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.     

chokh/rx3 specifies the retinal pigment epithelium fate independently of eye morphogenesis

Despite the importance of the retinal pigment epithelium (RPE) for vision, the molecular processes involved in its specification are poorly understood. We identified two new mutant alleles for the zebrafish gene chokh (chk), which display a reduction or absence of the RPE. Unexpectedly, the neural retina (NR) in chk is specified and laminated, indicating that the regulatory network leading to NR development is largely independent of the RPE. Genetic mapping and molecular characterization revealed that chk encodes Rx3. Expression analyses show that otx2 and mitfb are not expressed in the prospective RPE of chk, indicating that the retinal homeobox gene rx3 acts upstream of the molecular network controlling RPE specification. Cellular transplantations demonstrate that rx3 function is autonomously required to specify the prospective RPE. Though rx2 is also absent in chk, neither rx2 nor rx1 is required for RPE development. Thus, our data provide the first indication that, in addition to controlling optic lobe evagination and proliferation, chk/rx3 also determines cellular fate.     

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.     

Differential enhancement of neural and photoreceptor cell differentiation of cultured pineal cells by FGF-1, IGF-1, and EGF

There are several common features between the pineal organ and the lateral eye in their developmental and evolutionary aspects. The avian pineal is a photoendocrine organ that originates from the diencephalon roof and represents a transitional type between the photosensory organ of lower vertebrates and the endocrine gland of mammals. Previous cell culture studies have shown that embryonic avian pineal cells retain a wide spectrum of differentiative capacities, although little is known about the mechanisms involved in their fate determination. In the present study, we investigated the effects of various cell growth factors on the differentiation of photoreceptor and neural cell types using pineal cell cultures from quail embryos. The results show that IGF-1 promotes differentiation of rhodopsin-immunoreactive cells, but had no effect on neural cell differentiation. Simultaneous administration of EGF and IGF-1 further enhanced differentiation of rhodopsin-immunoreactive cells, although the mechanism of the synergistic effect is unknown. FGF-1 did not stimulate proliferation of neural progenitor cells, but intensively promoted and maintained expression of a neural cell phenotype. FGF-1 appeared to lead to the conversion from an epithelial (endocrinal) to a neuronal type. It also enhanced phenotypic expression of retinal ganglion cell markers but rather suppressed expression of an amacrine cell marker. These results indicate that growth factors are important regulatory cues for pineal cell differentiation and suggest that they play roles in determining the fate of the pineal organ and the eye. It can be speculated that the differences in environmental cues between the retina and pineal may result in the transition of the pineal primordium from a potentially ocular (retinal) organ to a photoendocrine organ.     

Sequential actions of Pax3 and Pax7 drive xanthophore development in zebrafish neural crest

The Pax3/7 gene family has a fundamental and conserved role during neural crest formation. In people, PAX3 mutation causes Waardenburg syndrome, and murine Pax3 is essential for pigment formation. However, it is unclear exactly how Pax3 functions within the neural crest. Here we show that pax3 is expressed before other pax3/7 members, including duplicated pax3b, pax7 and pax7b genes, early in zebrafish neural crest development. Knockdown of Pax3 protein by antisense morpholino oligonucleotides results in defective fate specification of xanthophores, with complete ablation in the trunk. Other pigment lineages are specified and differentiate. As a consequence of xanthophore loss, expression of pax7, a marker of the xanthophore lineage, is reduced in neural crest. Morpholino knockdown of Pax7 protein shows that Pax7 itself is dispensable for xanthophore fate specification, although yellow pigmentation is reduced. Loss of xanthophores after reduction of Pax3 correlates with a delay in melanoblast differentiation followed by significant increase in melanophores, suggestive of a Pax3-driven fate switch within a chromatophore precursor or stem cell. Analysis of other neural crest derivatives reveals that, in the absence of Pax3, the enteric nervous system is ablated from its inception. Therefore, Pax3 in zebrafish is required for specification of two specific lineages of neural crest, xanthophores and enteric neurons.     

XNGNR1-dependent neurogenesis mediates early neural cell death

Early neural cell death is programmed cell death occurring within proliferating and undifferentiated neural progenitors. Little is known about the regulation and role of early neural cell death. In Xenopus embryos, primary neurogenesis is disrupted following the inhibition of early neural cell death, indicating that it is required for normal primary neurogenesis. Here we show that early neural cell death is dependent on primary neurogenesis. Overexpression of XSoxD concomitantly reduced N-Tubulin expression and early neural cell death, as seen by reduced TUNEL staining in stage 15 embryos. Conversely, overexpression of XNgnr1 led to ectopic N-Tubulin expression and TUNEL staining. However, XNeuroD overexpression, which induces ectopic N-Tubulin expression downstream of XNgnr1, had no effect on early neural cell death. E1A12S differentially inhibits the differentiation pathway induced by XNGNR1 protein. E1A12S-mediated inhibition of XNGNR1 neurogenic activity resulted in the reduction of N-Tubulin expression and TUNEL staining. Taken together, our data establish that primary neurogenesis induced by XNGNR1 promotes early neural cell death. This indicates that XNgnr1 positively regulates early neural cell death. We propose that early neural cell death might eliminate cells with abnormally high levels of XNGNR1, which can result in pre-mature neuronal differentiation.     

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.     

Regulation of R7 and R8 differentiation by the spalt genes

Photoreceptor development begins in the larval eye imaginal disc, where eight distinct photoreceptor cells (R1-R8) are sequentially recruited into each of the developing ommatidial clusters. Final photoreceptor differentiation, including rhabdomere formation and rhodopsin expression, is completed during pupal life. During pupation, spalt was previously proposed to promote R7 and R8 terminal differentiation. Here we show that spalt is required for proper R7 differentiation during the third instar larval stage since the expression of several R7 larval markers (prospero, enhancer of split mdelta0.5, and runt) is lost in spalt mutant clones. In R8, spalt is not required for cell specification or differentiation in the larval disc but promotes terminal differentiation during pupation. We show that spalt is necessary for senseless expression in R8 and sufficient to induce ectopic senseless in R1-R6 during pupation. Moreover, misexpression of spalt or senseless is sufficient to induce ectopic rhodopsin 6 expression and partial suppression of rhodopsin 1. We demonstrate that spalt and senseless are part of a genetic network, which regulates rhodopsin 6 and rhodopsin 1. Taken together, our results suggest that while spalt is required for R7 differentiation during larval stages, spalt and senseless promote terminal R8 differentiation during pupal stages, including the regulation of rhodopsin expression.     

Near complete loss of retinal ganglion cells in the math5/brn3b double knockout elicits severe reductions of other cell types during retinal development

Retinal ganglion cells (RGCs) are the first cell type to differentiate during retinal histogenesis. It has been postulated that specified RGCs subsequently influence the number and fate of the remaining progenitors to produce the rest of the retinal cell types. However, several genetic knockout models have argued against this developmental role for RGCs. Although it is known that RGCs secrete cellular factors implicated in cell proliferation, survival, and differentiation, until now, limited publications have shown that reductions in the RGC number cause significant changes in these processes. In this study, we observed that Math5 and Brn3b double null mice exhibited over a 99% reduction in the number of RGCs during development. This severe reduction of RGCs is accompanied by a drastic loss in the number of all other retinal cell types that was never seen before. Unlike Brn3b null or Math5 null animals, mice null for both alleles lack an optic nerve and have severe retinal dysfunction. Results of this study support the hypothesis that RGCs play a pivotal role in the late phase of mammalian retina development.