Zebrafish Mutagenesis Yields Eye Morphological Mutants with Retinal and Lens Defects

Vision Research. Feb, 2002  |  Pubmed ID: 11853770

A chemical mutagenesis to identify zebrafish eye morphological mutants was performed by screening F(3) larvae at 5 and 7 days post-fertilization (dpf) for changes in eye or pupil size. Based on histological analysis, four different phenotypic classes were obtained. The two Class I and three Class II mutants are all characterized by small eyes and exhibit defects in early retinal development or unregulated cell death, respectively. The single Class III mutant has reduced ocular pigmentation. The three Class IV mutants display defects in the ocular lens, including one mutant line with normal sized eyes and pupils that develops lens opacity at 7 dpf.

Regulation of Light-dependent Gqalpha Translocation and Morphological Changes in Fly Photoreceptors

The EMBO Journal. Feb, 2003  |  Pubmed ID: 12554647

Heterotrimeric G-proteins relay signals between membrane-bound receptors and downstream effectors. Little is known, however, about the regulation of Galpha subunit localization within the natural endogenous environment of a specialized signaling cell. Here we show, using live Drosophila flies, that light causes massive and reversible translocation of the visual Gqalpha to the cytosol, associated with marked architectural changes in the signaling compartment. Molecular genetic dissection together with detailed kinetic analysis enabled us to characterize the translocation cycle and to unravel how signaling molecules that interact with Gqalpha affect these processes. Epistatic analysis showed that Gqalpha is necessary but not sufficient to bring about the morphological changes in the signaling organelle. Furthermore, mutant analysis indicated that Gqbeta is essential for targeting of Gqalpha to the membrane and suggested that Gqbeta is also needed for efficient activation of Gqalpha by rhodopsin. Our results support the 'two-signal model' hypothesis for membrane targeting in a living organism and characterize the regulation of both the activity-dependent Gq localization and the cellular architectural changes in Drosophila photoreceptors.

Novel Dominant Rhodopsin Mutation Triggers Two Mechanisms of Retinal Degeneration and Photoreceptor Desensitization

The Journal of Neuroscience : the Official Journal of the Society for Neuroscience. Mar, 2004  |  Pubmed ID: 15014127

A variety of rod opsin mutations result in autosomal dominant retinitis pigmentosa and congenital night blindness in humans. One subset of these mutations encodes constitutively active forms of the rod opsin protein. Some of these dominant rod opsin mutant proteins, which desensitize transgenic Xenopus rods, provide an animal model for congenital night blindness. In a genetic screen to identify retinal degeneration mutants in Drosophila, we identified a dominant mutation in the ninaE gene (NinaE(pp100)) that encodes the rhodopsin that is expressed in photoreceptors R1-R6. Deep pseudopupil analysis and histology showed that the degeneration was attributable to a light-independent apoptosis. Whole-cell recordings revealed that the NinaE(pp100) mutant photoreceptor cells were strongly desensitized, which partially masked their constitutive activity. This desensitization primarily resulted from both the persistent binding of arrestin (ARR2) to the NINAE(pp100) mutant opsin and the constitutive activity of the phototransduction cascade. Whereas mutations in several Drosophila genes other than ninaE were shown to induce photoreceptor cell apoptosis by stabilizing a rhodopsin-arrestin complex, NinaE(pp100) represented the first rhodopsin mutation that stabilized this protein complex. Additionally, the NinaE(pp100) mutation led to elevated levels of G(q)alpha in the cytosol, which mediated a novel retinal degeneration pathway. Eliminating both G(q)alpha and arrestin completely rescued the NinaE(pp100)-dependent photoreceptor cell death, which indicated that the degeneration is entirely dependent on both G(q)alpha and arrestin. Such a combination of multiple pathological pathways resulting from a single mutation may underlie several dominant retinal diseases in humans.

The Zebrafish Pard3 Ortholog is Required for Separation of the Eye Fields and Retinal Lamination

Developmental Biology. May, 2004  |  Pubmed ID: 15081374

The vertebrate retina develops from a sheet of neuroepithelial cells. Because adherens and tight junctions are critical for epithelial and neuronal differentiation in a variety of eukaryotic systems, we examined the role of Par-3, a PDZ scaffold protein that is critical in cellular membrane junction formation. We cloned the zebrafish Par-3 ortholog (pard3), which encodes two Pard3 proteins (150 and 180 kDa) that differ in their carboxyl-terminus. Immunohistochemistry revealed that Pard3 localized to the apical region of the retinal and brain neuroepithelium, partially overlapping the adherens junction-associated actin bundles. After retinal lamination, the Pard3 protein was restricted to the outer limiting membrane and the outer and inner plexiform layers in the retina. Reducing Pard3 expression with antisense morpholinos caused loss of the retinal pigmented epithelia, disruption of retinal lamination, and cell death in the ventral diencephalon, which resulted in cyclopia. Overexpressing Pard3 by injection of wild-type pard3 mRNA resulted in cyclopia and eyeless embryos. Thus, Pard3 plays a critical role in the origination and separation of zebrafish eye fields and retinal lamination.

Lens Opacity and Photoreceptor Degeneration in the Zebrafish Lens Opaque Mutant

Developmental Dynamics : an Official Publication of the American Association of Anatomists. May, 2005  |  Pubmed ID: 15765514

The zebrafish lens opaque (lop) mutant was identified in a chemical mutagenesis screen. The lop mutant, which develops normally through 4 days postfertilization (dpf), exhibits several signs of lens and retinal degeneration at 7 dpf. Histology revealed disrupted lens fibers and increased numbers of nucleated cells within the mutant lens and anterior chamber. The mutant lens also exhibited aberrant epithelial cell morphologies and lacked a definitive transition zone, which suggests that secondary fiber differentiation was interrupted. In addition, the mutant exhibits severely reduced photoreceptors and a reduction in the number of horizontal cells at 7 dpf. Other retinal cell classes appeared unaffected in the mutant. Transmission electron microscopy and opsin immunohistochemistry showed that the different photoreceptor types were generated at the retinal margin, but the rods and cones failed to mature and disappeared. The mutant lens and retina also displayed increased cell proliferation based on proliferating cell nuclear antigen immunolabeling, suggesting that the lens opacity was due to unregulated cell proliferation and undifferentiated cell accumulation within the mutant lens. The lop mutant phenotype supports recent studies showing the lens has a role in regulating teleost retinal development.

Cre-mediated Site-specific Recombination in Zebrafish Embryos

Developmental Dynamics : an Official Publication of the American Association of Anatomists. Aug, 2005  |  Pubmed ID: 15977183

Cre-mediated site-specific recombination has become an invaluable tool for manipulation of the murine genome. The ability to conditionally activate gene expression or to generate chromosomal alterations with this same tool would greatly enhance zebrafish genetics. This study demonstrates that the HSP70 promoter can be used to inducibly control expression of an enhanced green fluorescent protein (EGFP) -Cre fusion protein. The EGFP-Cre fusion protein is capable of promoting recombination between lox sites in injected plasmids or in stably inherited transgenes as early as 2 hr post-heat shock induction. Finally, the levels of Cre expression achieved in a transgenic fish line carrying the HSP70-EGFP-cre transgene are compatible with viability and both male and female transgenic fish are fertile subsequent to induction of EGFP-Cre expression. Hence, our data suggests that Cre-mediated recombination is a viable means of manipulating gene expression in zebrafish.

Expressed Sequence Tag Analysis of Zebrafish Eye Tissues for NEIBank

Molecular Vision. 2005  |  Pubmed ID: 16379021

To characterize gene expression patterns in various tissues of the zebrafish (Danio rerio) eye and identify zebrafish orthologs of human genes by expressed sequence tag (EST) analysis for NEIBank.

Zebrafish Foxe3: Roles in Ocular Lens Morphogenesis Through Interaction with Pitx3

Mechanisms of Development. Oct, 2006  |  Pubmed ID: 16963235

Foxe3 is a winged helix/forkhead domain transcription factor necessary for mammalian and amphibian lens development. Human FOXE3 mutations cause anterior segment dysgenesis and cataracts. The zebrafish foxe3 cDNA was PCR amplified from 24 h post-fertilization (hpf) embryo cDNA. The zebrafish foxe3 gene consists of a single exon on chromosome 8 and encodes a 422 amino acid protein. This protein possesses 44% and 67% amino acid identity with the human FOXE3 and Xenopus FoxE4 proteins, respectively. A polyclonal antiserum was generated against a bacterial fusion protein containing the Foxe3 carboxyl terminus. The purified antiserum detects zebrafish Foxe3 on immunoblots, in embryo wholemounts, and frozen tissue sections. The zebrafish Foxe3 protein is first detected in the lens at 31hpf and is restricted to the nucleated cell population, including the epithelial and elongating fiber cells. Knockdown of Foxe3 protein using an antisense morpholino results in small lenses with multilayered epithelial cells and fiber cell dysmorphogenesis. The morphants posses normal retinas, although retinal cell proteins, including rhodopsin, are abnormally expressed in the morphant lens tissue. Functional interactions between foxe3 and pitx3 during lens development were assessed by RT-PCR and comparison of Foxe3 and Pitx3 protein expression in both foxe3 and pitx3 morphants. Immunoblots and immunohistochemistry reveal Pitx3 is expressed in the foxe3 morphant lens, while Pitx3 knockdown results in the elimination of Foxe3 expression. These data demonstrate that Foxe3 is necessary for lens development in zebrafish and that foxe3 lies genetically downstream of pitx3 in a zebrafish lens development pathway.

Mutations in Laminin Alpha 1 Result in Complex, Lens-independent Ocular Phenotypes in Zebrafish

Developmental Biology. Nov, 2006  |  Pubmed ID: 16973147

We report phenotypic and genetic analyses of a recessive, larval lethal zebrafish mutant, bal(a69), characterized by severe eye defects and shortened body axis. The bal(a69) mutation was mapped to chromosome 24 near the laminin alpha 1 (lama1) gene. We analyzed the lama1 gene sequence within bal(a69) embryos and two allelic mutants, bal(arl) and bal(uw1). Missense (bal(a69)), nonsense (bal(arl)), and frameshift (bal(uw1)) alterations in lama1 were found to underlie the phenotypes. Extended analysis of bal(a69) ocular features revealed disrupted lens development with subsequent lens degeneration, focal cornea dysplasia, and hyaloid vasculature defects. Within the neural retina, the ganglion cells showed axonal projection defects and ectopic photoreceptor cells were noted at inner retinal locations. To address whether ocular anomalies were secondary to defects in lens differentiation, bal(a69) mutants were compared to embryos in which the lens vesicle was surgically removed. Our analysis suggests that many of the anterior and posterior ocular defects in bal(a69) are independent of the lens degeneration. Analysis of components of focal adhesion signaling complexes suggests that reduced focal adhesion kinase activation underlies the anterior segment dysgenesis in lama1 mutants. To assess adult ocular phenotypes associated with lama1 mutations, genetic mosaics were generated by transplanting labeled bal cells into ocular-fated regions of wild-type blastulas. Adult chimeric eyes displayed a range of defects including anterior segment dysgenesis and cataracts. Our analysis provides mechanistic insights into the developmental defects and ocular pathogenesis caused by mutations in laminin subunits.

Molecular Cloning of Three Zebrafish Lin7 Genes and Their Expression Patterns in the Retina

Experimental Eye Research. Jan, 2006  |  Pubmed ID: 16109407

The vertebrate retina develops from an undifferentiated sheet of neuroepithelial cells, whose differentiation requires the generation and maintenance of the correct cellular polarity. To examine the role of cell polarity in retinal development, we cloned three zebrafish lin7 genes (lin7a, lin7b, and lin7c), which each encodes a protein candidate that is required for generation/maintenance of neuroepithelial cell junctions. These three zebrafish Lin7 proteins share over 78% amino acid identity and contain both L27 and PDZ domains that are present in all Lin7 homologs. Immunoblots revealed that the Lin7b and Lin7c proteins were first expressed in the developing eye by 24hr postfertilization (hpf), while Lin7a was not detected in the eye until 72 hpf. At 33 hpf, the Lin7 proteins localized at, or slightly apical of, the actin-associated adherens junctions in the retinal neuroepithelium. This subcellular distribution required the expression of the Nok protein. In the absence of Nok, the Lin7 proteins failed to localize to either the ectopic adherens junctions or the cell membrane. At 4 days postfertilization, in situ hybridisation revealed that all three lin7 genes were expressed in both the ganglion cell layer and the bipolar cell region of the inner nuclear layer. The lin7a gene was also expressed in the amacrine and horizontal cell regions of the inner nuclear layer, while lin7c was also expressed in the outer nuclear layer. In the adult retina, where Lin7a is the predominant form expressed, the Lin7 proteins were localized to the outer and inner plexiform layers, the bipolar and horizontal cells of the inner nuclear layer, and the ganglion cells. These results suggest that the three zebrafish Lin7 proteins possess partially redundant, yet essential, roles in retinal development.

Retinal Regional Differences in Photoreceptor Cell Death and Regeneration in Light-lesioned Albino Zebrafish

Experimental Eye Research. Apr, 2006  |  Pubmed ID: 16199033

Teleost fish regenerate retinal cells from a population of inner nuclear layer (INL) stem cells. To characterize photoreceptor regeneration in zebrafish (Danio rerio), adult albino fish were subjected to constant intense light to cause photoreceptor cell death. Retinal morphometry was performed on histological sections of control and light-lesioned albino retinas to compare the extent of light damage in the ventral, central and dorsal retinal regions. In addition, opsin immunohistochemistry and TUNEL were used to compare photoreceptor cell death in these different retinal areas, while PCNA immunolabeling quantified the cell proliferation that precedes the photoreceptor regeneration. Transgenic albino; Tg(alpha1-tubulin:egfp) zebrafish were also exposed to the intense light in order to examine regeneration-related gene expression changes. The light-lesioned retinas are characterized by extensive rod and cone photoreceptor cell death in the central and dorsal regions. In contrast, many of the rods and cones survive in the ventral retina. The highest levels of INL cell proliferation, which occurs subsequent to photoreceptor death, correspond to the retinal regions that suffer the greatest levels of photoreceptor damage. In the ventral retina, where photoreceptor cell death is minimal, cell proliferation is confined to the ONL. In addition, EGFP expression from the alpha1-tubulin promoter is increased in Müller glial cells in the light-damaged central and dorsal retina, while transgene expression in the ventral retina is restricted to small, round INL cells. Furthermore, expression of the HuC/D neuronal antigen is detected in a subpopulation of the Müller cells in the light-damaged superior retinal region. These data demonstrate that adult albino zebrafish display retinal regional differences in photoreceptor cell death and in the regeneration-related INL cell proliferation response. The high levels of INL cell proliferation and alpha1-tubulin:egfp transgene expression in the Müller cells may be graded in response to the degree of photoreceptor cell death. This suggests that the levels of photoreceptor damage may directly influence cell responses in the underlying retinal layers.

Inhibition of Zebrafish Fin Regeneration Using in Vivo Electroporation of Morpholinos Against Fgfr1 and Msxb

Developmental Dynamics : an Official Publication of the American Association of Anatomists. Feb, 2006  |  Pubmed ID: 16273523

Increased interest in using zebrafish as a model organism has led to a resurgence of fin regeneration studies. This has allowed for the identification of a large number of gene families, including signaling molecules and transcription factors, which are expressed during regeneration. However, in cases where no specific inhibitor is available for the gene product of interest, determination of a functional role for these genes has been difficult. Here we demonstrate that in vivo electroporation of morpholino oligonucleotides is a feasible approach for protein knock-down during fin regeneration. Morpholino oligonucleotides against fgfr1 and msxb were utilized and knock-down of both proteins resulted in reduced fin outgrowth. Importantly, Fgfr1 knock-down phenocopied outgrowth inhibition obtained with an Fgfr1 inhibitor. Furthermore, this method provided direct evidence for a functional role for msxb in caudal fin regeneration. Finally, knock-down of Fgfr1, but not Msxb, affected the blastemal expression of msxc, suggesting this technique can be used to determine epistasis in genetic pathways affecting regeneration. Thus, this convenient reverse genetic approach allows researchers to quickly (1) assess the function of genes known to be expressed during fin regeneration, (2) screen genes for functional relevance during fin regeneration, and (3) assign genes to the molecular pathways underlying fin regeneration.

Two Different Transgenes to Study Gene Silencing and Re-expression During Zebrafish Caudal Fin and Retinal Regeneration

TheScientificWorldJournal. 2006  |  Pubmed ID: 17205188

We used the 500-bp Xenopus ef1-alpha promoter and the 2-kb zebrafish histone 2A.F/Z promoter to generate several independent transgenic zebrafish lines expressing EGFP. While both promoters drive ubiquitous EGFP expression in early zebrafish development, they are systematically silenced in several adult tissues, including the retina and caudal fin. However, EGFP expression is temporarily renewed in the adult during either caudal fin or retinal regeneration. In the Tg(H2A.F/Z:EGFP)nt line, EGFP is moderately expressed in both the wound epithelium and blastema of the regenerating caudal fin. In the Tg(ef1-alpha:EGFP)nt line, EGFP expression is reinitiated and restricted to the blastema of the regenerating caudal fin and colabels with BrdU, PCNA, and msxc-positive cells. Thus, these two ubiquitous promoters drive EGFP transgene expression in different cell populations during caudal fin regeneration. We further analyzed the ability of the ef1-alpha:EGFP transgene to label nonterminally differentiated cells during adult tissue regeneration. First, we demonstrated that the transgene is highly methylated in adult zebrafish caudal fin tissue, but not during fin regeneration, implicating methylation as a potential means of transgene silencing in this line. Next, we determined that the ef1-alpha:EGFP transgene is also re-expressed during adult retinal regeneration. Specifically, the ef1-alpha:EGFP transgene colabels with PCNA in the Müller glia, a specialized cell that is the source of neuronal progenitors during zebrafish retinal regeneration. Thus, we concluded that Tg(ef1-alpha:EGFP)nt line visually marks nonterminally differentiated cells in multiple adult regeneration environments and may prove to be a useful marker in tissue regeneration studies in zebrafish.

Regeneration of Inner Retinal Neurons After Intravitreal Injection of Ouabain in Zebrafish

The Journal of Neuroscience : the Official Journal of the Society for Neuroscience. Feb, 2007  |  Pubmed ID: 17301179

We examined the regenerative capacity of the adult zebrafish retina by intravitreal injection of a low ouabain concentration to rapidly damage the ganglion cell layer (GCL) and inner nuclear layer (INL) with minimal photoreceptor cell damage. By 24 h after ouabain injection, maximal numbers of terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling (TUNEL)-positive cells were detected in the INL and GCL, with low numbers of TUNEL-positive cells in the outer nuclear layer. Immunolabeling revealed that approximately 85% of the HuC/D-positive amacrine and ganglion cells were lost by 7 d post-ouabain injection (dpi). This ganglion cell loss was consistent with the small, but statistically significant, decrease in the optic nerve diameter. The regeneration response began within 1 dpi with increased proliferating cell nuclear antigen (PCNA) expression in both the INL and GCL. By 3 dpi, PCNA expression is primarily restricted to the Müller glia. By 5 dpi, most of the PCNA expression was localized to neuronal progenitors expressing the olig2:egfp transgene rather than the Müller glia. By 7 dpi, the neuronal progenitors began committing to the ganglion cell fate based on the coexpression of the atoh7:EGFP transgene and the zn5 antigen. The regeneration of ganglion and amacrine cells continued until 60 dpi, when they reached 75% of their uninjected control number. This demonstrates that inner retinal damage, without extensive photoreceptor damage, is sufficient to induce a regeneration response that is marked by the Müller glial cells reentering the cell cycle to produce neuronal progenitor cells that regenerate INL and ganglion cells in the zebrafish retina.

A Mirror-symmetric Cell Division That Orchestrates Neuroepithelial Morphogenesis

Nature. Apr, 2007  |  Pubmed ID: 17392791

The development of cell polarity is an essential prerequisite for tissue morphogenesis during embryogenesis, particularly in the development of epithelia. In addition, oriented cell division can have a powerful influence on tissue morphogenesis. Here we identify a novel mode of polarized cell division that generates pairs of neural progenitors with mirror-symmetric polarity in the developing zebrafish neural tube and has dramatic consequences for the organization of embryonic tissue. We show that during neural rod formation the polarity protein Pard3 is localized to the cleavage furrow of dividing progenitors, and then mirror-symmetrically inherited by the two daughter cells. This allows the daughter cells to integrate into opposite sides of the developing neural tube. Furthermore, these mirror-symmetric divisions have powerful morphogenetic influence: when forced to occur in ectopic locations during neurulation, they orchestrate the development of mirror-image pattern formation and the consequent generation of ectopic neural tubes.

Time Course Analysis of Gene Expression During Light-induced Photoreceptor Cell Death and Regeneration in Albino Zebrafish

Developmental Neurobiology. Jul, 2007  |  Pubmed ID: 17565703

Constant intense light causes apoptosis of rod and cone photoreceptors in adult albino zebrafish. The photoreceptors subsequently regenerate from proliferating inner nuclear layer (INL) progenitor cells that migrate to the outer nuclear layer (ONL) and differentiate into rods and cones. To identify gene expression changes during this photoreceptor regeneration response, a microarray analysis was performed at five time points during the light treatment. The time course included an early time point during photoreceptor death (16 h), later time points during progenitor cell proliferation and migration (31, 51, and 68 h) and a 96 h time point, which likely corresponds to the initial photoreceptor differentiation. Mean expression values for each gene were calculated at each time point relative to the control (0 h light exposure) and statistical analysis by one-way ANOVA identified 4567 genes exhibiting significant changes in gene expression along the time course. The genes within this data set were clustered based on their temporal expression patterns and proposed functions. Quantitative real-time PCR validated the microarray expression profiles for selected genes, including stat3 whose expression increased markedly during the light exposure. Based on immunoblots, both total and activated Stat3 protein expression also increased during the light treatment. Immunolocalization of Stat3 on retinal tissue sections demonstrated increased expression in photoreceptors and Müller glia by 16 h of light exposure. Some of the Stat3-positive Müller cells expressed PCNA at 31 h, suggesting that Stat3 may play a role in signaling a subset of Müller cells to proliferate during the regeneration response.

Genetic Determinants of Hyaloid and Retinal Vasculature in Zebrafish

BMC Developmental Biology. 2007  |  Pubmed ID: 17937808

The retinal vasculature is a capillary network of blood vessels that nourishes the inner retina of most mammals. Developmental abnormalities or microvascular complications in the retinal vasculature result in severe human eye diseases that lead to blindness. To exploit the advantages of zebrafish for genetic, developmental and pharmacological studies of retinal vasculature, we characterised the intraocular vasculature in zebrafish.

Mitogen-associated Protein Kinase- and Protein Kinase A-dependent Regulation of Rhodopsin Promoter Expression in Zebrafish Rod Photoreceptor Cells

Journal of Neuroscience Research. Feb, 2007  |  Pubmed ID: 17183589

Mitogen-associated protein kinase (MAPK)- and protein kinase A (PKA)-dependent signal transductions play important roles in the regulation of gene expression. Both MAPK and PKA pathways can be activated by light exposure. In this study, we investigated the effect of light on MAPK and PKA signal transduction and their roles in the regulation of rhodopsin promoter expression by using transgenic zebrafish [Tg(rhod::GFP)]. The Tg(rhod::GFP) fish express short half-life GFP that is under the transcriptional control of the zebrafish rhodopsin promoter and can therefore be used for in vivo studies of rhodopsin gene transcription in live cells. Blue light plays a role in the regulation of rhodopsin promoter expression via an MAPK-mediated signal transduction cascade. Blue light excites cryptochromes (CRY), which activate the downstream PKC-dependent MAPK signal pathway. White light, on the other hand, regulates rhodopsin promoter expression via a G-protein-coupled cAMP-dependent PKA pathway. White light promotes dopamine release in the retina, which activates dopamine receptors and the downstream PKA pathway. Blocking MAPK signaling diminishes the blue light-induced increases in rhodopsin promoter expression, but this treatment has no effect on white light-mediated rhodopsin promoter expression. Conversely, blocking the PKA pathway diminishes the white light-induced rhodopsin promoter expression but does not affect rhodopsin promoter expression regulated by blue light. Together, the data suggest that MAPK and PKA regulate rhodopsin transcription through parallel signal transduction pathways.

Generation and Characterization of Transgenic Zebrafish Lines Using Different Ubiquitous Promoters

Transgenic Research. Apr, 2008  |  Pubmed ID: 17968670

Two commonly used promoters to ubiquitously express transgenes in zebrafish are the Xenopus laevis elongation factor 1 alpha promoter (XlEef1a1) and the zebrafish histone variant H2A.F/Z (h2afv) promoter. Recently, transgenes utilizing these promoters were shown to be silenced in certain adult tissues, particularly the central nervous system. To overcome this limitation, we cloned the promoters of four zebrafish genes that likely are transcribed ubiquitously throughout development and into the adult. These four genes are the TATA box binding protein gene, the taube nuss-like gene, the eukaryotic elongation factor 1-gamma gene, and the beta-actin-1 gene. We PCR amplified approximately 2.5 kb upstream of the putative translational start site of each gene and cloned each into a Tol2 expression vector that contains the EGFP reporter transgene. We used these four Tol2 vectors to independently generate stable transgenic fish lines for analysis of transgene expression during development and in the adult. We demonstrated that all four promoters drive a very broad pattern of EGFP expression throughout development and the adult. Using the retina as a well-characterized component of the CNS, all four promoters appeared to drive EGFP expression in all neuronal and non-neuronal cells of the adult retina. In contrast, the h2afv promoter failed to express EGFP in the adult retina. When we examined EGFP expression in the various cells of the blood cell lineage, we observed that all four promoters exhibited a more heterogenous expression pattern than either the XlEef1a1 or h2afv promoters. While these four ubiquitous promoters did not express EGFP in all the adult blood cells, they did express EGFP throughout the CNS and in broader expression patterns in the adult than either the XlEef1a1 or h2afv promoters. For these reasons, these four promoters will be valuable tools for expressing transgenes in adult zebrafish.

Ganglion Cell Regeneration Following Whole-retina Destruction in Zebrafish

Developmental Neurobiology. Feb, 2008  |  Pubmed ID: 18000816

The retinas of adult teleost fish can regenerate neurons following injury. The current study provides the first documentation of functional whole retina regeneration in the zebrafish, Danio rerio, following intraocular injection of the cytotoxin, ouabain. Loss and replacement of laminated retinal tissue was monitored by analysis of cell death and cell proliferation, and by analysis of retina-specific gene expression patterns. The spatiotemporal process of retinal ganglion cell (RGC) regeneration was followed through the use of selective markers, and was found to largely recapitulate the spatiotemporal process of embryonic ganglion cell neurogenesis, over a more protracted time frame. However, the re-expression of some ganglion cell markers was not observed. The growth and pathfinding of ganglion cell axons was evaluated by measurement of the optic nerve head (ONH), and the restoration of normal ONH size was found to correspond to the time of recovery of two visually-mediated behaviors. However, some abnormalities were noted, including overproduction of RGCs, and progressive and excessive growth of the ONH at longer recovery times. This model system for whole-retina regeneration has provided an informative view of the regenerative process.

Inhibition of Müller Glial Cell Division Blocks Regeneration of the Light-damaged Zebrafish Retina

Developmental Neurobiology. Feb, 2008  |  Pubmed ID: 18161852

The adult zebrafish retina possesses a robust regenerative response. In the light-damaged retina, Müller glial cell divisions precede regeneration of rod and cone photoreceptors. Neuronal progenitors, which arise from the Müller glia, continue to divide and use the Müller glial cell processes to migrate to the outer nuclear layer and replace the lost photoreceptors. We tested the necessity of Müller glial cell division for photoreceptor regeneration. As knockdown tools were unavailable for use in the adult zebrafish retina, we developed a method to conditionally inhibit the expression of specific proteins by in vivo electroporation of morpholinos. We determined that two separate morpholinos targeted against the proliferating cell nuclear antigen (PCNA) mRNA reduced PCNA protein levels. Furthermore, injection and in vivo electroporation of PCNA morpholinos immediately prior to starting intense light exposure inhibited both Müller glial cell proliferation and neuronal progenitor marker Pax6 expression. PCNA knockdown additionally resulted in decreased expression of glutamine synthetase in Müller glia and Müller glial cell death, while amacrine and ganglion cells were unaffected. Finally, histological and immunological methods showed that long-term effects of PCNA knockdown resulted in decreased numbers of Müller glia and the failure to regenerate rod photoreceptors, short single cones, and long single cones. These data suggest that Müller glial cell division is necessary for proper photoreceptor regeneration in the light-damaged zebrafish retina and are consistent with the Müller glia serving as the source of neuronal progenitor cells in regenerating teleost retinas.

Fgf-dependent Depletion of MicroRNA-133 Promotes Appendage Regeneration in Zebrafish

Genes & Development. Mar, 2008  |  Pubmed ID: 18347091

Appendage regeneration is defined by rapid changes in gene expression that achieve dramatic developmental effects, suggesting involvement of microRNAs (miRNAs). Here, we find dynamic regulation of many miRNAs during zebrafish fin regeneration. In particular, miR-133 levels are high in uninjured fins but low during regeneration. When regeneration was blocked by Fibroblast growth factor (Fgf) receptor inhibition, high miR-133 levels were quickly restored. Experimentally increasing amounts of miR-133 attenuated fin regeneration. Conversely, miR-133 antagonism during Fgf receptor inhibition accelerated regeneration through increased proliferation within the regeneration blastema. The Mps1 kinase, an established positive regulator of blastemal proliferation, is an in vivo target of miR-133. Our findings identify miRNA depletion as a new regulatory mechanism for complex tissue regeneration.

Connexin43 (GJA1) is Required in the Population of Dividing Cells During Fin Regeneration

Developmental Biology. May, 2008  |  Pubmed ID: 18406403

In zebrafish, mutations in the gap junction gene connexin43 lead to short bony fin ray segments that give rise to the short fin phenotype. The sof(b123) mutant exhibits fins that are half the length of wild-type fins and have reduced levels of cx43 mRNA. We find that sof(b123) regenerating fins exhibit reduced levels of cell proliferation. Interestingly, the number of dividing cells per unit length of fin growth is similar between wild-type and mutant fins, suggesting that the number of cells that enter the cell cycle is specifically affected in sof(b123). Expression of cx43 is identified in mitotic cells, which further suggests that Cx43 may contribute to establishing or maintaining the population of dividing cells. Indeed, missense alleles exhibiting high or low levels of gap junctional communication reveal a correlation between defects in direct cell-cell communication, cell proliferation, and segment length. Finally, targeted gene knockdown of cx43 in adult regenerating fins recapitulates the sof(b123) phenotype, revealing that the loss of Cx43 is sufficient to reduce both cell proliferation and segment length. We hypothesize that the level of gap junctional intercellular communication among dividing cells regulates the level of cell proliferation and ultimately regulates bone growth.

Lengsin Expression and Function During Zebrafish Lens Formation

Experimental Eye Research. May, 2008  |  Pubmed ID: 18406404

A zebrafish ortholog of human lengsin was identified by EST analysis of an adult lens cDNA library. During zebrafish development, lengsin transcription is first detected at 24 h post-fertilization (hpf). Immunolocalization, using polyclonal antiserum generated against a Lengsin bacterial fusion protein, detects lens-specific protein in whole-mount embryos at 30 hpf. Lengsin expression in zebrafish follows the temporal expression of the alphaA- alphaB1- and betaB1-crystallin proteins in the lens. At 72 hpf, Lengsin is localized to a subpopulation of differentiating secondary fiber cells, while no expression is detected in the lens epithelial cells or central lens fibers. In the adult lens, Lengsin is restricted to a narrow band of cortical fibers and co-localizes with actin at the lateral faces of these interdigitating cells. Stable transgenic lines, using a 3 kb lengsin genomic fragment to regulate EGFP expression, recapitulate the Lengsin temporal and spatial expression patterns. Lengsin function in zebrafish lens formation was examined by antisense morpholino-mediated translation and mRNA splice inhibition. At 72 hpf, the lengsin morphant lenses are reduced in size and exhibit separations within the cortex due to defects in secondary fiber morphogenesis. The location of the morphant lens defects correlates with the Lengsin protein localization at this age. These results demonstrate Lengsin is required for proper fiber cell differentiation by playing roles in either cell elongation or the establishment of cell interactions.

The Tg(ccnb1:EGFP) Transgenic Zebrafish Line Labels Proliferating Cells During Retinal Development and Regeneration

Molecular Vision. 2008  |  Pubmed ID: 18509551

To create the Tg(ccnb1:EGFP)(nt18) zebrafish line that spatially and temporally labels retinal progenitor cells with enhanced green fluorescent protein (EGFP) during zebrafish retinal development and regeneration.

Characterization of Müller Glia and Neuronal Progenitors During Adult Zebrafish Retinal Regeneration

Experimental Eye Research. Nov, 2008  |  Pubmed ID: 18718467

The adult zebrafish retina exhibits a robust regenerative response following light-induced photoreceptor cell death. This response is initiated by the Müller glia proliferating in the inner nuclear layer (INL), which gives rise to neuronal progenitor cells that continue to divide and migrate to the outer nuclear layer (ONL), where they differentiate into rod and cone photoreceptors. We previously conducted a microarray analysis of retinal gene expression at 16, 31, 51, 68, and 96 h of constant intense-light treatment to identify genes and their corresponding proteins that may be involved in the generation and proliferation of the neuronal progenitor cells. We examined the expression of two candidate transcription factors, Pax6 and Ngn1, and one candidate transgene, olig2:EGFP, in the regenerating light-damaged retina. We compared the temporal and spatial expression patterns of these markers relative to PCNA (proliferating cell nuclear antigen), an established marker for proliferating cells in the zebrafish retina, and the Tg(gfap:EGFP) nt11 transgenic line that specifically labels Müller glial cells. We found that Müller glial cells dedifferentiate during regeneration, based on the loss of cell-specific markers such as GFAP (glial fibrillary acidic protein) and glutamine synthetase following their reentry into the cell cycle to produce neuronal progenitors. Pax6 expression was first detected in the proliferating neuronal progenitors by 51 h of constant light treatment, which is significantly after the Müller glia first reenter the cell cycle after 31h of light. This suggests that Pax6 expression increases in neuronal progenitors, rather than in the proliferating Müller glia. EGFP expression from the olig2 promoter was first detected by 68 h of constant light treatment in the dedifferentiated Müller glia, with Pax6 expressed in the closely associated proliferating neuronal progenitors migrating to the ONL. Both Pax6 and olig2 expression persisted until 3 days post-light treatment, when the neuronal progenitors begin differentiating into new rod and cone photoreceptors. Ngn1 protein expression was initially detected in proliferating neuronal progenitors at 68 h of light treatment. However, Ngn1 expression persisted in a subset of the INL nuclei until 17 days post-light treatment. Using the Tg(gfap:EGFP) nt11 transgenic line, Ngn1 was localized to the Müller glial nuclei that were reestablished following the regenerative response. These markers, therefore, can be used to identify different cell types at particular stages of retinal regeneration: neuronal progenitor formation, proliferation, and the reestablishment of the Müller glia cells. These markers will be important to further characterize the regeneration response in other retinal damage models and to elucidate the defects associated with mutants and morphants that disrupt the regeneration response.

Cellular Expression of Midkine-a and Midkine-b During Retinal Development and Photoreceptor Regeneration in Zebrafish

The Journal of Comparative Neurology. May, 2009  |  Pubmed ID: 19263476

In the retina of adult teleosts, stem cells are sustained in two specialized niches: the ciliary marginal zone (CMZ) and the microenvironment surrounding adult Müller glia. Recently, Müller glia were identified as the regenerative stem cells in the teleost retina. Secreted signaling molecules that regulate neuronal regeneration in the retina are largely unknown. In a microarray screen to discover such factors, we identified midkine-b (mdkb). Midkine is a highly conserved heparin-binding growth factor with numerous biological functions. The zebrafish genome encodes two distinct midkine genes: mdka and mdkb. Here we describe the cellular expression of mdka and mdkb during retinal development and the initial, proliferative phase of photoreceptor regeneration. The results show that in the embryonic and larval retina mdka and mdkb are expressed in stem cells, retinal progenitors, and neurons in distinct patterns that suggest different functions for the two molecules. Following the selective death of photoreceptors in the adult, mdka and mdkb are coexpressed in horizontal cells and proliferating Müller glia and their neurogenic progeny. These data reveal that Mdka and Mdkb are signaling factors present in the retinal stem cell niches in both embryonic and mature retinas, and that their cellular expression is actively modulated during retinal development and regeneration.

CNTF Induces Photoreceptor Neuroprotection and Müller Glial Cell Proliferation Through Two Different Signaling Pathways in the Adult Zebrafish Retina

Experimental Eye Research. Jun, 2009  |  Pubmed ID: 19450453

Ciliary neurotrophic factor (CNTF) acts in several processes in the vertebrate retina, including neuroprotection of photoreceptors in the stressed adult retina and regulation of neuronal progenitor cell proliferation during retinal development. However, the signaling pathway it utilizes (Jak/Stat, MAPK, or Akt) in these processes is ambiguous. Because dark-adapted albino zebrafish exhibit light-induced rod and cone cell death and subsequently regenerate the lost photoreceptor cells, zebrafish should be a useful model to study the role of CNTF in both neuroprotection and neuronal progenitor cell proliferation. We therefore investigated the potential roles of CNTF in both the undamaged and light-damaged adult zebrafish retinas. Intraocular injection of CNTF suppressed light-induced photoreceptor cell death, which then failed to exhibit the regeneration response that is marked by proliferating Müller glia and neuronal progenitor cells. Inhibiting the MAPK signaling pathway, but neither the Stat3 nor Akt pathways, significantly reduced the CNTF-mediated neuroprotection of light-induced photoreceptor cell death. Intraocular injection of CNTF into non-light-treated (undamaged) eyes mimicked constant intense light treatment by increasing Stat3 expression in Müller glia followed by increasing the number of proliferating Müller glia and neuronal progenitors. Knockdown of Stat3 expression in the CNTF-injected non-light-treated retinas significantly reduced the number of proliferating Müller glia, while coinjection of CNTF with either MAPK or Akt inhibitors did not inhibit the CNTF-induced Müller glia proliferation. Thus, CNTF utilizes a MAPK-dependant signaling pathway in neuroprotection of light-induced photoreceptor cell death and a Stat3-dependant signaling pathway to stimulate Müller glia proliferation.

Stepwise Maturation of Apicobasal Polarity of the Neuroepithelium is Essential for Vertebrate Neurulation

The Journal of Neuroscience : the Official Journal of the Society for Neuroscience. Sep, 2009  |  Pubmed ID: 19759292

During vertebrate neurulation, extensive cell movements transform the flat neural plate into the neural tube. This dynamic morphogenesis requires the tissue to bear a certain amount of plasticity to accommodate shape and position changes of individual cells as well as intercellular cohesiveness to maintain tissue integrity and architecture. For most of the neural plate-neural tube transition, cells are polarized along the apicobasal axis. The establishment and maintenance of this polarity requires many polarity proteins that mediate cell-cell adhesion either directly or indirectly. Intercellular adhesion reduces tissue plasticity and enhances tissue integrity. However, it remains unclear how apicobasal polarity is regulated to meet the opposing needs for tissue plasticity and tissue integrity during neurulation. Here, we show that N-Cad/ZO-1 complex-initiated apicobasal polarity is stabilized by the late-onsetting Lin7c/Nok complex after the extensive morphogenetic cell movements in neurulation. Loss of either N-Cad or Lin7c disrupts neural tube formation. Furthermore, precocious overexpression of Lin7c induces multiaxial mirror symmetry in zebrafish neurulation. Our data suggest that stepwise maturation of apicobasal polarity plays an essential role in vertebrate neurulation.

A Novel Model of Retinal Ablation Demonstrates That the Extent of Rod Cell Death Regulates the Origin of the Regenerated Zebrafish Rod Photoreceptors

The Journal of Comparative Neurology. Mar, 2010  |  Pubmed ID: 20058308

The adult zebrafish retina continuously produces rod photoreceptors from infrequent Müller glial cell division, yielding neuronal progenitor cells that migrate to the outer nuclear layer and become rod precursor cells that are committed to differentiate into rods. Retinal damage models suggested that rod cell death induces regeneration from rod precursor cells, whereas loss of any other retinal neurons activates Müller glia proliferation to produce pluripotent neuronal progenitors that can generate any other neuronal cell type in the retina. We tested this hypothesis by creating two transgenic lines that expressed the E. coli nitroreductase enzyme fused to EGFP (NTR-EGFP) in only rods. Treating transgenic adults with metronidazole resulted in two rod cell death models. First, killing all rods throughout the Tg(zop:nfsB-EGFP)(nt19) retina induced robust Müller glial proliferation, which yielded clusters of neuronal progenitor cells. In contrast, ablating only a subset of rods across the Tg(zop:nfsB-EGFP)(nt20) retina led to rod precursor, but not Müller glial, cell proliferation. We propose that two different criteria determine whether rod cell death will induce a regenerative response from the Müller glia rather than from the resident rod precursor cells in the ONL. First, there must be a large amount of rod cell death to initiate Müller glia proliferation. Second, the rod cell death must be acute, rather than chronic, to stimulate regeneration from the Müller glia. This suggests that the zebrafish retina possesses mechanisms to quantify the amount and timing of rod cell death.

The Zebrafish Galectin Drgal1-l2 is Expressed by Proliferating Müller Glia and Photoreceptor Progenitors and Regulates the Regeneration of Rod Photoreceptors

Investigative Ophthalmology & Visual Science. Jun, 2010  |  Pubmed ID: 20071673

The purpose of this study was to identify secreted proteins in the retina of the adult zebrafish that are induced by the selective death of photoreceptors and to test experimentally the function of these proteins during the regeneration of photoreceptors.

Pax6a and Pax6b Are Required at Different Points in Neuronal Progenitor Cell Proliferation During Zebrafish Photoreceptor Regeneration

Experimental Eye Research. May, 2010  |  Pubmed ID: 20152834

The light-damaged zebrafish retina results in the death of photoreceptor cells and the subsequent regeneration of the missing rod and cone cells. Photoreceptor regeneration initiates with asymmetric Müller glial cell division to produce neuronal progenitor cells, which amplify, migrate to the outer nuclear layer (ONL), and differentiate into both classes of photoreceptor cells. In this study, we examined the role of the Pax6 protein in regeneration. In zebrafish, there are two Pax6 proteins, one encoded by the pax6a gene and the other encoded by the pax6b gene. We intravitreally injected and electroporated morpholinos that were complementary to either the pax6a or pax6b mRNA to knockdown the translation of the corresponding protein. Loss of Pax6b expression did not affect Müller glial cell division, but blocked the subsequent first cell division of the neuronal progenitors. In contrast, the paralogous Pax6a protein was required for later neuronal progenitor cell divisions, which maximized the number of neuronal progenitors. Without neuronal progenitor cell amplification, proliferation of resident ONL rod precursor cells, which can only regenerate rods, increased inversely proportional to the number of INL neuronal progenitor cells. This confirmed that Müller glial-derived neuronal progenitor cells are necessary to regenerate cones and that distinct mechanisms selectively regenerate rod and cone photoreceptors. This work also defines distinct roles for Pax6a and Pax6b in regulating neuronal progenitor cell proliferation in the adult zebrafish retina and increases our understanding of the molecular pathways required for photoreceptor cell regeneration.

Zebrafish Class 1 Phosphatidylinositol Transfer Proteins: PITPbeta and Double Cone Cell Outer Segment Integrity in Retina

Traffic (Copenhagen, Denmark). Sep, 2010  |  Pubmed ID: 20545905

Phosphatidylinositol transfer proteins (PITPs) in yeast co-ordinate lipid metabolism with the activities of specific membrane trafficking pathways. The structurally unrelated metazoan PITPs (mPITPs), on the other hand, are an under-investigated class of proteins. It remains unclear what biological activities mPITPs discharge, and the mechanisms by which these proteins function are also not understood. The soluble class 1 mPITPs include the PITPalpha and PITPbeta isoforms. Of these, the beta-isoforms are particularly poorly characterized. Herein, we report the use of zebrafish as a model vertebrate for the study of class 1 mPITP biological function. Zebrafish express PITPalpha and PITPbeta-isoforms (Pitpna and Pitpnb, respectively) and a novel PITPbeta-like isoform (Pitpng). Pitpnb expression is particularly robust in double cone cells of the zebrafish retina. Morpholino-mediated protein knockdown experiments demonstrate Pitpnb activity is primarily required for biogenesis/maintenance of the double cone photoreceptor cell outer segments in the developing retina. By contrast, Pitpna activity is essential for successful navigation of early developmental programs. This study reports the initial description of the zebrafish class 1 mPITP family, and the first analysis of PITPbeta function in a vertebrate.

The Inhibitor of Phagocytosis, O-phospho-L-serine, Suppresses Müller Glia Proliferation and Cone Cell Regeneration in the Light-damaged Zebrafish Retina

Experimental Eye Research. Nov, 2010  |  Pubmed ID: 20696157

The damaged zebrafish retina replaces lost neurons through a regenerative response that initiates with the asymmetric cell division of Müller glia to produce neuronal progenitor cells that proliferate and migrate to the damaged retinal layer, where they differentiate into the lost neuronal cell types. Because Müller glia are known to phagocytose apoptotic retinal cells during development, we tested if Müller glia engulfed apoptotic rod cell bodies in light-damaged retinas. After 24h of constant intense light, damaged retinas revealed both a strong nuclear TUNEL signal in photoreceptors and a weak cytoplasmic TUNEL signal in Müller glia, although Müller glial apoptosis is not observed in the light-damaged retina. Light damage of a rod-specific transgenic reporter line, Tg(XlRho:EGFP)(fl1), resulted in some Müller glia containing both TUNEL signal and EGFP, which indicated that this subset of Müller glia engulfed apoptotic photoreceptor cell bodies. To determine if phagocytosis induced the Müller glial proliferative response in the light-damaged retina, we utilized O-phospho-l-serine (L-SOP), a molecule that mimics the phosphatidylserine head group and partially blocks microglial phagocytosis of apoptotic cells. Intravitreal injection of L-SOP immediately prior to beginning constant intense light treatment: i) did not significantly reduce light-induced photoreceptor cell death, ii) significantly reduced the number of PCNA-positive Müller glia, and iii) significantly reduced the number of cone photoreceptors in the regenerated retina relative to control retinas. Because L-SOP is also a specific group III metabotropic glutamate receptor (mGluR) agonist, we also tested if the more potent specific group III agonist, L-2-amino-4-phosphonobutyrate (L-AP4), the specific group III antagonist (RS)-α-Methylserine-O-phosphate (MSOP) or the specific group I antagonist, L-2-amino-3-phophonopropanoic acid (L-AP3) affected Müller glial proliferation. We found no changes with any of these factors compared to control retinas, revealing that metabotropic glutamate receptors were not necessary in the Müller glia proliferative response. Furthermore, ascl1a and stat3 expression were unaffected in either the L-SOP or MSOP-injected retinas relative to controls, suggesting L-SOP disrupts Müller glia proliferation subsequent to or in parallel with ascl1a and stat3 activation. This implies that at least one signaling mechanism, in addition to the process disrupted by L-SOP, is required to activate Müller glia proliferation in the light-damaged retina.

The Loss of Vacuolar Protein Sorting 11 (vps11) Causes Retinal Pathogenesis in a Vertebrate Model of Syndromic Albinism

Investigative Ophthalmology & Visual Science. May, 2011  |  Pubmed ID: 21330665

To establish the zebrafish platinum mutant as a model for studying vision defects caused by syndromic albinism diseases such as Chediak-Higashi syndrome, Griscelli syndrome, and Hermansky-Pudlak syndrome (HPS).

The Loss of Vacuolar Protein Sorting 11 (vps11) Causes Retinal Pathogenesis in a Vertebrate Model of Syndromic Albinism

Investigative Ophthalmology & Visual Science. May, 2011  |  Pubmed ID: 21622697

Purpose: To establish the zebrafish platinum mutant as a model for studying vision defects caused by syndromic albinism diseases such as Chediak-Higashi syndrome, Griscelli syndrome, and Hermansky-Pudlak syndrome (HPS). Methods: Bulked segregant analysis and candidate gene sequencing revealed that the zebrafish platinum mutation is a single nucleotide insertion in the vps11 (vacuolar protein sorting 11) gene. Expression of vps11 was determined by RT-PCR and in situ hybridization. Mutants were analyzed for pigmentation defects and retinal pathology by histology, immunohistochemistry, and transmission electron microscopy. Results: Phenocopy and rescue experiments determined that a loss of Vps11 results in the platinum phenotype. Expression of vps11 appeared ubiquitous during zebrafish development, with stronger expression in the developing retina and retinal pigmented epithelium (RPE). Zebrafish platinum mutants exhibited reduced pigmentation in the body and retinal pigmented epithelium (RPE), however, melanophore development, migration, and dispersion occurred normally. RPE, photoreceptors, and inner retinal neurons formed normally in zebrafish platinum mutants. However, a gradual loss of RPE, an absence of mature melanosomes and the subsequent degradation of RPE/photoreceptor interdigitation was observed. Conclusions: These data show that Vps11 is not required for normal retinal development or initiation of melanin biosynthesis, but is required for melanosome maturation and healthy maintenance of the RPE and photoreceptors.

Phosphatidylinositol Synthase is Required for Lens Structural Integrity and Photoreceptor Cell Survival in the Zebrafish Eye

Experimental Eye Research. Oct, 2011  |  Pubmed ID: 21722635

The zebrafish lens opaque (lop) mutant was previously isolated in a genetic screen and shown to lack rod and cone photoreceptors and exhibit lens opacity, or cataract, at 7 days post-fertilization (dpf). In this manuscript, we provide four different lines of evidence demonstrating that the lop phenotype results from a defect in the cdipt (phosphatidylinositol (PI) synthase; CDP-diacylglycerol-inositol 3-phosphatidyltransferase) gene. First, DNA sequence analysis revealed that the lop mutant contained a missense mutation in the lop open reading frame, which yields a nonconservative amino acid substitution (Ser-111-Cys) within the PI synthase catalytic domain. Second, morpholino-mediated knockdown of the cdipt-encoded PI synthase protein phenocopied the cdipt(lop/lop) mutant, with abnormal lens epithelial and secondary fiber cell morphologies and reduced numbers of photoreceptors. Third, microinjection of in vitro transcribed, wild-type cdipt mRNA into 1-4 cell stage cdipt(lop/lop) embryos significantly reduced the percentage of larvae displaying lens opacity at 7 dpf. Fourth, a cdipt retroviral-insertion allele, cdipt(hi559), exhibited similar lens and retinal abnormalities and failed to complement the cdipt(lop) mutant phenotype. To determine the initial cellular defects associated with the cdipt mutant, we examined homozygous cdipt(hi559/hi559) mutants prior to gross lens opacification at 6 dpf. The cdipt(hi559/hi559) mutants first exhibited photoreceptor layer disruption and photoreceptor cell death at 3 and 4 dpf, respectively, followed by lens dismorphogenesis by 5 dpf. RT-PCR revealed that the cdipt gene is maternally expressed and continues to be transcribed throughout development and into adulthood, in a wide variety of tissues. Using an anti-zebrafish PI synthase polyclonal antiserum, we localized the protein throughout the developing eye, including the photoreceptor layer and lens cortical secondary fiber cells. As expected, the polyclonal antiserum revealed that the PI synthase protein was reduced in amount in both the cdipt(lop/lop) and cdipt(hi559/hi559) mutants. Furthermore, we used a heterologous yeast phenotypic complementation assay to confirm that the wild-type zebrafish cdipt allele encodes functional PI synthase activity. Taken together, the cdipt-encoded PI synthase is required for survival of photoreceptor cells and lens epithelial and secondary cortical fiber cells. These zebrafish cdipt alleles represent excellent in vivo genetic tools to study the role of phosphatidylinositol and its phosphorylated derivatives in lens and photoreceptor development and maintenance.

FGF Signaling Regulates Rod Photoreceptor Cell Maintenance and Regeneration in Zebrafish

Experimental Eye Research. Nov, 2011  |  Pubmed ID: 21945172

Fgf signaling is required for many biological processes involving the regulation of cell proliferation and maintenance, including embryonic patterning, tissue homeostasis, wound healing, and cancer progression. Although the function of Fgf signaling is suggested in several different regeneration models, including appendage regeneration in amphibians and fin and heart regeneration in zebrafish, it has not yet been studied during zebrafish photoreceptor cell regeneration. Here we demonstrate that intravitreal injections of FGF-2 induced rod precursor cell proliferation and photoreceptor cell neuroprotection during intense light damage. Using the dominant-negative Tg(hsp70:dn-fgfr1) transgenic line, we found that Fgf signaling was required for homeostasis of rod, but not cone, photoreceptors. Even though fgfr1 is expressed in both rod and cone photoreceptors, we found that Fgf signaling differentially affected the regeneration of cone and rod photoreceptors in the light-damaged retina, with the dominant-negative hsp70:dn-fgfr1 transgene significantly repressing rod photoreceptor regeneration without affecting cone photoreceptors. These data suggest that rod photoreceptor homeostasis and regeneration is Fgf-dependent and that rod and cone photoreceptors in adult zebrafish are regulated by different signaling pathways.

Müller Glia As a Source of Neuronal Progenitor Cells to Regenerate the Damaged Zebrafish Retina

Advances in Experimental Medicine and Biology. 2012  |  Pubmed ID: 22183361

Characterization of Multiple Light Damage Paradigms Reveals Regional Differences in Photoreceptor Loss

Experimental Eye Research. Mar, 2012  |  Pubmed ID: 22425727

Zebrafish provide an attractive model to study the retinal response to photoreceptor apoptosis due to its remarkable ability to spontaneously regenerate retinal neurons following damage. There are currently two widely-used light-induced retinal degeneration models to damage photoreceptors in the adult zebrafish. One model uses constant bright light, whereas the other uses a short exposure to extremely intense ultraviolet light. Although both models are currently used, it is unclear whether they differ in regard to the extent of photoreceptor damage or the subsequent regeneration response. Here we report a thorough analysis of the photoreceptor damage and subsequent proliferation response elicited by each individual treatment, as well as by the concomitant use of both treatments. We show a differential loss of rod and cone photoreceptors with each treatment. Additionally, we show that the extent of proliferation observed in the retina directly correlates with the severity of photoreceptor loss. We also demonstrate that both the ventral and posterior regions of the retina are partially protected from light damage. Finally, we show that combining a short ultraviolet exposure followed by a constant bright light treatment largely eliminates the neuroprotected regions, resulting in widespread loss of rod and cone photoreceptors and a robust regenerative response throughout the retina.