Deborah L. Stenkamp

Extraretinal and retinal hedgehog signaling sequentially regulate retinal differentiation in zebrafish

Hedgehog (Hh) signaling is required for eye development in vertebrates; known roles in the zebrafish include regulation of eye morphogenesis and ganglion cell and photoreceptor differentiation. We employed a temporally selective Hh signaling knockdown strategy, by using antisense morpholino oligonucleotides or the teratogenic alkaloid cyclopamine, in order to dissect the separate roles of Hh signaling arising from specific sources. We also examined the eye phenotype of zebrafish slow muscle-omitted (smu) mutants, which lack a functional smoothened gene, encoding a component of the Hh signal transduction pathway. We find that Hh signaling from extraretinal sources is required for the initiation of retinal differentiation, but this involvement may be independent of the effects of Hh signaling on optic stalk development. We also find that Hh signals from ganglion cells participate in propagating expression of ath5, and we suggest that the effects of Hh signals from the retinal pigmented epithelium on photoreceptor differentiation may be mediated by the transcription factor rx1.

Neurogenesis in the Fish Retina

The retinas of teleost fish have long been of interest to developmental neurobiologists for their persistent plasticity during growth, life history changes, and response to injury. Because the vertebrate retina is a highly conserved tissue, the study of persistent plasticity in teleosts has provided insights into mechanisms for postembryonic retinal neurogenesis in mammals. In addition, in the past 10 years there has been an explosion in the use of teleost fish-zebrafish (Danio rerio) in particular-to understand the mechanisms of embryonic retinal neurogenesis in a model vertebrate with genetic resources. This review summarizes the key features of teleost retinal neurogenesis that make it a productive and interesting experimental system, and focuses on the contributions to our knowledge of retinal neurogenesis that uniquely required or significantly benefited from the use of a fish model system.

Molecular cloning and characterization of the putative ultraviolet-sensitive visual pigment of goldfish

A cDNA full length encoding a putative ultraviolet (UV)-sensitive visual pigment of goldfish was isolated. The deduced amino acid sequence shows 64% identity to those of human blue and chicken violet, and less identity (40-49%) to those of other vertebrate visual pigment. The mRNA is localized in the miniature short single cone cells, which are known to have a sensitivity maximum in the near UV-region.

Spatiotemporal coordination of rod and cone photoreceptor differentiation in goldfish retina

In this study, we have compared spatial and temporal aspects of development of new rods and cones in the adult goldfish by using a combination of bromodeoxyuridine immunocytochemistry and opsin in situ hybridization to determine the intervals between terminal mitosis (cell “birth”) and expression of opsin mRNA for each photoreceptor cell type. The goldfish opsins include rod opsin and four different cone opsins: red, green, blue, and ultraviolet. In a cohort of photoreceptors born at the same time, rods expressed opsin mRNA within 3 days of cell birth, while expression of cone opsin mRNA required at least 7 days. This temporal discrepancy in differentiation, coupled with a discordance in the site of cell genesis of rods and cones, allowed opsin expression to commence in both cell types in approximately the same retinal location. Commitment to the generic cone phenotype occurred within approximately 6 days throughout the cone cohort, as indicated by expression of interphotoreceptor retinoid‐binding protein (IRBP) mRNA, but expression of a specific spectral phenotype was delayed until rods differentiated nearby. Onset of expression of cone opsin mRNA followed a phenotype‐specific sequence: red, then green, then blue, and finally ultraviolet; in situ hybridization with two opsin probes confirmed that individual photoreceptors expressed only one type of opsin as they differentiated. This stepwise process of cone differentiation is consistent with the hypothesis that cell‐cell interactions among developing photoreceptors may coordinate selection of specific photoreceptor phenotypes. J. Comp. Neurol. 382:272‐284, 1997.

Embryonic retinal gene expression in sonic-you mutant zebrafish.

Hedgehog (Hh) signaling is required for proper eye development in vertebrates; known roles for Hh in the zebrafish include regulation of eye morphogenesis, ganglion cell neurogenesis, and photoreceptor differentiation. To gain insight into the mechanisms by which Hh signaling influences these developmental events, we have examined proliferation, cell death, and expression patterns of several retinal genes in the eyes of embryonic zebrafish lacking the sonic hedgehog gene. We find that features of the eye phenotype of the sonic‐you (syu) mutant are consistent with multiple roles for the Hh signal during retinal development. Most interestingly, half of the mutant retinas failed to initiate cell differentiation and, instead, retained a neuroepithelial appearance. In the other half of the mutants, retinal cell differentiation was initiated, but not fully propagated. We also find that Hh signaling is important for retinal cell proliferation and retinal cell survival; together, these functions provide an explanation for progressive microphthalmia in the syu‐/‐ mutant.

Evidence for two distinct mechanisms of neurogenesis and cellular pattern formation in regenerated goldfish retinas

After its destruction by intraocular injection of ouabain, the goldfish retina regenerates, but little is known about the histogenesis of the new tissue, including the structure and formation of regenerated cell mosaic patterns. In an effort to determine how retinal cells are generated and spatially organized within retina regenerated after ouabain injection, in situ hybridization and immunocytochemical techniques were combined with computational analyses of two‐dimensional spatial patterns of identified neurons. Labeling with specific opsin riboprobes revealed two distinct cone patterns in the ouabain‐injected eyes, each of which was different from the relatively orderly cone patterns of native retina. Central, regenerated regions had sparse aggregates of cones, and a relatively lower density of each cone type. Peripheral regions of experimental retina, likely derived from the circumferential germinal zone, had high densities of all cone types, each of which tended to be distributed randomly. The spatial patterns of inner retinal neurons in experimental eyes were also disorganized with respect to native retina. These results indicate that although some aspects of retinal regeneration resemble normal retinal development and growth, ouabain‐induced regeneration does not produce well‐organized mosaics of neurons, indicating a failure of the developmental interactions needed for proper pattern formation, which in turn could compromise visual recovery. Furthermore, the distinct cone patterns in different regions of experimental retina support the hypothesis that new goldfish retina arises via two spatially and cellularly distinct mechanisms after exposure to ouabain. J. Comp. Neurol. 431:363–381, 2001.

Mechanisms for persistent microphthalmia following ethanol exposure during retinal neurogenesis in zebrafish embryos

The exposure of the developing human embryo to ethanol results in a spectrum of disorders involving multiple organ systems, including the visual system. One common phenotype seen in humans exposed to ethanol in utero is microphthalmia. The objective of this study was to describe the effects of ethanol during retinal neurogenesis in a model organism, the zebrafish, and to pursue the potential mechanisms by which ethanol causes microphthalmia. Zebrafish embryos were exposed to 1% or 1.5% ethanol from 24 to 48 h after fertilization, a period during which the retinal neuroepithelium undergoes rapid proliferation and differentiation to form a laminated structure composed of different retinal cell types. Ethanol exposure resulted in significantly reduced eye size immediately following the treatment, and this microphthalmia persisted through larval development. This reduced eye size could not entirely be accounted for by the accompanying general delay in embryonic development. Retinal cell death was only slightly higher in ethanol-exposed embryos, although cell death in the lens was extensive in some of these embryos, and lenses were significantly reduced in size as compared to those of control embryos. The initiation of retinal neurogenesis was not affected, but the subsequent waves of cell differentiation were markedly reduced. Even cells that were likely generated after ethanol exposure--rod and cone photoreceptors and Müller glia--were delayed in their expression of cell-specific markers by at least 24 h. We conclude that ethanol exposure over the time of retinal neurogenesis resulted in persistent microphthalmia due to a combination of an overall developmental delay, lens abnormalities, and reduced retinal cell differentiation.

Retinal homeobox 1 is required for retinal neurogenesis and photoreceptor differentiation in embryonic zebrafish.

Retinal homeobox (Rx/Rax) genes are essential for the organogenesis of the vertebrate eye. These genes are dynamically expressed in a tissue-specific manner during eye development, suggesting pleiotropic roles. We use a temporally-selective gene knockdown approach to identify endogenous functions for the zebrafish rx genes, rx1 and rx2. Depletion of rx1 over the period of eye organogenesis resulted in severely reduced proliferation of retinal progenitors, the loss of expression of the transcription factor pax6, delayed retinal neurogenesis, and extensive retinal cell death. In contrast, depletion of rx2 over the same developmental time resulted in reduced expression of pax6 in the eye anlage, but only modest effects on retinal cell survival. Knockdown of rx1 specifically during photoreceptor development inhibited the expression of multiple photoreceptor-specific genes, while knockdown of rx2 over this time selectively inhibited the expression of a subset of these genes. Our findings support a function for rx2 in regulating pax6 within the optic primordia, a function for rx1 in maintaining the pluripotent, retinal progenitor cell state during retinal development, as well as selective functions for rx1 and rx2 in regulating photoreceptor differentiation.

The developmental sequence of gene expression within the rod photoreceptor lineage in embryonic zebrafish

In postembryonic zebrafish, rod photoreceptors are continuously generated from progenitors in the inner nuclear layer, which are derived from radial Müller glia that express the transcription factor pax6. We used BrdU incorporation, in combination with in situ hybridization for cell‐specific transcription factors, to establish the patterns of gene expression during rod lineage maturation in the embryonic zebrafish. Downregulation of pax6 expression was accompanied by sporadic upregulation of expression of the transcription factors NeuroD/nrd, rx1, crx, and Nr2e3/pnr. As cells of the rod lineage entered the outer nuclear layer, they became homogeneous, coordinately expressing NeuroD, rx1, crx, and Nr2e3. Postmitotic, maturing rods also expressed nrl, rod opsin, and rod transducin/gnat1. The presence of rx1 within the rod lineage and in maturing rods indicates that rx1 is not cone‐specific, as previously reported, and suggests a high degree of molecular similarity between rod and cone progenitor populations in the zebrafish. Developmental Dynamics 237:2903–1917, 2008.

The rod photoreceptor lineage of teleost fish

The retinas of postembryonic teleost fish continue to grow for the lifetime of the fish. New retinal cells are added continuously at the retinal margin, by stem cells residing at the circumferential germinal zone. Some of these retinal cells differentiate as Müller glia with cell bodies that reside within the inner nuclear layer. These glia retain some stem cell properties in that they carry out asymmetric cell divisions and continuously generate a population of transit-amplifying cells--the rod photoreceptor lineage--that are committed to rod photoreceptor neurogenesis. These rod progenitors progress through a stereotyped sequence of changes in gene expression as they continue to divide and migrate to the outer nuclear layer. Now referred to as rod precursors, they undergo terminal mitoses and then differentiate as rods, which are inserted into the existing array of rod and cone photoreceptors. The rod lineage displays developmental plasticity, as rod precursors can respond to the loss of rods through increased proliferation, resulting in rod replacement. The stem cells of the rod lineage, Müller glia, respond to acute damage of other retinal cell types by increasing their rate of proliferation. In addition, the Müller glia in an acutely damaged retina dedifferentiate and become multipotent, generating new, functional neurons. This review focuses on the cells of the rod lineage and includes discussions of experiments over the last 30 years that led to their identification and characterization, and the discovery of the stem cells residing at the apex of the lineage. The plasticity of cells of the rod lineage, their relationships to cone progenitors, and the applications of this information for developing future treatments for human retinal disorders will also be discussed.