Photoreceptor cell fate specification by the Glass transcription factor

描述（由申请人提供）：了解在正常发育中逐渐指定细胞命运的转录网络将提高我们再生受损细胞用于治疗目的的能力。基于果蝇中glass突变体的表型，Glass转录因子被鉴定为感光细胞命运的关键决定因素。在这些突变体中，眼原基中的细胞分化为神经元，但它们不能表达感光器特异性基因，产生异常的轴突投射，并在成年阶段之前死亡。使用自首次发现以来多年来开发的方法和概念对玻璃及其靶基因进行进一步分析，有可能揭示感光细胞命运决定是如何实施的。本文的第一个目的是探讨玻璃的功能和调节。将分析玻璃突变体表型，以确定玻璃如何影响发育早期不同感光细胞类型的逐步招募。为了研究光感受器分化中的后期事件，将使用引发剂半胱天冬酶突变来防止玻璃突变体中的细胞死亡，或者在分化开始后使用RNA干扰来耗尽玻璃。玻璃调节的调查将测试的假设，它整合从Pax6和其他视网膜决定转录因子的输入与输入信号通路，提供感光细胞分化的时间和空间控制。第二个目标将测试玻璃错误表达的影响，以确定它是否足以驱动感光细胞发育的任何方面。将通过表型和基因表达分析评价两种不同的Glass同种型将神经元或非神经元细胞转化为光感受器身份的能力。还将启动一个合作项目，以测试玻璃及其哺乳动物同源物将小鼠胚胎干细胞从神经元转化为感光细胞的能力。在第三个目标中，将研究单个Glass靶基因的作用。转基因RNA干扰将用于确定哪些基因可能被Glass直接激活，介导其对光感受器募集、分化、存活和轴突靶向的影响。除了消耗单个靶基因外， 可能起冗余作用的基因，包括Scratch家族的两个同源转录因子，将被同时去除。总之，这些实验将提高我们的理解光感受器身份的转录调控，并可能为再生医学提供有用的工具。

DESCRIPTION (provided by applicant): Understanding the transcriptional networks that progressively specify cell fates in normal development will improve our ability to regenerate damaged cells for therapeutic purposes. The Glass transcription factor was identified as a critical determinant of photoreceptor cell fate based on the phenotype of glass mutants in Drosophila. In these mutants, cells in the eye primordium differentiate as neurons, but they fail to express photoreceptor-specific genes, develop abnormal axonal projections, and die before the adult stage. Further analysis of Glass and its target genes, using methods and concepts that have been developed in the years since its initial discovery, has the potential to reveal how the photoreceptor fate decision is implemented. The first aim of this proposal will probe the function and regulation of Glass. The glass mutant phenotype will be analyzed to determine how Glass affects the progressive recruitment of distinct photoreceptor cell types early in development. To study later events in photoreceptor differentiation, an initiator caspase mutation will be used to prevent cell death in glass mutants, or glass will be depleted after differentiation has begun using RNA interference. Investigation of glass regulation will test the hypothesis that it integrates input from Pax6 and other retinal determination transcription factors with input from signaling pathways that provide temporal and spatial control of photoreceptor differentiation. The second aim will test the effects of Glass misexpression to determine whether it is sufficient to drive any aspects of photoreceptor development. The ability of two different isoforms of Glass to transform neuronal or non-neuronal cells towards a photoreceptor identity will be evaluated by phenotypic and gene expression analysis. A collaborative project to test the ability of Glass and its mammalian homologues to transform mouse embryonic stem cells from a neuronal to a photoreceptor identity will also be initiated. In the third aim, the role of individual Glass targe genes will be investigated. Transgenic RNA interference will be used to determine which of the genes likely to be directly activated by Glass mediate its effects on photoreceptor recruitment, differentiation, survival and axon targeting. In addition to depleting single target genes, related genes that might act redundantly, including two homologous transcription factors of the Scratch family will be removed simultaneously. Together, these experiments will improve our understanding of the transcriptional regulation of photoreceptor identity, and may provide useful tools for regenerative medicine.