Ganglion Cell Differentiation Research Articles

SWI/SNF Chromatin Remodeling ATPase Brm Regulates the Differentiation of Early Retinal Stem Cells/Progenitors by Influencing Brn3b Expression and Notch Signaling

Based on a variety of approaches, evidence suggests that different cell types in the vertebrate retina are generated by multipotential progenitors in response to interactions between cell intrinsic and cell extrinsic factors. The identity of some of the cellular determinants that mediate such interactions has emerged, shedding light on mechanisms underlying cell differentiation. For example, we know now that Notch signaling mediates the influence of the microenvironment on states of commitment of the progenitors by activating transcriptional repressors. Cell intrinsic factors such as the proneural basic helix-loop-helix and homeodomain transcription factors regulate a network of genes necessary for cell differentiation and maturation. What is missing from this picture is the role of developmental chromatin remodeling in coordinating the expression of disparate classes of genes for the differentiation of retinal progenitors. Here we describe the role of Brm, an ATPase in the SWI/SNF chromatin remodeling complex, in the differentiation of retinal progenitors into retinal ganglion cells. Using the perturbation of expression and function analyses, we demonstrate that Brm promotes retinal ganglion cell differentiation by facilitating the expression and function of a key regulator of retinal ganglion cells, Brn3b, and the inhibition of Notch signaling. In addition, we demonstrate that Brm promotes cell cycle exit during retinal ganglion cell differentiation. Together, our results suggest that Brm represents one of the nexus where diverse information of cell differentiation is integrated during cell differentiation.

Primitive Small Cell Tumor With Epithelial, Gangliocytic, Neuroendocrine, and Mesenchymal Differentiation: Report of 2 Cases

The authors describe 2 tumors that, to the best of their knowledge, are hitherto undescribed. The predominant cell type was small round to fusiform dark blue cells. The dark blue cells formed distinct epithelial cords with gland-like formations with mucicarmine-positive mucus. Another distinctive component of the tumors was a mesenchymal one. The mesenchymal areas appeared benign and could be likened to a fibroma having a densely collagenous stroma, or they had spindle cells set in the myxoid background, rendering a myxoma-like appearance. Another distinctive feature was ganglion cell differentiation. Mitotic figures, including atypical forms, were found only in the small cell component. All cells were immunohistochemically negative for actin, calponin, desmin, HMB45, neurofilament protein, CD99/MIC2, Melan A, tyrosinase, serotonin, CD56, Melan A, GFAP, and S-100 protein. Cytokeratin, synaptophysin, FLI1 protein, and chromogranin antibodies reacted only in the primitive small round cells, while all the other components were cytokeratin negative. Fluorescence in situ hybridization showed that the tumors are without the EWSR1 gene translocation and gain 12p. Ultrastructurally, the cells were endowed with well-formed intercellular desmosomes membrane-bound secretory in the cytoplasm. Granules were found in the cytoplasm. We suggest the name "primitive small cell tumor with epithelial, gangliocytic, neuroendocrine, and mesenchymal differentiation" for this neoplasm.

Characterization of early retinal progenitor microenvironment: Presence of activities selective for the differentiation of retinal ganglion cells and maintenance of progenitors

The maintenance and differentiation of retinal progenitors take place in the context of the microenvironment in which they reside at a given time during retinal histogenesis. To understand the nature of the microenvironment in the developing retina, we have examined the influence of activities present during the early stage of retinal histogenesis on enriched retinal progenitors, using the neurosphere model. Early and late retinal progenitors, enriched as neurospheres from embryonic day 14 (E14) and E18 rat retina, respectively, were cultured in embryonic day 3 (E3) chick retinal conditioned medium, simulating the microenvironment present during early retinal histogenesis. Examination of the differentiation and proliferation of retinal progenitors revealed that the early microenvironment contains at least three regulatory activities, which are partitioned in different size fractions of the conditioned medium with different heat sensitivity. First, it is characterized by activities, present in heat stable <30 kDa fraction, that promote the differentiation of retinal ganglion cells (RGCs), the early born neurons. Second, it contains activities, present in heat-sensitive >30 kDa fraction, that regulate the number of early born neurons and maintain the pool of retinal progenitors. Third, it possesses activities, present in heat-sensitive <30 kDa fraction, that prevent the premature differentiation of early retinal progenitors into the late born neurons. Thus, our observations demonstrate the regulatory influence of microenvironment on the maintenance and differentiation of retinal progenitors and establish neurospheres as a viable model system for the examination of such influences.

Zac1 promotes a Müller glial cell fate and interferes with retinal ganglion cell differentiation in Xenopus retina

The timing of cell cycle exit is tightly linked to cell fate specification in the developing retina. Accordingly, several tumor suppressor genes, which are key regulators of cell cycle exit in cancer cells, play critical roles in retinogenesis. Here we investigated the role of Zac1, a tumor suppressor gene encoding a zinc finger transcription factor, in retinal development. Strikingly, in gain-of-function assays in Xenopus, mouse Zac1 promotes proliferation and apoptosis at an intermediate stage of retinogenesis. Zac1 also influences cell fate decisions, preferentially promoting the differentiation of tumor-like clusters of abnormal neuronal cells in the ganglion cell layer, as well as inducing the formation of supernumerary Müller glial cells at the expense of other cell types. Thus Zac1 has the capacity to influence cell cycle exit, and cell fate specification and differentiation decisions by retinal progenitors, suggesting that further functional studies will uncover new insights into how retinogenesis is regulated.

Developmental changes in the fibre population of the optic nerve follow an avian/mammalian-like pattern in the turtle Mauremys leprosa

The changes in the axon and growth cone numbers in the optic nerve of the freshwater turtle Mauremys leprosa were studied by electron microscopy from the embryonic day 14 (E14) to E80, when the animals normally hatch, and from the first postnatal day (P0) to adulthood (5 years on). At E16, the first axons appeared in the optic nerve and were added slowly until E21. From E21, the fibre number increased rapidly, peaking at E34 (570,000 fibres). Thereafter, the axon number decreased sharply, and from E47 declined steadily until reaching the mature number (about 330,000). These observations indicated that during development of the retina there was an overproduction and later elimination of retinal ganglion cells. Growth cones were first observed in the optic nerve at as early as E16. Their number increased rapidly until E21 and continued to be high through E23 and E26. After E26, the number declined steeply and by E40 the optic nerve was devoid of growth cones. These results indicated that differentiation of the retinal ganglion cells occurred during the first half of the embryonic life. To examine the correlation between the loss of the fibres from the optic nerve and loss of the parent retinal ganglion cells, retinal sections were processed with the TUNEL technique. Apoptotic nuclei were detected in the ganglion cell layer throughout the period of loss of the optic fibres. Our results showed that the time course of the numbers of the fibres in the developing turtle optic nerve was similar to those found in birds and mammals.

Pax6 directly regulates the expression of Math5 during retinal ganglion cell differentiation

Pax6 directly regulates the expression of Math5 during retinal ganglion cell differentiation

Involvement of cytoskelatal proteins and growth factor receptors during development of the human eye

The spatial and temporal distribution of nestin, cytokeratins (CKs), vimentin, glial fibrillary acidic protein (GFAP), neurofilaments (NFs), beta-tubulin as well as fibroblast growth factor receptors (FGFRs) and platelet-derived growth factor receptor beta (PDGF-Rbeta) were investigated in the developing human eye in eight conceptuses of 5-9 postovulatory weeks using immunostaining. Nestin was found in the neuroglial precursors and the radial glial fibres of the optic nerve. In the pigmented retina, nestin was present only in the 5th week, while at later stages (6-9th week), co-expression of CKs and vimentin was seen. Nestin, CKs, vimentin, and GFAP were observed in the precursors to various cell types in the neural retina. Additionally, their expression was also apparent in the lens epithelium, showing its gradual fading following the lens fibre elongation. They appeared in the mesenchymal cells of the cornea, the choroid, the sclera, and the corpus vitreum, too. In the corneal epithelium, co-expression of nestin and CKs was detected. NFs and beta-tubulin were confined to the differentiating retinal neuroblasts. Growth factor receptors were seen in the retina, the lens epithelium while less intensely in the lens fibres, the corneal epithelium, and the mesenchymal cells. During the early eye development (5-9th week), IFs expressing normal pattern of distribution as well as acting in concert might contribute to the normal developmental processes occurring in certain parts of the human eye. Additionally, NFs and beta-tubulin seem to have an important role in the retinal ganglion cell differentiation, while FGFRs and PDGF-Rbeta may regulate the cell proliferation, differentiation, and survival in various parts of the developing eye.

Expression patterns of neurexin-1 and neuroligins in brain and retina of the chick embryo: Neuroligin-3 is absent in retina

Neuroligins (NLs) constitute a family of cell-surface proteins that interact with neurexins (beta-Nxs), another class of neuronal cell-surface proteins, one of each class functioning together in synapse formation. The localization of the various neurexins and neuroligins, however, has not yet been clarified in chicken. Therefore, we studied the expression patterns of neurexin-1 (Nx-1) and neuroligin-1 and -3 during embryonic development of the chick retina and brain by reverse-transcriptase polymerase chain reaction (RT-PCR) and in situ hybridization (ISH). While neurexin-1 increased continuously in both brain and retina, the expression of both neuroligins was more variable. As shown by ISH, Nx-1 is expressed in the inner half retina along with differentiation of ganglion and amacrine cells. Transcripts of NL-1 were detected as early as day 4 and increased with the maturation of the different brain regions. In different brain regions, NL-1 showed a different time regulation. Remarkably, neuroligin-3 was entirely absent in retina. This study indicates that synaptogenetic processes in brain and retina use different molecular machineries, whereby the neuroligins might represent the more distinctly regulated part of the neurexin–neuroligin complexes. Noticeably, NL-3 does not seem to be involved in the making of retinal synapses.

Pea3 expression is regulated by FGF signaling in developing retina

FGF signaling has been implicated as an important regulator of retinal development. As a first step in characterizing potential downstream targets of FGF signaling in the retina, we have analyzed expression of Pea3, a member of the Pea3 class of Ets-domain transcription factors, in the developing eye. We find that Pea3 is expressed in the developing retina, and its transcription is regulated by FGF receptor activation. In addition, FGF signaling activates Cath5, a gene necessary for retinal ganglion cell differentiation. These results suggest that FGF signaling via MAPK up-regulates transcription factors that in turn control retinal ganglion cell differentiation.

Forward Genetic Analysis of Visual Behavior in Zebrafish

The visual system converts the distribution and wavelengths of photons entering the eye into patterns of neuronal activity, which then drive motor and endocrine behavioral responses. The gene products important for visual processing by a living and behaving vertebrate animal have not been identified in an unbiased fashion. Likewise, the genes that affect development of the nervous system to shape visual function later in life are largely unknown. Here we have set out to close this gap in our understanding by using a forward genetic approach in zebrafish. Moving stimuli evoke two innate reflexes in zebrafish larvae, the optomotor and the optokinetic response, providing two rapid and quantitative tests to assess visual function in wild-type (WT) and mutant animals. These behavioral assays were used in a high-throughput screen, encompassing over half a million fish. In almost 2,000 F2 families mutagenized with ethylnitrosourea, we discovered 53 recessive mutations in 41 genes. These new mutations have generated a broad spectrum of phenotypes, which vary in specificity and severity, but can be placed into only a handful of classes. Developmental phenotypes include complete absence or abnormal morphogenesis of photoreceptors, and deficits in ganglion cell differentiation or axon targeting. Other mutations evidently leave neuronal circuits intact, but disrupt phototransduction, light adaptation, or behavior-specific responses. Almost all of the mutants are morphologically indistinguishable from WT, and many survive to adulthood. Genetic linkage mapping and initial molecular analyses show that our approach was effective in identifying genes with functions specific to the visual system. This collection of zebrafish behavioral mutants provides a novel resource for the study of normal vision and its genetic disorders.

Zebrafish R‐cadherin (Cdh4) controls visual system development and differentiation

In zebrafish, R-cadherin (cadherin-4 or Cdh4) is expressed in the retina and in retinorecipient brain regions, suggesting that Cdh4 functions during visual system development. Cdh4 function was examined during retinogenesis and retinal axon outgrowth using antisense morpholino oligonucleotides and mutant Cdh4 construct expression. In knockdowns, Cdh4 was reduced or absent, eyes were small, and retinae lacked discrete laminae. Increased cell death produced the small eye phenotype. Zn5-, Pax6-, and zpr-1-positive cells were reduced or absent in knockdown retinas but, when present, were in the correct laminae. Cdh4 knockdowns had sparse or absent retinal ganglion cell axons. When present, axons projected contralaterally but lacked fine branching and failed to reach the tectum or arborize the entire tectum. Mutant Cdh4 construct expression during retinal ganglion cell differentiation reduced or ablated neurite formation. Cdh4 is necessary for neural retina survival and differentiation, and required for normal retinotectal projection formation and tectal arborization.

Retinal growth hormone is an anti-apoptotic factor in embryonic retinal ganglion cell differentiation

Cells of the neural retina in the chick embryo undergo several waves of apoptosis during development, including peaks at approximately embryonic day (ED) 7 and 12. Prominent among the cells involved in these phases of cell death are the retinal ganglion cells (RGCs). We have previously shown that growth hormone (GH) is expressed in the neural retina, and particularly, in the RGCs. Here we study the ability of GH to rescue retinal cells from apoptosis, both in vitro and in vivo. When retinas from embryos at ED 6–8 are explanted on collagen gels, the application of recombinant GH, at 10 −6 m, significantly reduced the incidence of apoptotic cells in the cultures as judged by terminal deoxynucleotide transferase-mediated dUTP-biotin nick end labelling (TUNEL). GH was delivered to neural retinas in ovo, by microinjection into the eye cup at ED 2. When these embryos were examined at ED 6–8, no reduction in cell death was observed below the normal low control levels. However, when antibodies to GH were microinjected, the incidence of cell death increased significantly at ED 6, providing evidence that in vivo immunoneutralization of endogenous GH results in triggering of apoptotic signaling pathways. Evidence that RGCs are a particular target of this neuroprotective effect of GH was provided by examination of cultures enriched for RGCs by immunopanning. In serum-free culture, RGCs, identified by anti-Islet 1 immunolabelling, were found to be susceptible to the effect of GH immunoneutralization, which approximately quadrupled the incidence of apoptosis in the cultures. We propose that GH is a naturally occurring autocrine and/or paracrine neuroprotective agent in the developing retina which is involved in the regulation of retinal cell numbers during early embryogenesis.

The Organization of the Inner Retina in a Pure-Rod Deep-Sea Fish

The pure rod retina of a deep-sea eel species was used as a model system for the study of the differentiation of horizontal, bipolar, amacrine and ganglion cells. We wanted to test the hypothesis that the functional organization of the inner retina is less complex than in species with duplex, rod- and cone-containing retinae. We used immunocytochemistry, backfilling ganglion cells with fluorescent dextranes and microinjection of Lucifer Yellow, to visualize the micromorphology of the various cell types in a confocal microscope. The pure rod retina contains a single type of horizontal cell. The inner plexiform layer is 10–15 µm thick and shows three main sublayers. Bipolar terminals are found in all sublayers, but the majority are found in the inner sublamina b (PKC-immunoreactive cells, that in fish with duplex retinae receive a mixed rod-cone input). The neurochemical diversity of amacrine cells in terms of immunoreactivity does not differ from other teleosts; this similarity includes the pattern of dendritic stratification and ramification as revealed by microinjection. Ten different types of ganglion cells are distinguished based on the sizes of their perikaryon and dendritic field, and the stratification pattern in the inner plexiform layer. This is similar to the situation in catfish with retinae containing a single type of cone in addition to a majority of rods. In this respect, the differences between pure rod retinae and duplex retinae containing a single cone type were less obvious than hypothesized. In the deep-sea eel, the density of dendritic ramification in amacrine and ganglion cells was strongly reduced. This may be functionally related to the fact that vision in the deep sea environment relies exclusively on bioluminescence and is represented by burst-like emissions of point sources. This requires a mode of retinal signal processing that is less complex than in duplex retinae and involves a lower density of dendritic branching and synapses.

The hedgehog-PKA pathway regulates two distinct steps of the differentiation of retinal ganglion cells: the cell-cycle exit of retinoblasts and their neuronal maturation

In the developing zebrafish retina, neurogenesis is initiated in cells adjacent to the optic stalk and progresses to the entire neural retina. It has been reported that hedgehog (Hh) signalling mediates the progression of the differentiation of retinal ganglion cells (RGCs) in zebrafish. However, the progression of neurogenesis seems to be only mildly delayed by genetic or chemical blockade of the Hh signalling pathway. Here, we show that cAMP-dependent protein kinase (PKA) effectively inhibits the progression of retinal neurogenesis in zebrafish. Almost all retinal cells continue to proliferate when PKA is activated, suggesting that PKA inhibits the cell-cycle exit of retinoblasts. A cyclin-dependent kinase (cdk) inhibitor p27 inhibits the PKA-induced proliferation, suggesting that PKA functions upstream of cyclins and cdk inhibitors. Activation of the Wnt signalling pathway induces the hyperproliferation of retinal cells in zebrafish. The blockade of Wnt signalling inhibits the PKA-induced proliferation, but the activation of Wnt signalling promotes proliferation even in the absence of PKA activity. These observations suggest that PKA inhibits exit from the Wnt-mediated cell cycle rather than stimulates Wnt-mediated cell-cycle progression. PKA is an inhibitor of Hh signalling, and Hh signalling molecule morphants show severe defects in cell-cycle exit of retinoblasts. Together, these data suggest that Hh acts as a short-range signal to induce the cell-cycle exit of retinoblasts. The pulse inhibition of Hh signalling revealed that Hh signalling regulates at least two distinct steps of RGC differentiation: the cell-cycle exit of retinoblasts and RGC maturation. This dual requirement of Hh signalling in RGC differentiation implies that the regulation of a neurogenic wave is more complex in the zebrafish retina than in the Drosophila eye.

A gene network downstream of transcription factor Math5 regulates retinal progenitor cell competence and ganglion cell fate

Math5, a mouse homolog of the Drosophila proneural bHLH transcription factor Atonal, is essential in the developing retina to establish retinal progenitor cell competence for a ganglion cell fate. Elucidating the mechanisms by which Math5 influences progenitor cell competence is crucial for understanding how specification of neuronal cell fate occurs in the retina and it requires knowledge of the downstream target genes that depend on Math5 for their expression. To date, only a handful of genes downstream of Math5 have been identified. To better define the gene network operating downstream of Math5, we used custom-designed microarrays to examine the changes in embryonic retinal gene expression caused by deletion of math5. We identified 270 Math5-dependent genes, including those that were expressed specifically either in progenitor cells or differentiated ganglion cells. The ganglion cell-specific genes included both Brn3b-dependent and Brn3b-independent genes, indicating that Math5 regulates distinct branches of the gene network responsible for retinal ganglion cell differentiation. In math5-null progenitor cells, there was an up-regulation of the proneural genes math3, neuroD, and ngn2, indicating that Math5 suppresses the production of other cell types in addition to promoting retinal ganglion cell formation. The promoter regions of many Math5-dependent genes contained binding sites for REST/NRSF, suggesting that release from general repression in retinal progenitor cells is required for ganglion cell-specific gene activation. The identification of multiple roles for Math5 provides new insights into the gene network that defines progenitor cell competence in the embryonic retina.

Neuroblastoma Ganglion Cell Differentiation Following Chemotherapy

Neuroblastoma Ganglion Cell Differentiation Following Chemotherapy

Cerebral neurocytoma without ganglion cell differentiation

Neurocytomas are rare neuronal tumours, and are defined as tumours comprising cells with neuronal differentiation; they commonly arise in the ventricles. We report a case of a cerebral neurocytoma in a young man who presented with hemiparesis. The tumour was a radiologically a well-circumscribed large cyst with solid mass; the histopathology showed a well-differentiated lesion comprising uniform, round cells with perinuclear halos in a neuropil background, and immunohistochemically positive for neuronal markers (synaptophysin, neuron-specific enolase and neurofilaments). We suggest that cerebral neurocytomas should be considered among the uncommon causes of a large intra-axial cystic mass with a solid component in young adults.

Dlx1andDlx2function is necessary for terminal differentiation and survival of late-born retinal ganglion cells in the developing mouse retina

Dlx homeobox genes, the vertebrate homologs of Distal-less, play important roles in the development of the vertebrate forebrain, craniofacial structures and limbs. Members of the Dlx gene family are also expressed in retinal ganglion cells (RGC), amacrine and horizontal cells of the developing and postnatal retina. Expression begins at embryonic day 12.5 and is maintained until late embryogenesis for Dlx1, while Dlx2 expression extends to adulthood. We have assessed the retinal phenotype of the Dlx1/Dlx2 double knockout mouse, which dies at birth. The Dlx1/2 null retina displays a reduced ganglion cell layer (GCL), with loss of differentiated RGCs due to increased apoptosis, and corresponding thinning of the optic nerve. Ectopic expression of Crx, the cone and rod photoreceptor homeobox gene, in the GCL and neuroblastic layers of the mutants may signify altered cell fate of uncommitted RGC progenitors. However, amacrine and horizontal cell differentiation is relatively unaffected in the Dlx1/2 null retina. Herein, we propose a model whereby early-born RGCs are Dlx1 and Dlx2 independent, but Dlx function is necessary for terminal differentiation of late-born RGC progenitors.

The Role of CBFβ in T Cell Development.

Core binding factor β (Cbfβ) is a transcription factor that heterodimerizes with Runx (Cbfα) family members, thereby stabilizing the interaction between the Runx proteins and DNA. Genetically manipulated mouse models of Runx and Cbfb genes have demonstrated their critical functions in hematopoiesis (Runx1, Runx3 and Cbfb), bone formation (Runx2, Cbfb), proliferation of gastrointestinal epithelia (Runx3) and differentiation of dorsal root ganglion cells (Runx3). Studies on T cell development showed that Runx1 and Runx3 repress CD4 expression at different stages of development. In addition, Runx 1 and Runx 3 are required for CD8 T cell development during thymopoiesis. No defects were found when Runx2 was inactivated, even though it is expressed throughout T cell development.We have previously generated a knock-in mouse model expressing the Cbfb-MYH11 fusion gene (which is created by inv(16)(p13; q22) in human AML M4Eo). Heterozygous knock-in mice had a phenotype identical to that of the Cbfb and Runx1 null mice (embryonic lethality), suggesting that the fusion gene Cbfb-MYH11 functions in a dominant-negative manner. In order to study the function of Cbfb gene in T cell development, we used a mouse line with floxed exons 5 and 6 of Cbfb inserted 5′ to the Cbfb-MYH11 fusion cassette, which produced pseudo-normal mice (loxKI). By crossing the loxKI mice with mice expressing the Cre gene under the control of the T cell-specific Lck promoter (LckCre), we generated LckCre-loxKI double positive mice, in which the floxed exon 5 and 6 were deleted and Cbfb-MYH11 re-expressed only in the thymus when Lck started to express. The LckCre-loxKI mice were viable. However, their thymic development was severely impaired: The size of the thymuses in the mutant mice was about half the normal size, and the total number of thymocytes in the mutant mice was 10–20-fold reduced. FACS analysis of thymocytes from 4 to 12 week old mice showed a developmental blockade at the CD4/CD8-double negative (DN) stage, which was characterized by lower percentage of double positive cells and higher percentage of double negative cells. In addition, the CD4: CD8 ratio was altered. Furthermore, the mature T cell population size in the spleen of the mutant mice was lower than that of the control mice. Our preliminary data suggested that Cbfb plays an important role in T cell development. The mechanism through which Cbfb affects the T cell development is currently under investigation. It is likely that the phenotype reflects the combined effect of missing all three Runx genes, since the phenotype described here is more severe than either Runx1 or Runx3 null alone.

Zebrafish Gli3 functions as both an activator and a repressor in Hedgehog signaling

Hedgehog (Hh) signaling regulates cell differentiation and patterning in a wide variety of embryonic tissues. In vertebrates, at least three Gli transcription factors (Gli1, Gli2, and Gli3) are involved in Hh signal transduction. Comparative studies have revealed divergent requirements for Gli1 and Gli2 in zebrafish and mouse. Here, we address the question of whether Gli3 function has also diverged in zebrafish and analyze the regulatory interactions between Hh signaling and Gli activity. We find that zebrafish Gli3 has an early function as an activator of Hh target genes that overlaps with Gli1 activator function in the ventral neural tube. In vitro reporter analysis shows that Gli3 cooperates with Gli1 to activate transcription in the presence of high concentrations of Hh. During late somitogenesis stages, Gli3 is required as a repressor of the Hh response. Gli3 shares this repressor activity with Gli2 in the dorsal spinal cord, hindbrain, and midbrain, but not in the forebrain. Consistently, zebrafish Gli3 blocks Gli1-mediated activation of a reporter gene in the absence of Hh in vitro. In the eye, Gli3 is also required for proper ath5 expression and the differentiation of retinal ganglion cells (RGCs). These results reveal a conserved role for Gli3 in vertebrate development and uncover novel regional functions and regulatory interactions among gli genes.