Enhanced retinal ganglion cell differentiation by ath5 and NSCL1 coexpression.

Many aspects of the molecular regulation of retinal ganglion cell (RGC) differentiation remain unclear. Published studies have shown that genes encoding transcription factors of the homeodomain family1–4 and the basic helix-loop-helix (bHLH) family, including Ath5 and NSCL1, play a role in regulating gene expression during RGC development in the vertebrate retina. Ath5 is expressed in immature RGCs, and their expression becomes undetectable in mature RGCs5,6 (Ma et al., manuscript submitted). Chicken NSCL1, like its mammalian counterpart,7,8 is specifically expressed in the nervous system.9 In the developing retina, NSCL1 is first expressed in differentiating ganglion cells during early development and then in Muller glia during late development.10 Retroviral expression of NSCL1 in the developing chick retina causes premature cessation of mitotic activity, massive cell death, and microphthalmia.10 The function of ath5 in RGC development has been the subject of extensive investigation. Xenopus ath5 mRNA injected into Xenopus embryos at the two-cell stage promotes retinal progenitor cell differentiation into RGCs.11 In the embryonic chick retina, retroviral expression of chick and mouse ath5 increases the number of RGCs.6 Targeted deletion of ath5 in mouse results in a lack, or a dramatic reduction, of RGCs, indicating that ath5 is indispensable for RGC development.12,13 Kay et al.14 have reported a lack of RGCs in the zebrafish ath5 mutant, lackritz. Nonetheless, the role that ath5 plays during vertebrate retinal development is likely to be multifaceted. Brown et al.5 reported that lipofection of mouse ath5 into the frog retina resulted in an increase in bipolar cells. Through lineage tracing, Yang et al.15 found that photoreceptors and amacrine cells, in addition to RGCs, may express ath5 at certain times in their lives. Our recent RPE transdifferentiation assays indicate that chick ath5 (also referred to as cath5) can lead to the photoreceptor pathway (Ma et al., manuscript submitted). We have previously investigated the roles of ath5 and NSCL1 in RGC genesis using the RPE transdifferentiation assay we developed16–20 (Ma et al., manuscript submitted). The assay takes advantage of the simplicity and plasticity of RPE cells to allow us to examine the proneural activities of various genes or factors. The neural retina not only naturally expresses several proneural genes, but also contains different types of cells, creating molecularly and biochemically heterogeneous cellular contexts and intercellular environments. The non-neural RPE, in contrast, is a single-layered structure that lacks the expression of many proneural genes. Furthermore, the RPE is developmentally related to the retina. RPE tissue at early developmental stages may be triggered to transdifferentiate into a neural retina,21–24 and cultured RPE cells can be induced to undergo transdifferentiation into what closely resembles retinal neurons.16–19 An RPE cell transdifferentiation assay showed that ath5 and NSCL1 promote bFGF-initiated RPE transdifferentiation toward RGCs, but alone they are insufficient to induce this change; nonetheless, the extent of transdifferentiation is rather limited.20 In this study, we examined whether ath5 and NSCL1 act synergistically in advancing RGC differentiation. Ath5 and NSCL1 were coexpressed through an internal ribosomal entry site (IRES) and a replication-competent retrovirus. We found that retroviral coexpression of ath5 and NSCL1 in the developing chick retina increased the number of cells that expressed RGC markers more than the sum of the increases caused when the two genes were express separately. In RPE cell cultures, cells morphologically resembling RGCs were readily observed in dishes infected by retrovirus coexpressing ath5 and NSCL1. However, in the absence of bFGF, ath5 and NSCL1 coexpression did not alleviate the insufficiency in inducing RPE transdifferentiation toward RGCs.