Ruth A. Frey

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.

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.

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.

Ethanol-induced microphthalmia is not mediated by changes in retinoic acid or sonic hedgehog signaling during retinal neurogenesis.

BACKGROUND Microphthalmia (reduced eye size), generally accompanied by vision defects, is a hallmark of fetal alcohol spectrum disorder (FASD) in humans. In zebrafish, embryonic ethanol exposure over the time of retinal neurogenesis also results in microphthalmia. This microphthalmia is in part the consequence of reduced retinal cell differentiation, including photoreceptors. Here we pursue 2 signaling pathways implicated in other aspects of FASD pathogenesis: retinoic acid (RA) and Sonic hedgehog (Shh). METHODS We evaluated markers for RA and Shh signaling within the eyes of embryos treated with ethanol during the period of retinal neurogenesis. We also performed rescue experiments using administration of exogenous RA and microinjection of cholesterol, which augments Shh signaling. RESULTS Using sequential or co-treatments, RA did not rescue ethanol-induced microphthalmia at any concentration tested. In addition, RA itself caused microphthalmia, although the underlying mechanisms were distinct from those of ethanol. Interestingly, RA treatment appeared to recover photoreceptor differentiation in a concentration-dependent manner. This may be an independent effect of exogenous RA, as ethanol treatment alone did not alter RA signaling in the eye. Cholesterol injection also did not rescue ethanol-induced microphthalmia at any concentration tested, and ethanol treatments did not alter expression of shh, or of ptc-2, which is normally regulated by Shh signaling. CONCLUSIONS Together these findings indicate that, during the time of retinal neurogenesis, effects of ethanol on eye development are likely independent of the RA and Shh signaling pathways. These studies suggest that FASD intervention strategies based upon augmentation of RA or Shh signaling may not prevent ethanol-induced microphthalmia.

Retinal regeneration is facilitated by the presence of surviving neurons

Teleost fish regenerate their retinas after damage, in contrast to mammals. In zebrafish subjected to an extensive ouabain‐induced lesion that destroys all neurons and spares Müller glia, functional recovery and restoration of normal optic nerve head (ONH) diameter take place at 100 days postinjury. Subsequently, regenerated retinas overproduce cells in the retinal ganglion cell (RGC) layer, and the ONH becomes enlarged. Here, we test the hypothesis that a selective injury, which spares photoreceptors and Müller glia, results in faster functional recovery and fewer long‐term histological abnormalities. Following this selective retinal damage, recovery of visual function required 60 days, consistent with this hypothesis. In contrast to extensively damaged retinas, selectively damaged retinas showed fewer histological errors and did not overproduce neurons. Extensively damaged retinas had RGC axons that were delayed in pathfinding to the ONH, and showed misrouted axons within the ONH, suggesting that delayed functional recovery following an extensive lesion is related to defects in RGC axons exiting the eye and/or reaching their central targets. The atoh7, fgf8a, Sonic hedgehog (shha), and netrin‐1 genes were differentially expressed, and the distribution of hedgehog protein was disrupted after extensive damage as compared with selective damage. Confirming a role for Shh signaling in supporting rapid regeneration, shhat4+/‐ zebrafish showed delayed functional recovery after selective damage. We suggest that surviving retinal neurons provide structural/molecular information to regenerating neurons, and that this patterning mechanism regulates factors such as Shh. These factors in turn control neuronal number, retinal lamination, and RGC axon pathfinding during retinal regeneration.

Retinoic Acid Signaling Regulates Differential Expression of the Tandemly-Duplicated Long Wavelength-Sensitive Cone Opsin Genes in Zebrafish

The signaling molecule retinoic acid (RA) regulates rod and cone photoreceptor fate, differentiation, and survival. Here we elucidate the role of RA in differential regulation of the tandemly-duplicated long wavelength-sensitive (LWS) cone opsin genes. Zebrafish embryos were treated with RA from 48 hours post-fertilization (hpf) to 75 hpf, and RNA was isolated from eyes for microarray analysis. ~170 genes showed significantly altered expression, including several transcription factors and components of cellular signaling pathways. Of interest, the LWS1 opsin gene was strongly upregulated by RA. LWS1 is the upstream member of the tandemly duplicated LWS opsin array and is normally not expressed embryonically. Embryos treated with RA 48 hpf to 100 hpf or beyond showed significant reductions in LWS2-expressing cones in favor of LWS1-expressing cones. The LWS reporter line, LWS-PAC(H) provided evidence that individual LWS cones switched from LWS2 to LWS1 expression in response to RA. The RA signaling reporter line, RARE:YFP indicated that increased RA signaling in cones was associated with this opsin switch, and experimental reduction of RA signaling in larvae at the normal time of onset of LWS1 expression significantly inhibited LWS1 expression. A role for endogenous RA signaling in regulating differential expression of the LWS genes in postmitotic cones was further supported by the presence of an RA signaling domain in ventral retina of juvenile zebrafish that coincided with a ventral zone of LWS1 expression. This is the first evidence that an extracellular signal may regulate differential expression of opsin genes in a tandemly duplicated array.

Retinal proliferation response in the buphthalmic zebrafish, bugeye

The zebrafish retina regenerates in response to acute retinal lesions, replacing damaged neurons with new neurons. In this study we test the hypothesis that chronic stress to inner retinal neurons also triggers a retinal regeneration response in the bugeye zebrafish. Mutations in the lrp2 gene in zebrafish are associated with a progressive eye phenotype (bugeye) that models several risk factors for human glaucoma including buphthalmos (enlarged eyes), elevated intraocular pressure (IOP), and upregulation of genes related to retinal ganglion cell pathology. The retinas of adult bugeye zebrafish showed high rates of ongoing proliferation which resulted in the production of a small number of new retinal neurons, particularly photoreceptors. A marker of mechanical cell stress, Hsp27, was strongly expressed in inner retinal neurons and glia of bugeye retinas. The more enlarged eyes of individual bugeye zebrafish showed disrupted retinal lamination, and a persistent reduced density of neurons in the ganglion cell layer (GCL), although total numbers of GCL neurons were higher than in control eyes. Despite the presence of a proliferative response to damage, the adult bugeye zebrafish remained behaviorally blind. These findings suggest the existence of an unsuccessful regenerative response to a persistent pathological condition in the bugeye zebrafish.

Abnormal retinal development in Cloche mutant zebrafish.

Background: Functions for the early embryonic vasculature in regulating development of central nervous system tissues, such as the retina, have been suggested by in vitro studies and by in vivo manipulations that caused additional ocular vessels to develop. Here, we use an avascular zebrafish embryo, cloche−/− (clo−/−), to begin to identify necessary developmental functions of the ocular vasculature in regulating development and patterning of the neural retina, in vivo. These studies are possible in zebrafish embryos, which do not yet rely upon the vasculature for tissue oxygenation. Results: clo−/− embryos lacked early ocular vasculature and were microphthalmic, with reduced retinal cell proliferation and cell survival. Retinas of clo mutants were disorganized, with irregular synaptic layers, mispatterned expression domains of retinal transcription factors, morphologically abnormal Müller glia, reduced differentiation of specific retinal cell types, and sporadically distributed cone photoreceptors. Blockade of p53‐mediated cell death did not completely rescue this phenotype and revealed ectopic cones in the inner nuclear layer. clo−/− embryos did not upregulate a molecular marker for hypoxia. Conclusions: The disorganized retinal phenotype of clo−/− embryos is consistent with a neural and glial developmental patterning role for the early ocular vasculature that is independent of its eventual function in gas exchange. Developmental Dynamics 244:1439–1455, 2015.

Eye-specific gene expression following embryonic ethanol exposure in zebrafish: roles for heat shock factor 1.

The mechanisms through which ethanol exposure results in developmental defects remain unclear. We used the zebrafish model to elucidate eye-specific mechanisms that underlie ethanol-mediated microphthalmia (reduced eye size), through time-series microarray analysis of gene expression within eyes of embryos exposed to 1.5% ethanol. 62 genes were differentially expressed (DE) in ethanol-treated as compared to control eyes sampled during retinal neurogenesis (24-48 h post-fertilization). The EDGE (extraction of differential gene expression) algorithm identified >3000 genes DE over developmental time in ethanol-exposed eyes as compared to controls. The DE lists included several genes indicating a mis-regulated cellular stress response due to ethanol exposure. Combined treatment with sub-threshold levels of ethanol and a morpholino targeting heat shock factor 1 mRNA resulted in microphthalmia, suggesting convergent molecular pathways. Thermal preconditioning partially prevented ethanol-mediated microphthalmia while maintaining Hsf-1 expression. These data suggest roles for reduced Hsf-1 in mediating microphthalmic effects of embryonic ethanol exposure.