Transgenic Expression of a GFP-rhodopsin COOH-terminal Fusion Protein in Zebrafish Rod Photoreceptors

Visual Neuroscience. May-Jun, 2002  |  Pubmed ID: 12392175

To facilitate the identification and characterization of mutations affecting the retina and photoreceptors in the zebrafish, a transgene expressing green fluorescent protein (GFP) fused to the C-terminal 44 amino acids of Xenopus rhodopsin (Tam et al., 2000) under the control of the 1.3-kb proximal Xenopus opsin promoter was inserted into the zebrafish genome. GFP expression was easily observed in a ventral patch of retinal cells at 4 days postfertilization (dpf). Between 45-50% of the progeny from the F1, F2, and F3 generations expressed the transgene, consistent with a single integration event following microinjection. Immunohistochemical analysis demonstrated that GFP is expressed exclusively in rod photoreceptors and not in the UV, blue, or red/green double cones. Furthermore, GFP is localized to the rod outer segments with little to no fluorescence in the rod inner segments, rod cell bodies, or rod synapse regions, indicating proper targeting and transport of the GFP fusion protein. Application of exogenous retinoic acid (RA) increased the number of GFP-expressing cells throughout the retina, and possibly the level of expressed rhodopsin. When bred to a zebrafish rod degeneration mutant, fewer GFP-expressing rods were seen in living mutants as compared to wild-type siblings. This transgenic line will facilitate the search for recessive and dominant mutations affecting rod photoreceptor development and survival as well as proper rhodopsin expression, targeting, and transport.

Transgenic Expression of a GFP-rhodopsin COOH-terminal Fusion Protein in Zebrafish Rod Photoreceptors

Visual Neuroscience. Jul-Aug, 2002  |  Pubmed ID: 12511087

To facilitate the identification and characterization of mutations affecting the retina and photoreceptors in the zebrafish, a transgene expressing green fluorescent protein (GFP) fused to the C-terminal 44 amino acids of Xenopus rhodopsin (Tam et al., 2000) under the control of the 1.3-kb proximal Xenopus opsin promoter was inserted into the zebrafish genome. GFP expression was easily observed in a ventral patch of retinal cells at 4 days postfertilization (dpf). Between 45-50% of the progeny from the F1, F2, and F3 generations expressed the transgene, consistent with a single integration event following microinjection. Immunohistochemical analysis demonstrated that GFP is expressed exclusively in rod photoreceptors and not in the UV, blue, or red/green double cones. Furthermore, GFP is localized to the rod outer segments with little to no fluorescence in the rod inner segments, rod cell bodies, or rod synapse regions, indicating proper targeting and transport of the GFP fusion protein. Application of exogenous retinoic acid (RA) increased the number of GFP-expressing cells throughout the retina, and possibly the level of expressed rhodopsin. When bred to a zebrafish rod degeneration mutant, fewer GFP-expressing rods were seen in living mutants as compared to wild-type siblings. This transgenic line will facilitate the search for recessive and dominant mutations affecting rod photoreceptor development and survival as well as proper rhodopsin expression, targeting, and transport.

George Wald, 18 November 1906 - 12 April 1997

Proceedings of the American Philosophical Society. Dec, 2002  |  Pubmed ID: 12619664

A Morphological Classification of Ganglion Cells in the Zebrafish Retina

Visual Neuroscience. Nov-Dec, 2002  |  Pubmed ID: 12688671

We examined the distribution and morphological types of ganglion cells in the retina of the zebrafish, a model vertebrate genetic organism. Using cresyl violet and methylene blue staining, a prominent central area was observed in the ventral temporal retina. The density of ganglion cell layer neurons averaged from approximately 12,000/mm2 in the dorsal-nasal retina to a peak of approximately 37,000/mm2 in the ventral-temporal retina. Individual zebrafish ganglion cells were labeled by backfilling with DiI through the optic nerve followed by reconstruction using confocal microscopy. The dendritic stratification and branching pattern of each labeled ganglion cell was examined in relation to the borders of the inner plexiform layer (IPL). We identified 11 different morphological types of ganglion cell. The most commonly labeled ganglion cells were two types termed Type III or IV, which displayed highly stratified dendritic arborizations in their respective ON-, OFF-sublaminae of the IPL. Their dendritic branching patterns were highly asymmetric with many thorn-like varicosities that profusely filled the area of arborization. In contrast, Type V cells formed a small simply branching dendritic field in the innermost portion of the ON-sublamina of the IPL. Two large ganglion cell types (Types I and II) with wide monostratified dendritic fields were found in both the ON- and OFF-sublamina of the IPL. Three different types of multistratified/bistratified ganglion cells were found (Types, IX, X, and XI.) whose dendrites occupied different regions of the IPL. The multistratified dendrites of IX cells occupied the whole width of the IPL, while the dendrites of Type XI cells formed vertical claw-like endings in only the ON-sublamina of the IPL. We conclude that zebrafish ganglion cells display a rich variety of types and branching patterns. This study establishes a series of baseline measurements of zebrafish ganglion cells to facilitate examination of genes playing a role in the specification and stratification of ganglion cell types.

Positional Cloning of the Young Mutation Identifies an Essential Role for the Brahma Chromatin Remodeling Complex in Mediating Retinal Cell Differentiation

Proceedings of the National Academy of Sciences of the United States of America. May, 2003  |  Pubmed ID: 12748389

Zebrafish with the young (yng) mutation show a defect in retinal cell differentiation. Here we demonstrate that a mutation in a brahma-related gene (brg1) is responsible for the yng phenotype. Brahma homologues function as essential subunits for SWI/SNF-type chromatin remodeling complexes. Our analysis indicates that brg1 is required for the wave of mitogen-activated protein kinase activity that precedes retinal cell differentiation. Using specific inhibitors of the mitogen-activated protein kinase pathway we show this signal has a direct role in retinal cell differentiation. Lastly, through investigations of mutants in other chromatin remodeling subunits, we provide genetic evidence for gene and tissue specificity of the Brahma chromatin remodeling complex.

Lazy Eyes Zebrafish Mutation Affects Müller Glial Cells, Compromising Photoreceptor Function and Causing Partial Blindness

The Journal of Comparative Neurology. Aug, 2003  |  Pubmed ID: 12820161

A behavioral assay based on the optokinetic reflex was used to screen chemically mutagenized zebrafish larvae for deficits in visual function. A homozygous recessive mutation, lazy eyes (lze), was isolated based on the observation that 5-day postfertilization (dpf) mutants displayed weaker and less frequent eye movements than wild-type fish in response to moving stripes. Electroretinographic (ERG) recordings revealed that mutants had severely reduced a- and b-wave amplitudes relative to wild-type fish, indicating outer retinal dysfunction. Retinal lamination and cellular differentiation were normal in the lze retina; however, mutant photoreceptor cells had small outer segments and pyknotic nuclei were occasionally observed in the outer retina and the marginal zone of lze. Cone, rod, amacrine, bipolar, and Müller cell marker analyses indicated that the typical lze retina contained fewer rod photoreceptors and fewer Müller cells than wild-type fish at 5 dpf. At 3 dpf, however, mutant retinas had normal numbers of rod photoreceptors and Müller cells, suggesting that the initial differentiation of these cell types occurred normally. Rod photoreceptor histology was normal at this early stage, but Müller cells were often hypertrophied, suggesting that they were unhealthy. Constant light rearing of mutant animals accelerated the Müller cell degeneration, severely worsened the visual deficit, but had no obvious affect on the photoreceptors. When ERG responses and Müller cell degeneration from the same mutant animals were analyzed, the extent of the Müller cell loss matched closely the degree to which ERG responses were reduced. In summary, the lze gene appears to be required for Müller cell viability and normal visual function. The lze mutant may be a model for the study of the involvement of Müller cells in photoreceptor development and function.

Photoreceptor Structure and Development: Analyses Using GFP Transgenes

Methods in Cell Biology. 2004  |  Pubmed ID: 15602882

Glutamatergic Mechanisms in the Outer Retina of Larval Zebrafish: Analysis of Electroretinogram B- and D-waves Using a Novel Preparation

Zebrafish. 2004  |  Pubmed ID: 18248224

A new preparation is described for recording the electroretinogram (ERG) from larval zebrafish (5-8 days postfertilization) which has allowed the investigation of the pharmacology of cone photoreceptor inputs onto bipolar cells. By using a pharmacological cocktail to isolate the photoreceptors and bipolar cells from inhibitory influences, it was found that an excitatory amino acid transporter (EAAT) presumably linked to a Cl() channel mediates most of the synaptic transmission from the cone photoreceptors to the ON bipolar cells, although metabotropic glutamate receptors (presumably mGluR6) also make a small contribution. On the other hand, alpha-amino-3-hydroxy- 5-methyl-4-isoxazolepropionate (AMPA)/kainate receptors mediate synaptic transmission from cone photoreceptors to OFF bipolar cells. The glutamatergic input mechanisms underlying bipolar cell responses in the larval zebrafish are adultlike and similar to those in other teleost species.

Gene Expression Profiling of Zebrafish Embryonic Retina

Zebrafish. 2005  |  Pubmed ID: 18248185

A methodology for microdissecting intact retinas from zebrafish embryos at early developmental stages for expression profiling was developed in this study. Total RNA was extracted consistently and reproducibly from the dissected retinas using a customized extraction protocol. The results from microarray experiments indicated that the purified RNA samples faithfully represented the biological differences among different types of samples. Genes that were differentially expressed in a particular neuronal layer or region of the retina were detectable by microarray experiments. In conclusion, this methodology makes it possible to obtain retinal-specific total RNA for genomics research on retinal development in zebrafish.

Directional Asymmetries in the Optokinetic Response of Larval Zebrafish (Danio Rerio)

Zebrafish. 2005  |  Pubmed ID: 18248193

Photographic images of the optokinetic response (OKR) of larval zebrafish permitted the calculation of the amplitude and velocity of the response to gratings of various spatial frequencies rotating at different speeds. At low spatial frequencies, the amplitude of the OKR did not vary significantly for drum speeds ranging from 24 to 108 degrees/sec. Plotting the velocity of the OKR as a function of drum speed gave rise to a bell-shaped curve, with a maximum at about 48 degrees/sec. Interestingly, both eyes exhibited an asymmetric response to the rotating drum, that is, they were more responsive to temporal-to-nasal rotation than to nasal-to-temporal motion. Although this asymmetry persisted over the entire range of drum speeds tested, the situation was reversed when tested with gratings of higher spatial frequency (i.e., the eyes became more responsive to rotation in the nasal-to-temporal direction). The amplitude of the OKR for both eyes exhibited an inverse relation to increasing spatial frequency of the stimulus, whereas the velocity of the OKR showed a steep decline within the range of 0.08 to 0.14 cycles/degree. The data indicate that zebrafish are more responsive to objects with low spatial frequencies moving from behind the animal's head toward the frontal plane, and to high spatial frequencies of objects moving across the frontal plane (perpendicular to the anterior-posterior axis of the eye).

Identification of Zebrafish Insertional Mutants with Defects in Visual System Development and Function

Genetics. May, 2005  |  Pubmed ID: 15716491

Genetic analysis in zebrafish has been instrumental in identifying genes necessary for visual system development and function. Recently, a large-scale retroviral insertional mutagenesis screen, in which 315 different genes were mutated, that resulted in obvious phenotypic defects by 5 days postfertilization was completed. That the disrupted gene has been identified in each of these mutants provides unique resource through which the formation, function, or physiology of individual organ systems can be studied. To that end, a screen for visual system mutants was performed on 250 of the mutants in this collection, examining each of them histologically for morphological defects in the eye and behaviorally for overall visual system function. Forty loci whose disruption resulted in defects in eye development and/or visual function were identified. The mutants have been divided into the following phenotypic classes that show defects in: (1) morphogenesis, (2) growth and central retinal development, (3) the peripheral marginal zone, (4) retinal lamination, (5) the photoreceptor cell layer, (6) the retinal pigment epithelium, (7) the lens, (8) retinal containment, and (9) behavior. The affected genes in these mutants highlight a diverse set of proteins necessary for the development, maintenance, and function of the vertebrate visual system.

Tbx2b is Essential for Neuronal Differentiation Along the Dorsal/ventral Axis of the Zebrafish Retina

Proceedings of the National Academy of Sciences of the United States of America. Mar, 2005  |  Pubmed ID: 15755805

The mechanisms by which retinal neurons are patterned along the dorsal/ventral axis remain largely unknown, yet this patterning is integral for the topographic mapping of visual space. With an interest in elucidating the mechanisms that regulate the development of this retinal axis, we have characterized a T-box family transcription factor, Tbx2b, during zebrafish retinogenesis. Tbx2b is expressed throughout all phases of retinal development with a striking asymmetry of distribution highest dorsally to lowest ventrally. To examine Tbx2b function during retinal development, two morpholino antisense oligonucleotides were created; one blocking the translational start site of Tbx2b and the other interfering with Tbx2b mRNA splicing. Injection of either of these morpholinos resulted in profound defects in the development of the dorsal retina. By using molecular markers for neuronal subtypes, the ventral retina contained all cell types, whereas in the dorsal retina, only retinal ganglion cells expressed markers of differentiation. The cells of the dorsal retina were postmitotic, however, as demonstrated by a lack of BrdUrd incorporation during the normal periods of retinal differentiation. Markers for dorsal and ventral retinal compartments were also expressed normally in Tbx2b morphants. Combined, these observations suggest that the cellular mechanisms regulating neuronal differentiation within the retina are asymmetric about the dorsal/ventral axis and that Tbx2b mediates this process within the dorsal retina.

Retinal Bipolar Cell Input Mechanisms in Giant Danio. III. ON-OFF Bipolar Cells and Their Color-opponent Mechanisms

Journal of Neurophysiology. Jul, 2005  |  Pubmed ID: 15758056

Whole cell patch recording was performed from morphologically identified cone-driven on-off bipolar cells (Cabs) in giant danio retinal slices to study their glutamate receptors and light-evoked responses. Specific agonists were puffed in the presence of cobalt, picrotoxin, and strychnine to identify glutamate receptors on these cells. Most Cabs responded to both the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA)/kainate receptor agonist kainate and the excitatory amino acid transporter (EAAT) substrate D-aspartate, and both responses were localized to the dendrites. Kainate generated depolarizations whereas D-aspartate had E(rev) close to E(Cl) and generated hyperpolarizations, indicating that the AMPA/kainate receptors are sign-preserving, whereas the EAATs are sign-inverting. In response to white light, some Cabs gave on bipolar cell-like responses whereas others gave off bipolar cell-like ones, but many cells' responses had both on and off bipolar cell components. In response to appropriately colored center-selective stimuli, many Cabs responded to short and long wavelengths with opposite polarities and were thus double color-opponent. The depolarizing components of the responses to white or colored stimuli were suppressed by the EAAT blocker DL-threo-beta-benzyloxyaspartate (TBOA), whereas the hyperpolarizing components were reduced by the AMPA/kainate receptor antagonist 6,7-dinitroquinoxaline-2,3-dione (DNQX). These results are consistent with the hypothesis that both EAATs and AMPA/kainate receptors are involved in the generation of light-evoked responses in Cabs and that they confer these cells with on and off bipolar cell properties, respectively. Cabs can generate double color-opponent center responses by receiving inputs from certain cones through EAATs and from other cones through AMPA/kainate receptors.

Dazed Gene is Necessary for Late Cell Type Development and Retinal Cell Maintenance in the Zebrafish Retina

Developmental Dynamics : an Official Publication of the American Association of Anatomists. Jun, 2005  |  Pubmed ID: 15844196

Several molecules, such as growth factors and neurotrophic factors, are required both for the differentiation of specific retinal cell types and the long-term cell survival of all retinal neurons. As diffusible factors, these molecules act non-cell-autonomously. Here, we describe the loss of function phenotype for dazed (dzd), a gene that acts cell-autonomously for retinal cell survival and affects the differentiation of rod photoreceptors and the Muller glia. By 3 days after fertilization, dazed mutant embryos have small eyes and slight heart edema. Acridine orange staining indicated a significant degree of retinal cell death occurring by 48 hr after fertilization, and histological analysis revealed that dying cells were found in the inner and outer nuclear layers and near the marginal zones. Although molecular and morphological differentiation of the inner retina and cone photoreceptors occurred, rod photoreceptors failed to differentiate beyond a small patch in the ventral retina and rod precursors failed to respond to exogenously added retinoic acid, which normally potentiated rod differentiation. Mosaic analysis indicated that the dazed gene acts cell-autonomously for rod production and cell survival, as dazed clones failed to produce rods outside the ventral patch and dazed cells were not maintained in wild-type hosts. Raising mutants under constant light resulted in severe retinal degeneration, whereas raising embryos under constant darkness did not provide any additional protection from cell death. Behavioral analysis showed that a subpopulation of adult fish that were heterozygous for the dazed mutation had elevated visual thresholds and were night blind, suggesting that dazed may also be required for long-term dim-light vision. Taken together, our studies suggest a role for the dazed gene in rod and Muller cell development and overall retinal cell survival and maintenance.

Retinal Bipolar Cell Input Mechanisms in Giant Danio. I. Electroretinographic Analysis

Journal of Neurophysiology. Jan, 2005  |  Pubmed ID: 15229213

Electroretinograms (ERGs) were recorded from the giant danio (Danio aequipinnatus) to study glutamatergic input mechanisms onto bipolar cells. Glutamate analogs were applied to determine which receptor types mediate synaptic transmission from rods and cones to on and off bipolar cells. Picrotoxin, strychnine, and tetrodotoxin were used to isolate the effects of the glutamate analogs to the photoreceptor-bipolar cell synapse. Under photopic conditions, the group III metabotropic glutamate receptor (mGluR) antagonist (RS)-alpha-cyclopropyl-4-phosphonophenylglycine (CPPG) only slightly reduced the b-wave, whereas the excitatory amino acid transporter (EAAT) blocker dl-threo-beta-benzyl-oxyaspartate (TBOA) removed most of it. Complete elimination of the b-wave required both antagonists. The alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA)/kainate receptor antagonist 2,3-dioxo-6-nitro-1,2,3,4-tetrahydrobenzo[f]quinoxaline-7-sulfonamide (NBQX) blocked the d-wave. Under scotopic conditions, rod and cone inputs onto on bipolar cells were studied by comparing the sensitivities of the b-wave to photopically matched green and red stimuli. The b-wave was >1 log unit more sensitive to the green than to the red stimulus under control conditions. In CPPG or l-AP4 (l-(+)-2-amino-4-phosphonobutyric acid, a group III mGluR agonist), the sensitivity of the b-wave to the green stimulus was dramatically reduced and the b-waves elicited by the 2 stimuli became nearly matched. The d-wave elicited by dim green stimuli, which presumably could be detected only by the rods, was eliminated by NBQX. In conclusion: 1) cone signals onto on bipolar cells involve mainly EAATs but also mGluRs (presumably mGluR6) to a lesser extent; 2) rods signal onto on bipolars by mainly mGluR6; 3) off bipolar cells receive signals from both photoreceptor types by AMPA/kainate receptors.

Retinal Bipolar Cell Input Mechanisms in Giant Danio. II. Patch-clamp Analysis of on Bipolar Cells

Journal of Neurophysiology. Jan, 2005  |  Pubmed ID: 15229214

Glutamate receptors on giant danio retinal on bipolar cells were studied with whole cell patch clamping using a slice preparation. Cone-driven on bipolars (Cbs) and mixed-input on bipolars (Mbs) were identified morphologically. Most Cbs responded to the excitatory amino acid transporter (EAAT) substrate d-aspartate but not to the group III metabotropic glutamate receptor (mGluR) agonist l-(+)-2-amino-4-phosphonobutyric acid (l-AP4) or the AMPA/kainate receptor agonist kainate, suggesting EAATs are the primary glutamate receptors on Cbs. The EAAT inhibitor dl-threo-beta-benzyloxyasparate (TBOA) blocked all light-evoked responses of Cbs, suggesting these responses are mediated exclusively by EAATs. Conversely, all Mbs responded to d-aspartate and l-AP4 but not to kainate, indicating they have both EAATs and group III mGluRs (presumably mGluR6). The light responses of Mbs involve both receptors because they could be blocked by TBOA plus (RS)-alpha-cyclopropyl-4-phosphonophenylglycine (CPPG, a group III mGluR antagonist) but not by either alone. Under dark-adapted conditions, the responses of Mbs to green (rod-selective) stimuli were reduced by CPPG but enhanced by TBOA. In contrast, both antagonists reduced the responses to red (cone-selective) stimuli, although TBOA was more effective. Furthermore, under photopic conditions, TBOA failed to eliminate light-evoked responses of Mbs. Thus on Mbs, rod inputs are mediated predominantly by mGluR6, whereas cone inputs are mediated mainly by EAATs but also by mGluR6 to some extent. Finally, we explored the interactions between EAATs and mGluR6 in Mbs. Responses to d-aspartate were reduced by l-AP4 and vice versa. Therefore mGluR6 and EAATs suppress each other, and this might underlie mutual suppression between rod and cone signals in Mbs.

Effects of Ethanol on Photoreceptors and Visual Function in Developing Zebrafish

Investigative Ophthalmology & Visual Science. Oct, 2006  |  Pubmed ID: 17003456

Children born to mothers who have consumed alcohol during pregnancy have an array of retinal abnormalities and visual dysfunctions. In the past, rodent systems have been used to study the teratogenic effects of ethanol on vertebrate embryonic development. The exact developmental windows in which ethanol causes specific developmental defects have been difficult to determine because rodents and other mammals develop in utero. In this study, we characterized how ethanol affects the function and development of the visual system in an ex utero embryonic system, the zebrafish.

Gene Expression Profiling of Zebrafish Embryonic Retinal Pigment Epithelium in Vivo

Investigative Ophthalmology & Visual Science. Feb, 2007  |  Pubmed ID: 17251491

Eye development and photoreceptor maintenance is dependent on the retinal pigment epithelium (RPE), a thin layer of cells that underlies the neural retina. Despite its importance, development of RPE has not been studied by a genomic approach. In this study, a microarray expression-profiling methodology was established for studying RPE development.

OFF Ganglion Cells Cannot Drive the Optokinetic Reflex in Zebrafish

Proceedings of the National Academy of Sciences of the United States of America. Nov, 2007  |  Pubmed ID: 18025459

Whereas the zebrafish retina has long been an important model system for developmental and genetic studies, little is known about the responses of the inner retinal neurons. Here we report single-unit ganglion cell recordings from 5- to 6-day-old zebrafish larvae. In wild-type larvae we identify at least five subtypes of ganglion cell responses to full-field illumination, with ON-OFF and ON-type cells predominating. In the nrc mutant retina, in which the photoreceptor terminals develop abnormally, we observe normal OFF responses but abnormal ON-OFF responses and no ON responses. Previously characterized as blind, these mutants lack an optokinetic reflex (OKR), but in another behavioral assay nrc mutant fish have near-normal responses to the offset of light and slow and sluggish responses to the onset of light. Pharmacological block of the ON pathway mimics most of the nrc visual defects. We conclude that the abnormal photoreceptor terminals in nrc mutants predominantly perturb the ON pathway and that the ON pathway is necessary to drive the OKR in larval zebrafish.

Differential Expression of Duplicated VAL-opsin Genes in the Developing Zebrafish

Journal of Neurochemistry. Mar, 2008  |  Pubmed ID: 18036148

Non-visual opsins mediate various light-dependent physiological events. Our previous search for non-visual opsin genes in zebrafish led to the discovery of VAL-opsin (VAL-opsinA) in deep brain cells and retinal horizontal cells of the adult fish. In this study, we report the identification and characterization of its duplicated gene, VAL-opsinB, in zebrafish. A molecular phylogenetic analysis indicates that VAL-opsinB is orthologous to a previously reported salmon gene and that the duplication of the VAL-opsin gene occurred in the teleost lineage. The recombinant protein of zebrafish VAL-opsinB forms a green-sensitive photopigment when reconstituted with 11-cis-retinal. VAL-opsinB expression was detected in a limited number of cells of the brain and the eye, and the expression pattern is distinct from that of the VAL-opsinA gene. Such a differential expression pattern suggests that VAL-opsinA and VAL-opsinB are involved in different physiological events in zebrafish.

Probing Pineal-specific Gene Expression with Transgenic Zebrafish

Photochemistry and Photobiology. Jul-Aug, 2008  |  Pubmed ID: 18466202

The pineal gland of zebrafish (Danio rerio) contains light-sensitive photoreceptor cells and plays an important role in the neuroendocrine system. The zebrafish exorhodopsin gene encodes a pineal-specific photoreceptive protein, whose promoter region harbors a cis-acting element, pineal expression-promoting element (PIPE), directing pineal-specific gene expression. For in vivo genetic studies on PIPE-binding proteins and their regulatory mechanisms, we generated a transgenic zebrafish line, Tg(P(20)-rh/P:gfp), that expresses green fluorescent protein (GFP) under the control of the zebrafish rhodopsin promoter fused with 20 PIPE repeats. In Tg(P(20)-rh/P:gfp) fish, PIPE-dependent gene expression is visualized by GFP fluorescence in the pineal gland along with PIPE-independent GFP signals in the retinal rod photoreceptors. The transgenic fish exhibit detectable and reproducible GFP fluorescence in the larval pineal gland by 5 days postfertilization. Antisense morpholino-mediated knock-down of a pineal transcription factor gene, otx5, suppresses pineal GFP expression in the transgenic line. In a pilot screen of N-ethyl-N-nitrosourea-treated fish of the GFP transgenic line, we isolated potential dominant mutations that cause attenuation of pineal GFP fluorescence with a marginal effect on the retinal GFP signal. The results suggest that the Tg(P(20)-rh/P:gfp) line will be useful for detecting deficits in PIPE-dependent gene expression in the pineal gland.

Factorial Microarray Analysis of Zebrafish Retinal Development

Proceedings of the National Academy of Sciences of the United States of America. Sep, 2008  |  Pubmed ID: 18753621

In a zebrafish recessive mutant young (yng), retinal cells are specified to distinct cell classes, but they fail to morphologically differentiate. A null mutation in a brahma-related gene 1 (brg1) is responsible for this phenotype. To identify retina-specific Brg1-regulated genes that control cellular differentiation, we conducted a factorial microarray analysis. Gene expression profiles were compared from wild-type and yng retinas and stage-matched whole embryos at 36 and 52 hours postfertilization (hpf). From our analysis, three categories of genes were identified: (i) Brg1-regulated retinal differentiation genes (731 probesets), (ii) retina-specific genes independent of Brg1 regulation (3,038 probesets), and (iii) Brg1-regulated genes outside the retina (107 probesets). Biological significance was confirmed by further analysis of components of the Cdk5 signaling pathway and Irx transcription factor family, representing genes identified in category 1. This study highlights the utility of factorial microarray analysis to efficiently identify relevant regulatory pathways influenced by both specific gene products and normal developmental events.

Zebrafish: a Model System for the Study of Eye Genetics

Progress in Retinal and Eye Research. Jan, 2008  |  Pubmed ID: 17962065

Over the last decade, the use of the zebrafish as a genetic model has moved beyond the proof-of-concept for the analysis of vertebrate embryonic development to demonstrated utility as a mainstream model organism for the understanding of human disease. The initial identification of a variety of zebrafish mutations affecting the eye and retina, and the subsequent cloning of mutated genes have revealed cellular, molecular and physiological processes fundamental to visual system development. With the increasing development of genetic manipulations, sophisticated techniques for phenotypic characterization, behavioral approaches and screening strategies, the identification of novel genes or novel gene functions will have important implications for our understanding of human eye diseases, pathogenesis, and treatment.

Morphological Types and Connectivity of Horizontal Cells Found in the Adult Zebrafish (Danio Rerio) Retina

The Journal of Comparative Neurology. Jan, 2008  |  Pubmed ID: 18022944

We describe here different types of horizontal cells in the zebrafish retina and how they connect to photoreceptors. To label horizontal cells, crystals of DiI were placed onto the tips of pulled glass pipettes and inserted into the inner nuclear layer of fixed whole-mount retinas. The DiI-labeled horizontal cells were imaged by confocal microscopy and analyzed according to dendritic arborization, cell depth, dendritic terminal morphology, and connectivity with photoreceptors. Three types of horizontal cells were unequivocally identified: two cone-connecting (H1/2 and H3) and one rod-related cell. H1/2 cells have dendritic terminals that are arranged in "rosette" clusters and that connect to cone photoreceptors without any apparent specificity. H3 cells are larger and have dendritic terminal doublets arranged in a rectilinear pattern. This pattern corresponds to the mosaic of the single cones in the zebrafish photoreceptor mosaic and indicates that H3 cells connect specifically to either the blue-sensitive (long-single) or ultraviolet-sensitive (short-single) cones. Thus, H3 cells are likely to be chromaticity-type cells that process specific color information, whereas H1/2 cells are probably luminosity-type cells that process luminance information. Rod horizontal cells were identified by their shape and dendritic pattern, and they connect with numerous rod photoreceptors via small spherical terminals.

Specificity of the Horizontal Cell-photoreceptor Connections in the Zebrafish (Danio Rerio) Retina

The Journal of Comparative Neurology. Oct, 2009  |  Pubmed ID: 19655401

Horizontal cells (HCs) are involved in establishing the center-surround receptive field organization of photoreceptor and bipolar cells. In many species, HCs respond differentially to colors and may play a role in color vision. An earlier study from our laboratory suggested that four types of HCs exist in the zebrafish retina: three cone HCs (H1, H2 and H3) and one rod HC. In this study, we describe their photoreceptor connections. Cones are arranged in a mosaic in which rows of alternating blue (B)- and ultraviolet (UV)-sensitive single cones alternate with rows of red (R)- and green (G)-sensitive double cones; the G cones are adjacent to UV cones and B cones adjacent to R cones. Two small-field (H1 and H2) and two large-field (H3 and rod HC) cells were observed. The cone HC dendritic terminals connected to cones with single boutons, doublets, or rosettes, whereas the rod HCs connected to rods with single boutons. The single boutons/doublets/rosettes of cone HCs were arranged in double rows separated by single rows for H1 cells, in pairs and singles for H2 cells, and in a rectilinear pattern for H3 cells. These connectivity patterns suggest that H1 cells contact R, G, and B cones, H2 cells G, B, and UV cones, and H3 cells B and UV cones. These predictions were confirmed by applying the DiI method to SWS1-GFP retinas whose UV cones express green fluorescent protein. Each rod HC was adjacent to the soma or axon of a DiI-labeled cone HC and connected to 50-200 rods.

Zebrafish Larvae Lose Vision at Night

Proceedings of the National Academy of Sciences of the United States of America. Mar, 2010  |  Pubmed ID: 20224035

Darkness serves as a stimulus for vertebrate photoreceptors; they are actively depolarized in the dark and hyperpolarize in the light. Here, we show that larval zebrafish essentially turn off their visual system at night when they are not active. Electroretinograms recorded from larval zebrafish show large differences between day and night; the responses are normal in amplitude throughout the day but are almost absent after several hours of darkness at night. Behavioral testing also shows that larval zebrafish become unresponsive to visual stimuli at night. This phenomenon is largely circadian driven as fish show similar dramatic changes in visual responsiveness when maintained in continuous darkness, although light exposure at night partially restores the responses. Visual responsiveness is decreased at night by at least two mechanisms: photoreceptor outer segment activity decreases and synaptic ribbons in cone pedicles disassemble.

Larval Zebrafish Turn off Their Photoreceptors at Night

Communicative & Integrative Biology. Sep, 2010  |  Pubmed ID: 21057632

Studies in several vertebrate species have shown that visual sensitivity and a number of other retinal phenomena are regulated by circadian mechanisms. For example, ultra-structural studies of 5 day old zebrafish larvae have shown that synaptic ribbons in photoreceptor terminals undergo dramatic diurnal alterations. These synaptic ribbons are very prominent during the day, but are almost completely absent at night. The implications of this circadian driven process on visual function are not well understood. We recently showed that larval zebrafish essentially lose visual responsiveness at night. This shut-down of retinal function at night is regulated by at least two mechanisms: the disassembly of synaptic ribbons in cone pedicles and a decrease of outer segment activity. Here, we summarize our recently reported observations and further discuss our hypothesis on how this phenomenon of shutting-down retinal function at night may provide a means for zebrafish larvae to conserve energy.

The Bugeye Mutant Zebrafish Exhibits Visual Deficits That Arise with the Onset of an Enlarged Eye Phenotype

Investigative Ophthalmology & Visual Science. Jun, 2011  |  Pubmed ID: 21460263

The bugeye mutant has an enlarged eye phenotype, presumably because of elevated intraocular pressure. Since elevated intraocular pressure is a significant risk factor for glaucoma, the bugeye zebrafish mutant may be a model organism for the disease.

Cellular Expression of Smarca4 (Brg1)-regulated Genes in Zebrafish Retinas

BMC Developmental Biology. 2011  |  Pubmed ID: 21756345

In a recent genomic study, Leung et al. used a factorial microarray analysis to identify Smarca4 (Brg1)-regulated genes in micro-dissected zebrafish retinas. Two hundred and fifty nine genes were grouped in three-way ANOVA models which carried the most specific retinal change. To validate the microarray results and to elucidate cellular expression patterns of the significant genes for further characterization, 32 known genes were randomly selected from this group. In situ hybridization of these genes was performed on the same types of samples (wild-type (WT) and smarca4a50/a50 (yng) mutant) at the same stages (36 and 52 hours post-fertilization (hpf)) as in the microarray study.