Stem-loop binding protein is required for retinal cell proliferation,neurogenesis, and intraretinal axon pathfinding in zebrafish

In the developing retina, neurogenesis and cell differentiation are coupled with cell proliferation. However, molecular mechanisms that coordinate cell proliferation and differentiation are not fully understood. In this study, we found that retinal neurogenesis is severely delayed in the zebrafish stem-loop binding protein (slbp) mutant. SLBP binds to a stem-loop structure at the 3′-end of histone mRNAs, and regulates a replication-dependent synthesis and degradation of histone proteins. Retinal cell proliferation becomes slower in the slbp1 mutant, resulting in cessation of retinal stem cell proliferation. Although retinal stem cells cease proliferation by 2 days postfertilization (dpf) in the slbp mutant, retinal progenitor cells in the central retina continue to proliferate and generate neurons until at least 5 dpf. We found that this progenitor proliferation depends on Notch signaling, suggesting that Notch signaling maintains retinal progenitor proliferation when faced with reduced SLBP activity. Thus, SLBP is required for retinal stem cell maintenance. SLBP and Notch signaling are required for retinal progenitor cell proliferation and subsequent neurogenesis. We also show that SLBP1 is required for intraretinal axon pathfinding, probably through morphogenesis of the optic stalk, which expresses attractant cues. Taken together, these data indicate important roles of SLBP in retinal development.     

Identification of Arhgef12 and Prkci as genetic modifiers of retinal dysplasia in the Crb1rd8 mouse model

Mutations in the apicobasal polarity gene CRB1 lead to diverse retinal diseases, such as Leber congenital amaurosis, cone-rod dystrophy, retinitis pigmentosa (with and without Coats-like vasculopathy), foveal retinoschisis, macular dystrophy, and pigmented paravenous chorioretinal atrophy. Limited correlation between disease phenotypes and CRB1 alleles, and evidence that patients sharing the same alleles often present with different disease features, suggest that genetic modifiers contribute to clinical variation. Similarly, the retinal phenotype of mice bearing the Crb1 retinal degeneration 8 (rd8) allele varies with genetic background. Here, we initiated a sensitized chemical mutagenesis screen in B6.Cg-Crb1^rd8/Pjn, a strain with a mild clinical presentation, to identify genetic modifiers that cause a more severe disease phenotype. Two models from this screen, Tvrm266 and Tvrm323, exhibited increased retinal dysplasia. Genetic mapping with high-throughput exome and candidate-gene sequencing identified causative mutations in Arhgef12 and Prkci, respectively. Epistasis analysis of both strains indicated that the increased dysplastic phenotype required homozygosity of the Crb1^rd8 allele. Retinal dysplastic lesions in Tvrm266 mice were smaller and caused less photoreceptor degeneration than those in Tvrm323 mice, which developed an early, large diffuse lesion phenotype. At one month of age, Müller glia and microglia mislocalization at dysplastic lesions in both modifier strains was similar to that in B6.Cg-Crb1^rd8/Pjn mice but photoreceptor cell mislocalization was more extensive. External limiting membrane disruption was comparable in Tvrm266 and B6.Cg-Crb1^rd8/Pjn mice but milder in Tvrm323 mice. Immunohistological analysis of mice at postnatal day 0 indicated a normal distribution of mitotic cells in Tvrm266 and Tvrm323 mice, suggesting normal early development. Aberrant electroretinography responses were observed in both models but functional decline was significant only in Tvrm323 mice. These results identify Arhgef12 and Prkci as modifier genes that differentially shape Crb1-associated retinal disease, which may be relevant to understanding clinical variability and underlying disease mechanisms in humans.     

Multiple domains in the Crumbs Homolog 2a (Crb2a) protein are required for regulating rod photoreceptor size

Background  

Vertebrate retinal photoreceptors are morphologically complex cells that have two apical regions, the inner segment and the outer segment. The outer segment is a modified cilium and is continuously regenerated throughout life. The molecular and cellular mechanisms that underlie vertebrate photoreceptor morphogenesis and the maintenance of the outer segment are largely unknown. The Crumbs (Crb) complex is a key regulator of apical membrane identity and size in epithelia and in Drosophila photoreceptors. Mutations in the human gene CRUMBS HOMOLOG 1 (CRB1) are associated with early and severe vision loss. Drosophila Crumbs and vertebrate Crb1 and Crumbs homolog 2 (Crb2) proteins are structurally similar, all are single pass transmembrane proteins with a large extracellular domain containing multiple laminin- and EGF-like repeats and a small intracellular domain containing a FERM-binding domain and a PDZ-binding domain. In order to begin to understand the role of the Crb family of proteins in vertebrate photoreceptors we generated stable transgenic zebrafish in which rod photoreceptors overexpress full-length Crb2a protein and several other Crb2a constructs engineered to lack specific domains.     

Crumbs limits oxidase-dependent signaling to maintain epithelial integrity and prevent photoreceptor cell death

Drosophila melanogaster Crumbs (Crb) and its mammalian orthologues (CRB1–3) share evolutionarily conserved but poorly defined roles in regulating epithelial polarity and, in photoreceptor cells, morphogenesis and stability. Elucidating the molecular mechanisms of Crb function is vital, as mutations in the human CRB1 gene cause retinal dystrophies. Here, we report that Crb restricts Rac1–NADPH oxidase-dependent superoxide production in epithelia and photoreceptor cells. Reduction of superoxide levels rescued epithelial defects in crb mutant embryos, demonstrating that limitation of superoxide production is a crucial function of Crb and that NADPH oxidase and superoxide contribute to the molecular network regulating epithelial tissue organization. We further show that reduction of Rac1 or NADPH oxidase activity or quenching of reactive oxygen species prevented degeneration of Crb-deficient retinas. Thus, Crb fulfills a protective role during light exposure by limiting oxidative damage resulting from Rac1–NADPH oxidase complex activity. Collectively, our results elucidate an important mechanism by which Crb functions in epithelial organization and the prevention of retinal degeneration.     

Expression of a novel secretory form (Crb1s) of mouse Crumbs homologue Crb1 in skin development

Drosophila Crumbs and the mammalian homologues encoded by the Crb genes are transmembrane proteins required for determination of retinal cell polarity. We cloned a novel variant of mouse Crb1 and termed it Crb1s. Since the 3'-end of exon 6 remained unspliced, Crb1s coded for a short secretory protein lacking the transmembrane and cytoplasmic domains required for the function of Crb1. The Crb1 expression was confined to brain and eye, whereas Crb1s was detectable in various tissues including skin, lung, and kidney in adult mice. Active expression of Crb1s, but not Crb1, was observed during the skin development, in which localization of the Crb1s protein was altered from the basal layer to the upper layers. Cultured mouse keratinocytes synthesized the Crb1s protein and secreted a 80 kDa processed form to the supernatant. After Ca(2+)-induced differentiation, Crb1s became associated with focal adhesions and cell-cell contacts. Crb1s may play a role distinct from that of Crb1 in epidermal tissue morphogenesis.     

Histone deacetylase 1 regulates retinal neurogenesis in zebrafish by suppressing Wnt and Notch signaling pathways

In the developing vertebrate retina, progenitor cells initially proliferate but begin to produce postmitotic neurons when neuronal differentiation occurs. However, the mechanism that determines whether retinal progenitor cells continue to proliferate or exit from the cell cycle and differentiate is largely unknown. Here, we report that histone deacetylase 1 (Hdac1) is required for the switch from proliferation to differentiation in the zebrafish retina. We isolated a zebrafish mutant, ascending and descending (add), in which retinal cells fail to differentiate into neurons and glial cells but instead continue to proliferate. The cloning of the add gene revealed that it encodes Hdac1. Furthermore, the ratio of the number of differentiating cells to that of proliferating cells increases in proportion to Hdac activity, suggesting that Hdac proteins regulate a crucial step of retinal neurogenesis in zebrafish. Canonical Wnt signaling promotes the proliferation of retinal cells in zebrafish, and Notch signaling inhibits neuronal differentiation through the activation of a neurogenic inhibitor, Hairy/Enhancer-of-split (Hes). We found that both the Wnt and Notch/Hes pathways are activated in the add mutant retina. The cell-cycle progression and the upregulation of Hes expression in the add mutant retina can be inhibited by the blockade of Wnt and Notch signaling, respectively. These data suggest that Hdac1 antagonizes these pathways to promote cell-cycle exit and the subsequent neurogenesis in zebrafish retina. Taken together, these data suggest that Hdac1 functions as a dual switch that suppresses both cell-cycle progression and inhibition of neurogenesis in the zebrafish retina.     

Retinal neurons regulate proliferation of postnatal progenitors and Müller glia in the rat retina via TGF beta signaling

The number of proliferating cells in the rodent retina declines dramatically after birth. To determine if extrinsic factors in the retinal micro-environment are responsible for this decline in proliferation, we established cultures of retinal progenitors or Muller glia, and added dissociated retinal neurons from older retinas. The older cells inhibited proliferation of progenitor cells and Muller glia. When these experiments were performed in the presence of TGF(beta)RII-Fc fusion protein, an inhibitor of TGF(beta) signaling, proliferation was restored. This suggests a retina-derived TGF(beta) signal is responsible for the developmental decline in retinal proliferation. TGFbeta receptors I and II are expressed in the retina and are located in nestin-positive progenitors early in development and glast-positive Muller glia later in development. RT-PCR and immunofluorescence data show TGF(beta)2 is the most highly expressed TGF(beta)ligand in the postnatal retina, and it is expressed by inner retinal neurons. Addition of either TGF(beta)1 or TGF(beta)2 to postnatal day 4 retinas significantly inhibited progenitor proliferation, while treatment of explanted postnatal day 6 retinas with TGF(beta) signaling inhibitors resulted in increased proliferation. Last, we tested the effects of TGF(beta) in vivo by injections of TGF(beta) signaling inhibitors: when TGF(beta) signaling is inhibited at postnatal day 5.5, proliferation is increased in the central retina; and when co-injected with EGF at postnatal day 10, TGF(beta)inhibitors stimulate Muller glial proliferation. In sum, these results show that retinal neurons produce a cytostatic TGF(beta) signal that maintains mitotic quiescence in the postnatal rat retina.     

c-kit marks late retinal progenitor cells and regulates their differentiation in developing mouse retina

Retinal progenitor cells are believed to display altered proliferation and differentiation during retinal development, suggesting that retinal progenitor cell populations are not homogeneous. However, the composition of progenitor cell populations is not known, due in part to the lack of known surface markers identifying distinct stages of retinal progenitor cells. We found a dramatic change in the expression profile of the cell surface antigens c-kit and stage-specific embryonic antigen-1 (SSEA-1) in retinal progenitor cells during development. While SSEA-1 was expressed early in development, c-kit expression peaked in late stage progenitor cells. The identification of these developmental markers enabled us to characterize distinct sub-populations of retinal progenitor cells. Progenitor cell subpopulations expressing either SSEA-1, c-kit, or both showed different proliferation and differentiation abilities. Although SSEA-1-positive cells were augmented by beta-catenin signaling, c-kit-positive cells were positively regulated by Notch signaling. Taken together, our data suggest that c-kit and SSEA-1 can be used to spatiotemporally differentiate retinal progenitor populations that have intrinsically distinct characteristics. Prolonged expression of c-kit by a retrovirus resulted in the promotion of proliferation and the appearance of nestin-positive cells in the presence of the c-kit ligand, stem cell factor (SCF). This suggests a role for c-kit, Notch, and the beta-catenin signaling network in retinal development.     

Vsx2/Chx10 ensures the correct timing and magnitude of Hedgehog signaling in the mouse retina

Vertebrate retinal progenitor cells (RPCs) undergo a robust proliferative expansion to produce enough cells for the retina to form appropriately. Vsx2 (formerly Chx10), a homeodomain protein expressed in RPCs, is required for sufficient proliferation to occur. Sonic Hedgehog protein (SHH), secreted by retinal ganglion cells (RGCs), activates Hedgehog (Hh) signaling in RPCs and is also required for sufficient proliferation to occur. Therefore, we sought to determine if reduced Hh signaling is a contributing factor to the proliferation changes that occur in the absence of Vsx2. To do this, we examined Shh expression and Hh signaling activity in the homozygous ocular retardation J (orJ) mouse, which harbors a recessive null allele in the Vsx2 gene. We found that Shh expression and Hh signaling activity are delayed during early retinal development in orJ mice and this correlates with a delay in the onset of RGC differentiation. At birth, reduced expression of genes regulated by Hh signaling was observed despite the production of SHH ligand. orJ RPCs respond to pre-processed recombinant SHH ligand (SHH-N) in explant culture as evidenced by increased proliferation and expression of Hh target genes. Interestingly, proliferation in the orJ retina is further inhibited by cyclopamine, an antagonist of Hh signaling. Our results suggest that reduced Hh signaling contributes to the reduced level of RPC proliferation in the orJ retina, thereby revealing a role for Vsx2 in mediating mitogen signaling.     

Mutations in N-cadherin and a Stardust homolog,Nagie oko,affect cell-cycle exit in zebrafish retina

It has been reported that the loss of apicobasal cell polarity and the disruption of adherens junctions induce hyperplasia in the mouse developing brain. However, it is not fully understood whether hyperplasia is caused by an enhanced cell proliferation, an inhibited neurogenesis, or both. In this study, we found that the ratio of the number of proliferating progenitor cells to the total number of retinal cells increases in the neurogenic stages in zebrafish n-cadherin (ncad) and nagie oko (nok) mutants, in which the apicobasal cell polarity and adherens junctions in the retinal epithelium are disrupted. The cell-cycle progression was not altered in the ncad and nok mutants. Rather, the ratio of the number of cells undergoing neurogenic cell division to the total number of cells undergoing mitosis decreased in the ncad and nok mutant retinas, suggesting that the switching from proliferative cell division to neurogenic cell division was compromised in these mutant retinas. These findings suggest that the inhibition of neurogenesis is a primary defect that causes hyperplasia in the ncad and nok mutant retinas. The Hedgehog-protein kinase A signaling pathway and the Notch signaling pathway regulate retinal neurogenesis in zebrafish. We found that both signaling pathways are involved in the generation of neurogenic defects in the ncad and nok mutant retinas. Taken together, these findings suggest that apicobasal cell polarity and epithelial integrity are essential for retinal neurogenesis in zebrafish.     

In the absence of Sonic hedgehog, p53 induces apoptosis and inhibits retinal cell proliferation, cell-cycle exit and differentiation in zebrafish

Background

Sonic hedgehog (Shh) signaling regulates cell proliferation during vertebrate development via induction of cell-cycle regulator gene expression or activation of other signalling pathways, prevents cell death by an as yet unclear mechanism and is required for differentiation of retinal cell types. Thus, an unsolved question is how the same signalling molecule can regulate such distinct cell processes as proliferation, cell survival and differentiation.

Methodology/Principal Findings

Analysis of the zebrafish shh ^−/− mutant revealed that in this context p53 mediates elevated apoptosis during nervous system and retina development and interferes with retinal proliferation and differentiation. While in shh ^−/− mutants there is activation of p53 target genes and p53-mediated apoptosis, an increase in Hedgehog (Hh) signalling by over-expression of dominant-negative Protein Kinase A strongly decreased p53 target gene expression and apoptosis levels in shh ^−/− mutants. Using a novel p53 reporter transgene, I confirm that p53 is active in tissues that require Shh for cell survival. Proliferation assays revealed that loss of p53 can rescue normal cell-cycle exit and the mitotic indices in the shh ^−/− mutant retina at 24, 36 and 48 hpf. Moreover, generation of amacrine cells and photoreceptors was strongly enhanced in the double p53 ^−/− shh ^−/− mutant retina suggesting the effect of p53 on retinal differentiation.

Conclusions

Loss of Shh signalling leads to the p53-dependent apoptosis in the developing nervous system and retina. Moreover, Shh-mediated control of p53 activity is required for proliferation and cell cycle exit of retinal cells as well as differentiation of amacrine cells and photoreceptors.     

VEGF activates divergent intracellular signaling components to regulate retinal progenitor cell proliferation and neuronal differentiation

During vertebrate neurogenesis, multiple extracellular signals influence progenitor cell fate choices. The process by which uncommitted progenitor cells interpret and integrate signals is not well understood. We demonstrate here that in the avascular chicken retina, vascular endothelial growth factor (VEGF) secreted by postmitotic neurons acts through the FLK1 receptor present on progenitor cells to influence cell proliferation and commitment. Augmenting VEGF signals increases progenitor cell proliferation and decreases retinal ganglion cell genesis. Conversely, absorbing endogenous VEGF ligand or disrupting FLK1 activity attenuates cell proliferation and enhances retinal ganglion cell production. In addition, we provide evidence that VEGF signals transmitted by the FLK1 receptor activate divergent intracellular signaling components, which regulate different responses of progenitor cells. VEGF-induced proliferation is influenced by the MEK-ERK pathway, as well as by the basic helix-loop-helix factor HES1. By contrast, VEGF-dependent ganglion cell suppression does not require MEK-ERK activation, but instead relies on VEGF-stimulated HES1 activity, which is independent of NOTCH signaling. Moreover, elevated HES1 expression promotes progenitor cell proliferation and prevents overproduction of retinal ganglion cells owing to the loss of VEGF or sonic hedgehog (SHH), another signal that suppresses ganglion cell development. Based on previous and current findings, we propose that HES1 serves as a convergent signaling node within early retinal progenitor cells to integrate various cell-extrinsic cues, including VEGF and SHH, in order to control cell proliferation and neuronal specification.     

The expression of NOTCH2, HES1 and SOX9 during mouse retinal development

Notch signaling is an important regulator of both developmental and post-developmental processes. In the developing retina, Notch1 is required for the maintenance of retinal progenitor cells and for inhibiting photoreceptor cell fate, while Notch3 is required for inhibiting ganglion cell fate. Here we used immunolabeling coupled with a knock-in reporter approach to obtain a detailed spatiotemporal expression pattern of Notch2 during mouse retinal development. Although previous in situ hybridization studies did not reveal appreciable levels of Notch2 in the developing retina, we detected NOTCH2 protein and reporter expression in early embryonic retinal progenitors that also expressed the Notch downstream gene, HES1. In the postnatal retina, NOTCH2, as well as the Notch downstream genes, HES1 and SOX9, were detected in VSX2/Cyclin D1/SOX2-expressing cells in the postnatal retina, and in the mature retina NOTCH2 was most abundant in Müller glia. Our findings indicate a potential role for Notch2 in the developing and mature retina.     

GFAP-Driven GFP Expression in Activated Mouse Müller Glial Cells Aligning Retinal Blood Vessels Following Intravitreal Injection of AAV2/6 Vectors

Background

Müller cell gliosis occurs in various retinal pathologies regardless of the underlying cellular defect. Because activated Müller glial cells span the entire retina and align areas of injury, they are ideal targets for therapeutic strategies, including gene therapy.

Methodology/Principal Findings

We used adeno-associated viral AAV2/6 vectors to transduce mouse retinas. The transduction pattern of AAV2/6 was investigated by studying expression of the green fluorescent protein (GFP) transgene using scanning-laser ophthalmoscopy and immuno-histochemistry. AAV2/6 vectors transduced mouse Müller glial cells aligning the retinal blood vessels. However, the transduction capacity was hindered by the inner limiting membrane (ILM) and besides Müller glial cells, several other inner retinal cell types were transduced. To obtain Müller glial cell-specific transgene expression, the cytomegalovirus (CMV) promoter was replaced by the glial fibrillary acidic protein (GFAP) promoter. Specificity and activation of the GFAP promoter was tested in a mouse model for retinal gliosis. Mice deficient for Crumbs homologue 1 (CRB1) develop gliosis after light exposure. Light exposure of Crb1^−/− retinas transduced with AAV2/6-GFAP-GFP induced GFP expression restricted to activated Müller glial cells aligning retinal blood vessels.

Conclusions/Significance

Our experiments indicate that AAV2 vectors carrying the GFAP promoter are a promising tool for specific expression of transgenes in activated glial cells.     

SSEA-1 marks regionally restricted immature subpopulations of embryonic retinal progenitor cells that are regulated by the Wnt signaling pathway

Identification and expansion of retinal progenitor cells are critical issues from both scientific and clinical aspects. Here, we identified SSEA-1 (CD15) as a novel surface antigen that can be used to define immature retinal progenitor cells. SSEA-1-expressing retinal cells were found in the peripheral region of the early embryonic mouse retina, and then their number dramatically disappeared along with retinal development. FACS analysis showed that the cells strongly positive for SSEA-1 co-expressed Ki67 proliferation antigen in all the developmental stages examined. The SSEA-1-expressing cells formed larger colonies than the non-expressing ones in retinal re-aggregation cultures. Moreover, late onset of rhodopsin expression was observed in SSEA-1-positive progenitor cells, supporting the idea that these cells have an intrinsically immature character. Differential expression of Wnt signal-related genes between SSEA-1-positive and -negative subpopulations of retina cells was revealed, and the expression of constitutively active forms of Wnt signaling molecules resulted in a greater number of SSEA-1-positive cells. In light of all of the data taken together, we propose SSEA-1 to be a surface marker to define a regionally restricted immature subset of progenitor cells of mouse neural retina, with SSEA-1 expression by them positively regulated by Wnt signals.