Msx2 alters the timing of retinal ganglion cells fate commitment and differentiation

Timing of cell fate commitment determines distinct retinal cell types, which is believed to be controlled by a tightly coordinated regulatory program of proliferation, cell cycle exit and differentiation. Although homeobox protein Msx2 could induce apoptosis of optic vesicle, it is unclear whether Msx2 regulates differentiation and cell fate commitment of retinal progenitor cells (RPCs) to retinal ganglion cells (RGCs). In this study, we show that overexpression of Msx2 transiently suppressed the expression of Cyclin D1 and blocked cell proliferation. Meanwhile, overexpression of Msx2 delayed the expression of RGC-specific differentiation markers (Math5 and Brn3b), which showed that Msx2 could affect the timing of RGCs fate commitment and differentiation by delaying the timing of cell cycle exit of retinal progenitors. These results indicate Msx2 possesses dual regulatory functions in controlling cell cycle progression of retinal RPCs and timing of RGCs differentiation.     

Modeling activity and target-dependent developmental cell death of mouse retinal ganglion cells ex vivo

Programmed cell death is widespread during the development of the central nervous system and serves multiple purposes including the establishment of neural connections. In the mouse retina a substantial reduction of retinal ganglion cells (RGCs) occurs during the first postnatal week, coinciding with the formation of retinotopic maps in the superior colliculus (SC). We previously established a retino-collicular culture preparation which recapitulates the progressive topographic ordering of RGC projections during early post-natal life. Here, we questioned whether this model could also be suitable to examine the mechanisms underlying developmental cell death of RGCs. Brn3a was used as a marker of the RGCs. A developmental decline in the number of Brn3a-immunolabelled neurons was found in the retinal explant with a timing that paralleled that observed in vivo. In contrast, the density of photoreceptors or of starburst amacrine cells increased, mimicking the evolution of these cell populations in vivo. Blockade of neural activity with tetrodotoxin increased the number of surviving Brn3a-labelled neurons in the retinal explant, as did the increase in target availability when one retinal explant was confronted with 2 or 4 collicular slices. Thus, this ex vivo model reproduces the developmental reduction of RGCs and recapitulates its regulation by neural activity and target availability. It therefore offers a simple way to analyze developmental cell death in this classic system. Using this model, we show that ephrin-A signaling does not participate to the regulation of the Brn3a population size in the retina, indicating that eprhin-A-mediated elimination of exuberant projections does not involve developmental cell death.     

Developmental wave of Brn3b expression leading to RGC fate specification is synergistically maintained by miR‐23a and miR‐374

Differential regulation of Brn3b is essential for the Retinal Ganglion Cell (RGC) development in the two phases of retinal histogenesis. This biphasic Brn3b regulation is required first, during early retinal histogenesis for RGC fate specification and secondly, during late histogenesis, where Brn3b is needed for RGC axon guidance and survival. Here, we have looked into how the regulation of Brn3b at these two stages happens. We identified two miRNAs, miR‐23a and miR‐374, as regulators of Brn3b expression, during the early stage of RGC development. Temporal expression pattern of miR‐23a during E10–19, PN1–7, and adult retina revealed an inverse relation with Brn3b expression. Though miR‐374 did not show such a pattern, its co‐expression with miR‐23a evidently inhibited Brn3b. We further substantiated these findings by ex vivo overexpression of these miRNAs in E14 mice retina and found that miR‐23a and miR‐374 together brings about a change in Brn3b expression pattern in ganglion cell layer (GCL) of the developing retina. From our results, it appears that the combined expression of these miRNAs could be regulating the timing of the wave of Brn3b expression required for early ganglion cell fate specification and later for its survival and maturation into RGCs. Taken together, here we provide convincing evidences for the existence of a co‐ordinated mechanism by miRNAs to down regulate Brn3b that will ultimately regulate the development of RGCs from their precursors. © 2014 Wiley Periodicals, Inc. Develop Neurobiol 74: 1155–1171, 2014     

bHLH genes cath5 and cNSCL1 promote bFGF-stimulated RPE cells to transdifferentiate toward retinal ganglion cells

The molecular mechanism of retinal ganglion cell (RGC) genesis and development is not well understood. Published data suggest that the process may involve two bHLH genes, ath5 and NSCL1. Gain-of-function studies show that ath5 increases RGC production in the developing retina. We examined whether two chick genes, cath5 and cNSCL1, can guide retinal pigment epithelial (RPE) cells to transdifferentiate toward RGCs. Ectopic expression of cath5 and cNSCL1 in cultured chick RPE cells was achieved through retroviral transduction. cath5 alone was unable to induce de novo expression of early RGC markers, such as RA4 antigen, neurofilament (160 kDa), and a neurofilament-associated antigen. However, cath5 induced the expression of these proteins when the RPE cells were cultured with medium supplemented with bFGF. Since bFGF alone can induce only RA4 antigen, the expression of the additional RGC markers reflects a synergism between cath5 and bFGF in promoting RPE transdifferentiation toward RGCs. Morphologically, the RA4(+) cells in bFGF + cath5 cultures appeared more neuron-like than those generated by bFGF alone. cNSCL1 also promoted bFGF-stimulated RPE cells to transdifferentiate toward RGCs that expressed RA4 antigen, N-CAM, Islet-1, neurofilament, and neurofilament-associated antigen. We found that cath5 induced cNSCL1 expression, but not vice versa. Our data suggest that cath5 or cNSCL1 alone was insufficient to induce RPE transdifferentiation into RGCs, but could further neural differentiation initiated by bFGF. We propose that intrinsic factors act synergistically with extrinsic factors during RGC genesis and development.     

Rescue of Retinal Function by BDNF in a Mouse Model of Glaucoma

Vision loss in glaucoma is caused by progressive dysfunction of retinal ganglion cells (RGCs) and optic nerve atrophy. Here, we investigated the effectiveness of BDNF treatment to preserve vision in a glaucoma experimental model. As an established experimental model, we used the DBA/2J mouse, which develops chronic intraocular pressure (IOP) elevation that mimics primary open-angle glaucoma (POAG). IOP was measured at different ages in DBA/2J mice. Visual function was monitored using the steady-state Pattern Electroretinogram (P-ERG) and visual cortical evoked potentials (VEP). RGC alterations were assessed using Brn3 immunolabeling, and confocal microscope analysis. Human recombinant BDNF was dissolved in physiological solution (0.9% NaCl); the effects of repeated intravitreal injections and topical eye BDNF applications were independently evaluated in DBA/2J mice with ocular hypertension. BDNF level was measured in retinal homogenate by ELISA and western blot. We found a progressive decline of P-ERG and VEP responses in DBA/2J mice between 4 and 7 months of age, in relationship with the development of ocular hypertension and the reduction of Brn3 immunopositive RGCs. Conversely, repeated intravitreal injections (BDNF concentration = 2 µg/µl, volume = 1 µl, for each injection; 1 injection every four days, three injections over two weeks) and topical eye application of BDNF eye-drops (12 µg/µl, 5 µl eye-drop every 48 h for two weeks) were able to rescue visual responses in 7 month DBA/2J mice. In particular, BDNF topical eye treatment recovered P-ERG and VEP impairment increasing the number of Brn3 immunopositive RGCs. We showed that BDNF effects were independent of IOP reduction. Thus, topical eye treatment with BDNF represents a promisingly safe and feasible strategy to preserve visual function and diminish RGC vulnerability to ocular hypertension.     

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.     

Comparative Evaluation of Methods for Estimating Retinal Ganglion Cell Loss in Retinal Sections and Wholemounts

To investigate the reliability of different methods of quantifying retinal ganglion cells (RGCs) in rat retinal sections and wholemounts from eyes with either intact optic nerves or those axotomised after optic nerve crush (ONC). Adult rats received a unilateral ONC and after 21 days the numbers of Brn3a⁺, βIII-tubulin⁺ and Islet-1⁺ RGCs were quantified in either retinal radial sections or wholemounts in which FluoroGold (FG) was injected 48 h before harvesting. Phenotypic antibody markers were used to distinguish RGCs from astrocytes, macrophages/microglia and amacrine cells. In wholemounted retinae, counts of FG⁺ and Brn3a⁺ RGCs were of similar magnitude in eyes with intact optic nerves and were similarly reduced after ONC. Larger differences in RGC number were detected between intact and ONC groups when images were taken closer to the optic nerve head. In radial sections, Brn3a did not stain astrocytes, macrophages/microglia or amacrine cells, whereas βIII-tubulin and Islet-1 did localize to amacrine cells as well as RGCs. The numbers of βIII-tubulin⁺ RGCs was greater than Brn3a⁺ RGCs, both in retinae from eyes with intact optic nerves and eyes 21 days after ONC. Islet-1 staining also overestimated the number of RGCs compared to Brn3a, but only after ONC. Estimates of RGC loss were similar in Brn3a-stained radial retinal sections compared to both Brn3a-stained wholemounts and retinal wholemounts in which RGCs were backfilled with FG, with sections having the added advantage of reducing experimental animal usage.     

Retinal ganglion cell type, size, and spacing can be specified independent of homotypic dendritic contacts

In Brn3b(-/-) mice, where 80% of retinal ganglion cells degenerate early in development, the remaining 20% include most or all ganglion cell types. Cells of the same type cover the retinal surface evenly but tile it incompletely, indicating that a regular mosaic and normal dendritic field size can be maintained in the absence of contact among homotypic cells. In Math5(-/-) mice, where only approximately 5% of ganglion cells are formed, the dendritic arbors of at least two types among the residual ganglion cells are indistinguishable from normal in shape and size, even though throughout development they are separated by millimeters from the nearest neighboring ganglion cell of the same type. It appears that the primary phenotype of retinal ganglion cells can develop without homotypic contact; dendritic repulsion may be an end-stage mechanism that fine-tunes the dendritic arbors for more efficient coverage of the retinal surface.     

Rewiring the retinal ganglion cell gene regulatory network: Neurod1 promotes retinal ganglion cell fate in the absence of Math5

Retinal progenitor cells (RPCs) express basic helix-loop-helix (bHLH) factors in a strikingly mosaic spatiotemporal pattern, which is thought to contribute to the establishment of individual retinal cell identity. Here, we ask whether this tightly regulated pattern is essential for the orderly differentiation of the early retinal cell types and whether different bHLH genes have distinct functions that are adapted for each RPC. To address these issues, we replaced one bHLH gene with another. Math5 is a bHLH gene that is essential for establishing retinal ganglion cell (RGC) fate. We analyzed the retinas of mice in which Math5 was replaced with Neurod1 or Math3, bHLH genes that are expressed in another RPC and are required to establish amacrine cell fate. In the absence of Math5, Math5Neurod1-KI was able to specify RGCs, activate RGC genes and restore the optic nerve, although not as effectively as Math5. By contrast, Math5Math3-KI was much less effective than Math5Neurod1-KI in replacing Math5. In addition, expression of Neurod1 and Math3 from the Math5Neurod1-KI/Math3-KI allele did not result in enhanced amacrine cell production. These results were unexpected because they indicated that bHLH genes, which are currently thought to have evolved highly specialized functions, are nonetheless able to adjust their functions by interpreting the local positional information that is programmed into the RPC lineages. We conclude that, although Neurod1 and Math3 have evolved specialized functions for establishing amacrine cell fate, they are nevertheless capable of alternative functions when expressed in foreign environments.