Proper patterning of the optic fissure requires the sequential activity of BMP7 and SHH

The optic disc develops at the interface between optic stalk and retina, and enables both the exit of visual fibres and the entrance of mesenchymal cells that will form the hyaloid artery. In spite of the importance of the optic disc for eye function, little is known about the mechanisms that control its development. Here, we show that in mouse embryos, retinal fissure precursors can be recognised by the expression of netrin 1 and the overlapping distribution of both optic stalk (Pax2, Vax1) and ventral neural retina markers (Vax2, Raldh3). We also show that in the absence of Bmp7, fissure formation is not initiated. This absence is associated with a reduced cell proliferation and apoptosis in the proximoventral quadrant of the optic cup, lack of the hyaloid artery, optic nerve aplasia, and intra-retinal misrouting of RGC axons. BMP7 addition to organotypic cultures of optic vesicles from Bmp7-/- embryos rescues Pax2 expression in the ventral region, while follistatin, a BMP7 antagonist, prevents it in early, but not in late, optic vesicle cultures from wild-type embryos. The presence of Pax2-positive cells in late optic cup is instead abolished by interfering with Shh signalling. Furthermore, SHH addition re-establishes Pax2 expression in late optic cups derived from ocular retardation (or) embryos, where optic disc development is impaired owing to the near absence of SHH-producing RGC. Collectively, these data indicate that BMP7 is required for retinal fissure formation and that its activity is needed, before SHH signalling, for the generation of PAX2-positive cells at the optic disc.     

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.     

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.     

Pushing the envelope of retinal ganglion cell genesis: context dependent function of Math5 (Atoh7)

The basic-helix-loop helix factor Math5 (Atoh7) is required for retinal ganglion cell (RGC) development. However, only 10% of Math5-expressing cells adopt the RGC fate, and most become photoreceptors. In principle, Math5 may actively bias progenitors towards RGC fate or passively confer competence to respond to instructive factors. To distinguish these mechanisms, we misexpressed Math5 in a wide population of precursors using a Crx BAC or 2.4 kb promoter, and followed cell fates with Cre recombinase. In mice, the Crx cone-rod homeobox gene and Math5 are expressed shortly after cell cycle exit, in temporally distinct, but overlapping populations of neurogenic cells that give rise to 85% and 3% of the adult retina, respectively. The Crx>Math5 transgenes did not stimulate RGC fate or alter the timing of RGC births. Likewise, retroviral Math5 overexpression in retinal explants did not bias progenitors towards the RGC fate or induce cell cycle exit. The Crx>Math5 transgene did reduce the abundance of early-born (E15.5) photoreceptors two-fold, suggesting a limited cell fate shift. Nonetheless, retinal histology was grossly normal, despite widespread persistent Math5 expression. In an RGC-deficient (Math5 knockout) environment, Crx>Math5 partially rescued RGC and optic nerve development, but the temporal envelope of RGC births was not extended. The number of early-born RGCs (before E13) remained very low, and this was correlated with axon pathfinding defects and cell death. Together, these results suggest that Math5 is not sufficient to stimulate RGC fate. Our findings highlight the robust homeostatic mechanisms, and role of pioneering neurons in RGC 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.     

Co-ordinating retinal histogenesis: early cell cycle exit enhances early cell fate determination in the Xenopus retina

The laminar arrays of distinct cell types in the vertebrate retina are built by a histogenic process in which cell fate is correlated with birth order. To explore this co-ordination mechanistically, we altered the relative timing of cell cycle exit in the developing Xenopus retina and asked whether this affected the activity of neural determinants. We found that Xath5, a bHLH proneural gene that promotes retinal ganglion cell (RGC) fate, ( Kanekar, S., Perron, M., Dorsky, R., Harris, W. A., Jan, L. Y., Jan, Y. N. and Vetter, M. L. (1997) Neuron 19, 981-994), does not cause these cells to be born prematurely. To drive cells out of the cell cycle early, therefore, we misexpressed the cyclin kinase inhibitor, p27Xic1. We found that early cell cycle exit potentiates the ability of Xath5 to promote RGC fate. Conversely, the cell cycle activator, cyclin E1, which inhibits cell cycle exit, biases Xath5-expressing cells toward later neuronal fates. We found that Notch activation in this system caused cells to exit the cell cycle prematurely, and when it is misexpressed with Xath5, it also potentiates the induction of RGCs. The potentiation is counteracted by co-expression of cyclin E1. These results suggest a model of histogenesis in which the activity of factors that promote early cell cycle exit enhances the activity of factors that promote early cellular fates.     

Effect of p75NTR on the regulation of naturally occurring cell death and retinal ganglion cell number in the mouse eye

Neurotrophins induce neural cell survival and differentiation during retinal development and regeneration through the high-affinity tyrosine kinase (Trk) receptors. On the other hand, nerve growth factor (NGF) binding to the low-affinity neurotrophin receptor p75 (p75(NTR)) might induce programmed cell death (PCD) in the early phase of retinal development. In the present study, we examined the retinal cell types that experience p75(NTR)-induced PCD and identify them to be postmitotic retinal ganglion cells (RGCs). However, retinal morphology, RGC number, and BrdU-positive cell number in p75(NTR) knockout (KO) mouse were normal after embryonic day 15 (E15). In chick retina, migratory RGCs express p75(NTR), whereas layered RGCs express the high-affinity NGF receptor TrkA, which may switch the pro-apoptotic signaling of p75(NTR) into a neurotrophic one. In contrast to the chick model, migratory RGCs express TrkA, while stratified RGCs express p75(NTR) in mouse retina. However, RGC number in TrkA KO mouse was also normal at birth. We next examined the expression of transforming growth factor beta (TGFbeta) receptor, which modulates chick RGC number in combination with p75(NTR), but was absent in mouse RGCs. p75(NTR) and TrkA seem to be involved in the regulation of mouse RGC number in the early phase of retinal development, but the number may be later adjusted by other molecules. These results suggest the different mechanism of RGC number control between mouse and chick retina.     

Sonic hedgehog is indirectly required for intraretinal axon pathfinding by regulating chemokine expression in the optic stalk

Successful axon pathfinding requires both correct patterning of tissues, which will later harbor axonal tracts, and precise localization of axon guidance cues along these tracts at the time of axon outgrowth. Retinal ganglion cell (RGC) axons grow towards the optic disc in the central retina, where they turn to exit the eye through the optic nerve. Normal patterning of the optic disc and stalk and the expression of guidance cues at this choice point are necessary for the exit of RGC axons out of the eye. Sonic hedgehog (Shh) has been implicated in both patterning of ocular tissue and direct guidance of RGC axons. Here, we examine the precise spatial and temporal requirement for Hedgehog (Hh) signaling for intraretinal axon pathfinding and show that Shh acts to pattern the optic stalk in zebrafish but does not guide RGC axons inside the eye directly. We further reveal an interaction between the Hh and chemokine pathways for axon guidance and show that cxcl12a functions downstream of Shh and depends on Shh for its expression at the optic disc. Together, our results support a model in which Shh acts in RGC axon pathfinding indirectly by regulating axon guidance cues at the optic disc through patterning of the optic stalk.     

Autonomous and non-autonomous Shh signalling mediate the in vivo growth and guidance of mouse retinal ganglion cell axons

In non-mammalian vertebrates, the relatively homogeneous population of retinal ganglion cells (RGCs) differentiates and projects entirely to the contralateral side of the brain under the influence of sonic hedgehog (Shh). In mammals, by contrast, there are two different RGC types: the Zic2-positive ipsilateral projecting and the Isl2-positive contralateral projecting. We asked whether the axons of these two populations respond to Shh and if their response differs. We have also analysed whether midline- and RGC-derived Shh contributes to the growth of the axons in the proximal visual pathway. We show that these two RGC types are characterised by a differential expression of Shh signalling components and that they respond differently to Shh when challenged in vitro. In vivo blockade of Shh activity, however, alters the path and distribution mostly of the contralateral projecting RGC axons at the chiasm, indicating that midline-derived Shh participates in funnelling contralateral visual fibres in this region. Furthermore, interference with Shh signalling in the RGCs themselves causes abnormal growth and navigation of contralateral projecting axons in the proximal portion of the pathway, highlighting a novel cell-autonomous mechanism by which Shh can influence growth cone behaviour.     

Kinase independent function of EphB receptors in retinal axon pathfinding to the optic disc from dorsal but not ventral retina

Optic nerve formation requires precise retinal ganglion cell (RGC) axon pathfinding within the retina to the optic disc, the molecular basis of which is not well understood. At CNS targets, interactions between Eph receptor tyrosine kinases on RGC axons and ephrin ligands on target cells have been implicated in formation of topographic maps. However, studies in chick and mouse have shown that both Eph receptors and ephrins are also expressed within the retina itself, raising the possibility that this receptor-ligand family mediates aspects of retinal development. Here, we more fully document the presence of specific EphB receptors and B-ephrins in embryonic mouse retina and provide evidence that EphB receptors are involved in RGC axon pathfinding to the optic disc. We find that as RGC axons begin this pathfinding process, EphB receptors are uniformly expressed along the dorsal-ventral retinal axis. This is in contrast to the previously reported high ventral-low dorsal gradient of EphB receptors later in development when RGC axons map to CNS targets. We show that mice lacking both EphB2 and EphB3 receptor tyrosine kinases, but not each alone, exhibit increased frequency of RGC axon guidance errors to the optic disc. In these animals, major aspects of retinal development and cellular organization appear normal, as do the expression of other RGC guidance cues netrin, DCC, and L1. Unexpectedly, errors occur in dorsal but not ventral retina despite early uniform or later high ventral expression of EphB2 and EphB3. Furthermore, embryos lacking EphB3 and the kinase domain of EphB2 do not show increased errors, consistent with a guidance role for the EphB2 extracellular domain. Thus, while Eph kinase function is involved in RGC axon mapping in the brain, RGC axon pathfinding within the retina is partially mediated by EphB receptors acting in a kinase-independent manner.     

Label-Free Density Measurements of Radial Peripapillary Capillaries in the Human Retina

Radial peripapillary capillaries (RPCs) comprise a unique network of capillary beds within the retinal nerve fibre layer (RNFL) and play a critical role in satisfying the nutritional requirements of retinal ganglion cell (RGC) axons. Understanding the topographical and morphological characteristics of these networks through in vivo techniques may improve our understanding about the role of RPCs in RGC axonal health and disease. This study utilizes a novel, non-invasive and label-free optical imaging technique, speckle variance optical coherence tomography (svOCT), for quantitatively studying RPC networks in the human retina. Six different retinal eccentricities from 16 healthy eyes were imaged using svOCT. The same eccentricities were histologically imaged in 9 healthy donor eyes with a confocal scanning laser microscope. Donor eyes were subject to perfusion-based labeling techniques prior to retinal dissection, flat mounting and visualization with the microscope. Capillary density and diameter measurements from each eccentricity in svOCT and histological images were compared. Data from svOCT images were also analysed to determine if there was a correlation between RNFL thickness and RPC density. The results are as follows: (1) The morphological characteristics of RPC networks on svOCT images are comparable to histological images; (2) With the exception of the nasal peripapillary region, there were no significant differences in RPC density measurements between svOCT and histological images; (3) Capillary diameter measurements were significantly greater in svOCT images compared to histology; (4) There is a positive correlation between RPC density and RNFL thickness. The findings in this study suggest that svOCT is a reliable modality for analyzing RPC networks in the human retina. It may therefore be a valuable tool for aiding our understanding about vasculogenic mechanisms that are involved in RGC axonopathies. Further work is required to explore the reason for some of the quantitative differences between svOCT and histology.     

Regulation of retinal ganglion cell production by Sonic hedgehog

Previous work has shown that production of retinal ganglion cells is in part regulated by inhibitory factors secreted by ganglion cell themselves; however, the identities of these molecules are not known. Recent studies have demonstrated that the signaling molecule Sonic hedgehog (Shh) secreted by differentiated retinal ganglion cells is required to promote the progression of ganglion cell differentiation wave front and to induce its own expression. We present evidence that Shh signals play a role to negatively regulate ganglion cell genesis behind the differentiation wave front. Higher levels of Shh expression are detected behind the wave front as ganglion cells accumulate, while the Patched 1 receptor of Shh is expressed in adjacent retinal progenitor cells. Retroviral-mediated overexpression of Shh results in reduced ganglion cell proportions in vivo and in vitro. Conversely, inhibiting endogenous Shh activity by anti-Shh antibodies leads to an increased production of ganglion cells. Shh signals modulate ganglion cell production within the normal period of ganglion cell genesis in vitro without significantly affecting cell proliferation or cell death. Moreover, Shh signaling affects progenitor cell specification towards the ganglion cell fate during or soon after their last mitotic cycle. Thus, Shh derived from differentiated ganglion cells serves as a negative regulator behind the differentiation wave front to control ganglion cell genesis from the competent progenitor pool. Based on these results and other recent findings, we propose that Shh signals secreted by early-differentiated retinal neurons play dual roles at distinct concentration thresholds to orchestrate the progression of retinal neurogenic wave and the emergence of new neurons.     

Pax-6 expression during retinal regeneration in the adult newt

The present study examined the expression of Pax-6 during retinal regeneration in adult newts using in situ hybridization. In a normal retina, Pax-6 is expressed in the ciliary marginal zone, the inner part of the inner nuclear layer, and the ganglion cell layer. After surgical removal of the neural retina, retinal pigment epithelial cells proliferate into retinal precursor cells and regenerate a fully functional retina. At the beginning of retinal regeneration, Pax-6 was expressed in all retinal precursor cells. As regeneration proceeded, differentiating cells appeared at the scleral and vitreal margins of the regenerating retina, which had no distinct plexiform layers. In this stage, the expression of Pax-6 was localized in a strip of cells along the vitreal margin of the regenerating retina. In the late stage of regeneration, when the layer structure was completed, the expression pattern of Pax-6 became similar to that of a normal retina. It was found that Pax-6 is expressed in the retinal precursor cells in the early regenerating retina and that the expression pattern of Pax-6 changed as cell differentiation proceeded during retinal regeneration.