Retinal Ganglion Cell Differentiation Research Articles

The guanine nucleotide exchange factor Vav3 regulates differentiation of progenitor cells in the developing mouse retina.

The seven main cell types in the mammalian retina arise from multipotent retinal progenitor cells, a process that is tightly regulated by intrinsic and extrinsic signals. However, the molecular mechanisms that control proliferation, differentiation and cell-fate decisions of retinal progenitor cells are not fully understood yet. Here, we report that the guanine nucleotide exchange factor Vav3, a regulator of Rho-GTPases, is involved in retinal development. We demonstrate that Vav3 is expressed in the mouse retina during the embryonic period. In order to study the role of Vav3 in the developing retina, we generate Vav3-deficient mice. The loss of Vav3 results in an accelerated differentiation of retinal ganglion cells and cone photoreceptors during early and late embryonic development. We provide evidence that more retinal progenitor cells express the late progenitor marker Sox9 in Vav3-deficient mice than in wild-types. This premature differentiation is compensated during the postnatal period and late-born cell types such as bipolar cells and Müller glia display normal numbers. Taken together, our data imply that Vav3 is a regulator of retinal progenitor cell differentiation, thus highlighting a novel role for guanine nucleotide exchange factors in retinogenesis.

Zebrafish retinal defects induced by ethanol exposure are rescued by retinoic acid and folic acid supplement

Fetal Alcohol Spectrum Disorder (FASD) is caused by prenatal alcohol exposure, producing craniofacial, sensory, motor, and cognitive defects. FASD is highly prevalent in low socioeconomic populations, which are frequently accompanied by malnutrition. FASD-associated ocular pathologies include microphthalmia, optic nerve hypoplasia, and cataracts. The present study characterizes specific retinal tissue defects, identifies ethanol-sensitive stages during retinal development, and dissects the effect of nutrient supplements, such as retinoic acid (RA) and folic acid (FA) on ethanol-induced retinal defects. Exposure to pathophysiological concentrations of ethanol (during midblastula transition through somitogenesis; 2–24 h post fertilization [hpf]) altered critical transcription factor expression involved in retinal cell differentiation, and produced severe retinal ganglion cell, photoreceptor, and Müller glial differentiation defects. Ethanol exposure did not alter retinal cell differentiation induction, but increased retinal cell death and proliferation. RA and FA nutrient co-supplementation rescued retinal photoreceptor and ganglion cell differentiation defects. Ethanol exposure during retinal morphogenesis stages (16–24 hpf) produced retinal defects like those seen with ethanol exposure between 2 and 24 hpf. Significantly, during an ethanol-sensitive time window (16–24 hpf), RA co-supplementation moderately rescued these defects, whereas FA co-supplementation showed significant rescue of optic nerve and photoreceptor differentiation defects. Interestingly, RA, but not FA, supplementation after ethanol exposure could reverse ethanol-induced optic nerve and photoreceptor differentiation defects. Our results indicate that various ethanol-sensitive events underlie FASD-associated retinal defects. Nutrient supplements like retinoids and folate were effective in alleviating ethanol-induced retinal defects.

Differentiation of retinal ganglion cells and photoreceptor precursors from mouse induced pluripotent stem cells carrying an Atoh7/Math5 lineage reporter.

The neural retina is a critical component of the visual system, which provides the majority of sensory input in humans. Various retinal degenerative diseases can result in the permanent loss of retinal neurons, especially the light-sensing photoreceptors and the centrally projecting retinal ganglion cells (RGCs). The replenishment of lost RGCs and the repair of optic nerve damage are particularly challenging, as both RGC specification and their subsequent axonal growth and projection involve complex and precise regulation. To explore the developmental potential of pluripotent stem cell-derived neural progenitors, we have established mouse iPS cells that allow cell lineage tracing of progenitors that have expressed Atoh7/Math5, a bHLH transcription factor required for RGC production. These Atoh7 lineage reporter iPS cells encode Cre to replace one copy of the endogenous Atoh7 gene and a Cre-dependent YFP reporter in the ROSA locus. In addition, they express pluripotent markers and are capable of generating teratomas in vivo. Under anterior neural induction and neurogenic conditions in vitro, the Atoh7-Cre/ROSA-YFP iPS cells differentiate into neurons that co-express various RGC markers and YFP, indicating that these neurons are derived from Atoh7-expressing progenitors. Consistent with previous in vivo cell lineage studies, the Atoh7-Cre/ROSA-YFP iPS cells also give rise to a subset of Crx-positive photoreceptor precursors. Furthermore, inhibition of Notch signaling in the iPSC cultures results in a significant increase of YFP-positive RGCs and photoreceptor precursors. Together, these results show that Atoh7-Cre/ROSA-YFP iPS cells can be used to monitor the development and survival of RGCs and photoreceptors from pluripotent stem cells.

Methods of Retinal Ganglion Cell Differentiation From Pluripotent Stem Cells

Methods of Retinal Ganglion Cell Differentiation From Pluripotent Stem Cells

Transcriptome of Atoh7 retinal progenitor cells identifies new Atoh7-dependent regulatory genes for retinal ganglion cell formation.

The bHLH transcription factor ATOH7 (Math5) is essential for establishing retinal ganglion cell (RGC) fate. However, Atoh7-expressing retinal progenitor cells (RPCs) can give rise to all retinal cell types, suggesting that other factors are involved in specifying RGCs. The basis by which a subpopulation of Atoh7-expressing RPCs commits to an RGC fate remains uncertain but is of critical importance to retinal development since RGCs are the earliest cell type to differentiate. To better understand the regulatory mechanisms leading to cell-fate specification, a binary genetic system was generated to specifically label Atoh7-expressing cells with green fluorescent protein (GFP). Fluorescence-activated cell sorting (FACS)-purified GFP(+) and GFP(-) cells were profiled by RNA-seq. Here, we identify 1497 transcripts that were differentially expressed between the two RPC populations. Pathway analysis revealed diminished growth factor signaling in Atoh7-expressing RPCs, indicating that these cells had exited the cell cycle. In contrast, axon guidance signals were enriched, suggesting that axons of Atoh7-expressing RPCs were already making synaptic connections. Notably, many genes enriched in Atoh7-expressing RPCs encoded transcriptional regulators, and several were direct targets of ATOH7, including, and unexpectedly, Ebf3 and Eya2. We present evidence for a Pax6-Atoh7-Eya2 pathway that acts downstream of Atoh7 but upstream of differentiation factor Pou4f2. EYA2 is a protein phosphatase involved in protein-protein interactions and posttranslational regulation. These properties, along with Eya2 as an early target gene of ATOH7, suggest that EYA2 functions in RGC specification. Our results expand current knowledge of the regulatory networks operating in Atoh7-expressing RPCs and offer new directions for exploring the earliest aspects of retinogenesis.

Methods of Retinal Ganglion Cell Differentiation From Pluripotent Stem Cells.

Glaucoma, the worldwide leading cause of irreversible blindness, is characterized by progressive degeneration of the optic nerve and loss of retinal ganglion cells. Research into glaucoma pathogenesis has been hampered by difficulties in isolating and culturing retinal ganglion cells in vitro. However, recent improvements in laboratory techniques have enabled the generation of a variety of mature cell types from pluripotent stem cells, including retinal ganglion cells. Indeed, stem cell-based approaches have the potential to revolutionize the field by providing an unlimited source of cells for replacement therapies and by enabling development of in vitro disease models for drug screening and research. Consequently, research aimed at directing pluripotent stem cells to differentiate into retinal ganglion cells has expanded dramatically during the past decade, resulting in significant advances in technique and efficiency. In this paper, we review the methodology for retinal ganglion cell differentiation from pluripotent stem cells of both mouse and human origin and summarize how these techniques have opened up new avenues for modelling glaucoma. Generation of stem cell-derived retinal ganglion cells will have significant translational values, providing an in vitro platform to study the mechanisms responsible for pathogenesis and for drug screening to improve treatment options, as well as for the development of cell therapies for optic neuropathies such as glaucoma.

A homozygous SIX6 mutation is associated with optic disc anomalies and macular atrophy and reduces retinal ganglion cell differentiation

A homozygous <i><scp>SIX6</scp></i> mutation is associated with optic disc anomalies and macular atrophy and reduces retinal ganglion cell differentiation

Isl1 and Pou4f2 Form a Complex to Regulate Target Genes in Developing Retinal Ganglion Cells

Precise regulation of gene expression during biological processes, including development, is often achieved by combinatorial action of multiple transcription factors. The mechanisms by which these factors collaborate are largely not known. We have shown previously that Isl1, a Lim-Homeodomain transcription factor, and Pou4f2, a class IV POU domain transcription factor, co-regulate a set of genes required for retinal ganglion cell (RGC) differentiation. Here we further explore how these two factors interact to precisely regulate gene expression during RGC development. By GST pulldown assays, co-immunoprecipitation, and electrophoretic mobility shift assays, we show that Isl1 and Pou4f2 form a complex in vitro and in vivo, and identify the domains within these two proteins that are responsible for this interaction. By luciferase assay, in situ hybridization, and RNA-seq, we further demonstrate that the two factors contribute quantitatively to gene expression in the developing RGCs. Although each factor alone can activate gene expression, both factors are required to achieve optimal expression levels. Finally, we discover that Isl1 and Pou4f2 can interact with other POU and Lim-Homeodomain factors respectively, indicating the interactions between these two classes of transcription factors are prevalent in development and other biological processes.

Chemically Induced Specification of Retinal Ganglion Cells From Human Embryonic and Induced Pluripotent Stem Cells

The loss of retinal ganglion cells (RGCs) is the primary pathological change for many retinal degenerative diseases. Although there is currently no effective treatment for this group of diseases, cell transplantation to replace lost RGCs holds great potential. However, for the development of cell replacement therapy, better understanding of the molecular details involved in differentiating stem cells into RGCs is essential. In this study, a novel, stepwise chemical protocol is described for the differentiation of human embryonic stem cells and induced pluripotent stem cells into functional RGCs. Briefly, stem cells were differentiated into neural rosettes, which were then cultured with the Notch inhibitor N-[N-(3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl ester (DAPT). The expression of neural and RGC markers (BRN3A, BRN3B, ATOH7/Math5, γ-synuclein, Islet-1, and THY-1) was examined. Approximately 30% of the cell population obtained expressed the neuronal marker TUJ1 as well the RGC markers. Moreover, the differentiated RGCs generated action potentials and exhibited both spontaneous and evoked excitatory postsynaptic currents, indicating that functional and mature RGCs were generated. In combination, these data demonstrate that a single chemical (DAPT) can induce PAX6/RX-positive stem cells to undergo differentiation into functional RGCs.

Nel positively regulates the genesis of retinal ganglion cells by promoting their differentiation and survival during development

For correct functioning of the nervous system, the appropriate number and complement of neuronal cell types must be produced during development. However, the molecular mechanisms that regulate the production of individual classes of neurons are poorly understood. In this study, we investigate the function of the thrombospondin-1-like glycoprotein, Nel (neural epidermal growth factor [EGF]-like), in the generation of retinal ganglion cells (RGCs) in chicks. During eye development, Nel is strongly expressed in the presumptive retinal pigment epithelium and RGCs. Nel overexpression in the developing retina by in ovo electroporation increases the number of RGCs, whereas the number of displaced amacrine cells decreases. Conversely, knockdown of Nel expression by transposon-mediated introduction of RNA interference constructs results in decrease in RGC number and increase in the number of displaced amacrine cells. Modifications of Nel expression levels do not appear to affect proliferation of retinal progenitor cells, but they significantly alter the progression rate of RGC differentiation from the central retina to the periphery. Furthermore, Nel protects RGCs from apoptosis during retinal development. These results indicate that Nel positively regulates RGC production by promoting their differentiation and survival during development.

Chemically-induced specification of retinal ganglion cells from human embryonic and induced pluripotent stem cells

The loss of retinal ganglion cells (RGCs) is the primary pathological change for many retinal degenerative diseases. Although there is currently no effective treatment for this group of diseases, cell transplantation to replace lost RGCs holds great potential. However, for the development of cell replacement therapy, better understanding of the molecular details involved in differentiating stem cells into RGCs is essential. In this study, a novel, stepwise chemical protocol is described for the differentiation of human embryonic stem cells and induced pluripotent stem cells into functional RGCs. Briefly, stem cells were differentiated into neural rosettes, which were then cultured with the Notch inhibitor N-[N-(3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl ester (DAPT). The expression of neural and RGC markers (BRN3A, BRN3B, ATOH7/Math5, γ-synuclein, Islet-1, and THY-1) was examined. Approximately 30% of the cell population obtained expressed the neuronal marker TUJ1 as well the RGC markers. Moreover, the differentiated RGCs generated action potentials and exhibited both spontaneous and evoked excitatory postsynaptic currents, indicating that functional and mature RGCs were generated. In combination, these data demonstrate that a single chemical (DAPT) can induce PAX6/RX-positive stem cells to undergo differentiation into functional RGCs.

Protection by an Oral Disubstituted Hydroxylamine Derivative against Loss of Retinal Ganglion Cell Differentiation following Optic Nerve Crush

Thy-1 is a cell surface protein that is expressed during the differentiation of retinal ganglion cells (RGCs). Optic nerve injury induces progressive loss in the number of RGCs expressing Thy-1. The rate of this loss is fastest during the first week after optic nerve injury and slower in subsequent weeks. This study was undertaken to determine whether oral treatment with a water-soluble N-hydroxy-2,2,6,6-tetramethylpiperidine derivative (OT-440) protects against loss of Thy-1 promoter activation following optic nerve crush and whether this effect targets the earlier quick phase or the later slow phase. The retina of mice expressing cyan fluorescent protein under control of the Thy-1 promoter (Thy1-CFP mice) was imaged using a blue-light confocal scanning laser ophthalmoscope (bCSLO). These mice then received oral OT-440 prepared in cream cheese or dissolved in water, or plain vehicle, for two weeks and were imaged again prior to unilateral optic nerve crush. Treatments and weekly imaging continued for four more weeks. Fluorescent neurons were counted in the same defined retinal areas imaged at each time point in a masked fashion. When the counts at each time point were directly compared, the numbers of fluorescent cells at each time point were greater in the animals that received OT-440 in cream cheese by 8%, 27%, 52% and 60% than in corresponding control animals at 1, 2, 3 and 4 weeks after optic nerve crush. Similar results were obtained when the vehicle was water. Rate analysis indicated the protective effect of OT-440 was greatest during the first two weeks and was maintained in the second two weeks after crush for both the cream cheese vehicle study and water vehicle study. Because most of the fluorescent cells detected by bCSLO are RGCs, these findings suggest that oral OT-440 can either protect against or delay early degenerative responses occurring in RGCs following optic nerve injury.

ADAM10 mediatesN-cadherin ectodomain shedding during retinal ganglion cell differentiation in primary cultured retinal cells from the developing chick retina

Here, we examined the role of ADAM10 during retinal cell differentiation in retinal sections and in vitro cultures of developing chick retinal cells from embryonic day 6 (ED6). Immunohistochemistry showed that ADAM10 is abundantly expressed in the inner zone of neuroblastic layer at ED5, and it becomes more highly expressed in the ganglion cell layer at ED7 and ED9. Western blotting confirmed that ADAM10 was expressed as an inactive pro-form that was processed to a shorter, active form in control cultured cells, but in cultures treated with an ADAM10 inhibitor (GI254023X) and ADAM10-specific siRNA, the level of mature ADAM10 decreased. Phase-contrast microscopy showed that long neurite extensions were present in untreated cultures 24 h after plating, whereas cultures treated with GI254023X showed significant decreases in neurite extension. Immunofluorescence staining revealed that there were far fewer differentiated ganglion cells in ADAM10 siRNA and GI254023X-treated cultures compared to controls, whereas the photoreceptor cells were unaltered. The Pax6 protein was more strongly detected in the differentiated ganglion cells of control cultures compared to ADAM10 siRNA and GI254023X-treated cultures. N-cadherin ectodomain shedding was apparent in control cultures after 24 h, when ganglion cell differentiation was observed, but ADAM10 siRNA and GI254023X treatment inhibited these processes. In contrast, N-cadherin staining was strongly detected in photoreceptor cells regardless of ADAM10 siRNA and GI254023X treatment. Taken together, these data indicate that the inhibition of ADAM10 can inhibit Pax6 expression and N-cadherin ectodomain shedding in retinal cells, possibly affecting neurite outgrowth and ganglion cell differentiation.

Inhibition of histone deacetylases 1 and 3 protects injured retinal ganglion cells.

Thy-1 is a marker of retinal ganglion cell (RGC) differentiation. Optic nerve injury triggers reduction of Thy-1 promoter activation followed by retinal ganglion cell (RGC) death. This study determined whether MS-275, an inhibitor of the histone deacetylases 1 and 3, can inhibit these changes. Mice expressing cyan fluorescent protein (CFP) under control of the Thy-1 promoter received MS-275 (subcutaneous) or vehicle three times per week starting 1 week before optic nerve crush and continuing for 6 weeks. The same retinal area was imaged using the blue-light confocal scanning laser ophthalmoscope before and after optic nerve crush every week, and fluorescent spots were counted manually. The eyes were then processed for histopathologic analysis. The mean proportions of fluorescent retinal neurons remaining in the vehicle group following optic nerve crush were 36 ± 8, 18 ± 6, 13 ± 10, 12 ± 4, 13 ± 5, and 13 ± 5% at weeks 1 through 6, respectively (n = 6). In contrast, the mean proportions of fluorescent retinal neurons remaining in the group treated with MS-275 were 59 ± 19, 39 ± 11, 34 ± 12, 33 ± 15, 32 ± 13, and 27 ± 15% at weeks 1 through 6, respectively (n = 7, P < 0.05 at weeks 1 through 5). Rate analysis showed that MS-275 slowed the rate of loss during the first 2 weeks by 23% (P < 0.05) and subsequently was similar. Histopathologic analysis revealed 27 ± 13% greater ganglion cell layer (GCL) neurons in the eyes from mice that received MS-275 treatment (P < 0.02). These results indicate that treatment with MS-275 protects against the loss of RGC differentiation and promotes RGC survival following optic nerve injury.

An in vitro comparison study: The effects of fetal bovine serum concentration on retinal progenitor cell multipotentiality

Retinal progenitor cells (RPCs) are an excellent resource for retinal replacement therapy, because they show enormous potential to differentiate into retinal-specific cell types. While the differentiating influence of serum has long been appreciated, the effects of serum concentration on RPC differentiation into specified retinal neural cells have not been investigated. Using cultured murine RPCs, this study compared the effects of different levels of fetal bovine serum (FBS) (1%, 5%, 10% and 20%) on RPC differentiation in vitro. RPC multipotentiality was assessed by using quantitative polymerase chain reaction (qPCR) to determine the relative expression levels of 10 genes involved in retinal development. In addition, analyses of cell morphology and retinal development-related protein expression were performed using microscopy and immunocytochemistry. The data revealed that 1% FBS-induced cultures preferentially generated rhodopsin- and PKC-α-positive cells. Calbindin and AP2α expression levels were greater in 5% FBS-induced cultures. Brn3a was expressed at similar levels in 1%, 5% and 10% FBS treatment conditions but diminished in 20% FBS conditions. Twenty percent FBS induced more glial fibrillary acid protein (GFAP)-immunoreactive cells corresponding to glia populations. These findings suggest that the concentration of FBS plays an important role in RPC differentiation in vitro. Treatment with low levels of FBS favors differentiation of rhodopsin-positive photoreceptors, interneurons and retinal ganglion cells (RGCs), while high FBS concentrations preferentially induce differentiation of glia cells. These results are expected to facilitate research in the treatment of neurodegenerative retinal diseases.

Deterministic or Stochastic Choices in Retinal Neuron Specification

There are two views on vertebrate retinogenesis: a deterministic model dependent on fixed lineages and a stochastic model in which choices of division modes and cell fates cannot be predicted. In this issue of Neuron, He etal. (2012) address this question in zebrafish using live imaging and mathematical modeling.

Generating human retinal ganglion cells from human induced pluripotent cells in feeder and feeder‐free conditions

AbstractPurpose Glaucoma, and other optic neuropathies, results in the loss of retinal ganglion cells (RGCs) and vision dysfunction. Therefore, cell replacement therapy may offer promising treatments to protect the degenerating retina and potentially restore vision function. Although significant progress has been made to generate photoreceptors from stem cells, there has been little advancement in efficiently generating RGCs. Therefore, we have been investigating the optimal conditions for differentiating RGCs from stem cells for cell replacement therapy.Methods Human induced pluripotent stem cells (iPSCs) were maintained and expanded on feeder layers using previously published conditions. During embryoid body formation, genes and proteins were tested for effects on increasing RGC differentiation efficiency. Differentiated RGCs were identified and quantified using RGC‐specific markers including Brn3. Feeder‐free conditions were examined for functional equivalence.Results iPSCs that express stem cell‐specific markers were passaged in proliferative conditions and differentiated into RGCs in vitro. Feeder‐free cell culture conditions allowed iPSC maintenance without the presence of mouse immunogenicity. iPSCs were found to respond to pro‐RGC differentiation signals in similar fashion to rodent embryonic retinal progenitors.Conclusion These findings will provide valuable insight in our understanding of RGC differentiation and will pave the way to cure retinal diseases. The transplantation of these cells in vivo will elucidate the integration potential of these cells in degenerating retina.

Retinal ganglion cell differentiation and protection using neuronally differentiated human dental pulp stem cells

AbstractPurpose To investigate whether the neuronally differentiated human dental pulp stem cells (NDhDPSCs) can differentiate into retinal ganglion cells (RGCs) and whether the NDhDPSCs can protect primary mouse RGCs against oxidative stress injury.Methods Human dental pulp stem cells were harvested and neuronally differentiated using various conditioned media. By immunohistochemistry, western immunoblots, and real‐time RT‐PCR, their cellular characteristics were evaluated and the best culture condition was determined for differentiation into RGC/glia‐like NDhDPSCs. Regarding glial‐like NDhDPSCs, they were cocultured with primary mouse RGCs under oxidative stress injury.Results In specialized conditioned media, human dental pulp stem cells were differentiated into RGC/glia‐like NDhDPSCs. They expressed RGC/glia‐specfic markers as well as neuronal stem cell markers. When the glia‐like NDhDPSCs were indirectly cocultured with primary mouse RGCs, they protect the RGCs against oxidative stress injury as determined by TUNEL assay.Conclusion The human dental pulp stem cells may differentiate into RGC‐like NDhDPSCs in a suitable culture condition. And the glia‐like NDhDPSCs can protect primary mouse RGCs against oxidative stress injury in vitro.

Math5 (Atoh7) gene dosage limits retinal ganglion cell genesis

The basic helix-loop-helix factor Math5 (Atoh7) is critical for the determination of retinal ganglion cell (RGC) fate in mice. Recently, genome-wide association studies have identified the ATOH7 locus as a major determinant of variation in the human optic disc area, which is directly correlated with the RGC number. These studies suggest that the level of Math5 expression may determine the ultimate number of RGCs. To test this hypothesis, we systematically compared optic nerve area and RGC axon number in C57BL/6J congenic Math5+/- and +/+ mice at young adult and neonatal ages by transmission electron microscopy. Optic disc area and RGC abundance were not significantly different in adults, but heterozygotes had thinner optic nerves and 25-30% fewer RGCs at birth than wild-type littermates (P<0.05). Our results suggest that Math5 dosage is important for the genesis, but not the ultimate number, of RGCs. Our findings highlight the importance of ganglion cell culling as a compensatory mechanism for retinal homeostasis, and support a quantitative role for Math5 in RGC specification.

Staurosporine induces ganglion cell differentiation in part by stimulating urokinase-type plasminogen activator expression and activation in the developing chick retina

Here, we investigated whether staurosporine-mediated urokinase-type plasminogen activator (uPA) activation is involved in retinal ganglion cell (RGC) differentiation. Retinal cells were isolated from developing chick retinas at embryonic day 6 (E6). Relatively few control cells grown in serum-free medium started to form processes by 12h. In contrast, staurosporine-treated cells had processes within 3h, and processes were evident at 8h. Immunofluorescence staining showed that Tuj-1-positive cells with shorter neurites could be detected in control cultures at 18h, whereas numerous Tuj-1 positive ganglion cells with longer neuritic extensions were seen in staurosporine-treated cultures. BrdU-positive proliferating cells were more numerous in control cultures than in staurosporine-treated cultures, and the BrdU staining was not detected in post-mitotic Tuj-1 positive ganglion cells. Western blotting of cell lysates showed that staurosporine induced high levels of the active form of uPA. The staurosporine-induced uPA signal was localized predominantly in the soma, neurites and axons of Tuj-1-positive ganglion cells. Amiloride, an inhibitor of uPA, markedly reduced staurosporine-induced Tuj-1 staining, neurite length, neurite number, and uPA staining versus controls. In developing retinas in ovo, amiloride administration remarkably reduced the staurosporine-induced uPA staining and RGC differentiation. Taken together, our in vitro and in vivo data collectively indicate that uPA plays a role in the staurosporine-mediated stimulation of RGC differentiation.