In mice, all of the six retinal neuron types are generated from common multipotent retinal progenitors, and their differentiation from progenitors is regulated by both extrinsic and intrinsic factors. Previously, we showed that targeted deletion of the atonal (ato) homologue math5 blocked the differentiation of most retinal ganglion cells (RGCs), revealing an essential role for math5 in RGC differentiation. In this study, we used the Cre-loxP recombination system to trace the fate of math5-expressing cells in retina. Our results demonstrated that math5 expression was associated with the differentiation of multiple retinal neuron types, including RGCs, photoreceptor, horizontal, and amacrine cells, implying that math5 expression alone is not sufficient to determine the RGC fate. Math5 expression was restricted to postmitotic cells in developing retina, suggesting that cell fate commitment of retinal neurons occurs after the terminal mitosis. The insufficiency of and requirement for math5 in RGC differentiation indicates that, like ato in the development of Drosophila R8 photoreceptors, math5 plays a role in determining the RGC competence state of retinal progenitors and that additional positive and negative factors are required in determining RGC fate. Furthermore, we show that loss of Math5 function severely reduced the RGC expression of the transcription factors Brn-3b, Gfi-1, Isl-1, Isl-2, Nscl-1, Nscl-2, and RPF-1, suggesting that Math5 expression is required to activate a comprehensive transcription network of RGC differentiation.

*LIM-homeodomain (HD) and POU-HD transcription factors play crucial roles in neurogenesis. However, it remains largely unknown how they cooperate in this process and what downstream target genes they regulate. Here, we show that ISL1, a LIM-HD protein, is co-expressed with BRN3B, a POU-HD factor, in nascent post-mitotic retinal ganglion cells (RGCs). Similar to the Brn3b-null retinas, retina-specific deletion of Isl1 results in the apoptosis of a majority of RGCs and in RGC axon guidance defects. The Isl1 and Brn3b double null mice display more severe retinal abnormalities with a near complete loss of RGCs, indicating the synergistic functions of these two factors. Furthermore, we show that both Isl1 and Brn3b function downstream of Math5 to regulate the expression of a common set of RGC-specific genes. Whole-retina chromatin immunoprecipitation and in vitro transactivation assays reveal that ISL1 and BRN3B concurrently bind to and synergistically regulate the expression of a common set of RGC-specific genes. Thus, our results uncover a novel regulatory mechanism of BRN3B and ISL1 in RGC differentiation.

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Several basic helix-loop-helix (bHLH) genes have been shown to be essential for the generation of the auditory sensory hair cells or the spiral ganglion (SG) neurons that innervate the hair cells in the cochlea, as well as a variety of cell types in the other nervous systems. However, it remains elusive what cellular context-dependent mechanisms confer the inner ear-specific neuronal or sensory competency/identities. We explored the possibility that one of the mechanisms responsible for generating cellular diversity in the nervous system through cooperative action of bHLH and LIM-homeodomain (LIM-HD) transcriptional factors might also contribute to the inner ear-specific sensory and/or neuronal competency. Here, we show that Islet1 (Isl1), a LIM-HD protein, is expressed early in the otocyst in the region that gives rise to both the auditory sensory organ, the organ of Corti, and SG neurons. Subsequently, the expression of Isl1 is maintained in SG neurons but is transitory in the sensory lineage. At embryonic day 12 (E12) in mice, the expression of Isl1 marks distinctively the ventral portion of the nascent cochlear epithelium encompassing the primordial organ of Corti. At E13, Isl1 is maintained at relatively high levels in the sensory primordium while down-regulated in the other regions of the cochlear duct. As the sensory epithelium starts to differentiate, it is down-regulated in the entire cochlear epithelium. The expression of Isl1 in the developing inner ear reveals an early and likely a common step in the development of both sensory and neuronal lineages of the inner ear, and suggests its potential role in the inner ear-specific sensory and neuronal cell development.