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为治疗目的再生受损细胞的能力。基于果蝇玻璃突变体的表型，玻璃转录因子被确定为光感受器细胞命运的关键决定因素。在这些突变体中，眼原基细胞分化为神经元，但它们不能表达光受体特异性基因，产生异常的轴突投射，并在成体阶段之前死亡。对谷歌眼镜及其靶基因的进一步分析，使用自首次发现以来多年来发展起来的方法和概念，有可能揭示光感受器命运决定是如何实现的。本提案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渐募集。为了研究光感受器分化的后期事件，将使用启动caspase突变来防止玻璃突变体中的细胞死亡，或者在分化开始使用RNA干扰后玻璃将被耗尽。对玻璃调节的研究将验证一种假设，即它将Pax6和其他视网膜决定转录因子的输入与提供光感受器分化时空控制的信号通路的输入整合在一起。第二个目标将测试Glass错误表达的影响，以确定它是否足以驱动感光器发育的任何方面。两种不同的Glass异构体转化神经元或非神经元细胞的能力将通过表型和基因表达分析进行评估。一个合作项目也将启动，以测试谷歌眼镜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可能被Glass直接激活的基因介导其对光感受器募集、分化、存活和轴突靶向的影响。除了消耗单个目标基因，相关的

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.