Neuhauss SC, Schier AF, Solnica-Krezel L, Stemple DL, Stainier DY, Abdelilah S, Zwartkruis F, Rangini Z, Driever W. Mutations affecting development of the zebrafish retina. Development 1996; 123: 263–73. Fadool JM, Brockerhoff SE, Hyatt GA, Dowling JE. Mutations affecting eye morphology in the developing zebrafish (Danio rerio). Dev Genet 1997; 20: 288–95. Vihtelic TS, Hyde DR. Zebrafish mutagenesis yields eye morphological mutants with retinal and lens defects. Vision Res 2002; 42: 535–40. Malicki J. Harnessing the power of forward genetics – analysis of neuronal diversity and patterning in the zebrafish retina. Trends Neurosci 2000; 23: 531–41. Neuhauss SCF, Biehlmaier O, Seeliger MW, Das T, Kohler K, Harris WA, Baier H. Genetic disorders of vision revealed by a behavioral screen of 400 essential loci in zebrafish. J Neurosci 1999; 19: 8603–15. Kelsh RN, Brand M, Jiang YJ, Heisenberg CP, Lin S, Haffter P, Odenthal J, Mullins MC, van Eeden FJ, Furutani-Seiki M, Granato M, Hammerschmidt M, Kane DA, Warga RM, Beuchle D, Vogelsang L, Nusslein-Volhard C. Zebrafish pigmentation mutations and the processes of neural crest development. Development 1996; 123: 369–89. Mullins MC, Hammerschmidt M, Haffter P, Nusslein-Volhard C. Large-scale mutagenesis in the zebrafish: in search of genes controlling development in a vertebrate. Curr Biol 1994; 4: 189–202. Gavrieli Y, Sherman Y, Ben-Sasson SA. Identification of programmed cell death in situ via specific labeling of nuclear DNA fragmentation. J Cell Biol 1992; 119: 493–501. Easter SS Jr, Nicola GN. The development of eye movements in the zebrafish (Danio rerio). Dev Psychobiol 1997; 31: 267– 76. Biehlmaier O, Neuhauss SC, Kohler K. Onset and time course of apoptosis in the developing zebrafish retina. Cell Tissue Res 2001; 306: 199–207. Haffter P, Granato M, Brand M, Mullins MC, Hammerschmidt M, Kane DA, Odenthal J, van Eeden FJ, Jiang YJ, Heisenberg CP, Kelsh RN, Furutani-Seiki M, Vogelsang E, Beuchle D, Schach U, Fabian C, Nusslein-Volhard C. The identification of genes with unique and essential functions in the development of the zebrafish, Danio rerio. Development 1996; 123: 1–36. Xu GZ, Li WW, Tso MO. Apoptosis in human retinal degenerations. Trans Am Ophthalmol Soc 1996; 94: 411–30.

78 20. Reme CE, Grimm C, Hafezi F, Wenzel A, Williams TP. Apoptosis in the retina: the silent death of vision. News Physiol Sci 2000; 15: 120–24. 21. Chang GQ, Hao Y, Wong F. Apoptosis: final common pathway of photoreceptor death in rd, rds, and rhodopsin mutant mice. Neuron 1993; 11: 595–605. 22. Doerre G, Malicki J. Genetic analysis of photoreceptor cell development in the zebrafish retina. Mech Dev 2002; 110: 125–38.

23.

Oetting WS. Albinism. Curr Opin Pediatr 1999; 11: 565–71.

Address for correspondence: S. Neuhauss, Brain Research Institute, University of Zurich and Swiss Federal Institute of Technology, Winterthurerstrasse 190, CH-8057 Zurich, Switzerland Phone +41-1-635 3288; Fax +41-1-635 3303; E-mail: [email protected]