Identifying the transcriptional core logic that determines photoreceptor cell specification

 描述（由申请人提供）：细胞命运规范由激活细胞类型特异性基因表达模式的转录调节因子施加。许多这些主调节器是保守的从无脊椎动物到脊椎动物，使果蝇的眼睛一个很好的模型系统，在其中解决这一过程。该建议将调查果蝇锌指转录因子玻璃（Gl），这似乎是一个关键的决定因素，在控制从神经元前体感光细胞类型的发育开关。在gl突变体中，眼祖细胞启动神经元分化，如通过神经元标记物的存在所检测到的，但是不能表达光感受器特异性基因，异常地将轴突延伸到脑中，并且在达到成年之前死亡。我们假设，表征Gl在感光细胞分化中的作用将揭示眼睛发育的新分子特征。这些发现将对开发用于视网膜退行性疾病患者的基于干细胞的疗法具有转化意义。第一个目的是鉴定需要GI功能的感光器发育的特定阶段。过早的细胞死亡已经阻止了对这种需要的仔细分析，并且目前尚不知道在2000年是否需要Gl。 后期用于光感受器的分化和维持，或者如果其唯一的作用是作为激活光感受器转录网络的早期决定子AC。为了解决这个问题，我们将在晚期阶段抑制gl表达，并检查在gl突变背景下不能诱导细胞死亡的细胞的命运。第二个目标将确定GI是否足够 诱导果蝇或哺乳动物细胞中感光细胞的发育。使用遗传工具，我们将在苍蝇组织中错误表达Gl，并通过表型分析、免疫组织化学和基因表达谱确定它是否可以诱导光感受器特异性基因或性状。研究表明，Gl与脊椎动物视紫红质启动子结合，使其成为诱导哺乳动物细胞基因表达变化的潜在有力工具。Gl与未表征的人锌指蛋白ZNF 500共享同源性，所述锌指蛋白ZNF 500也在视网膜中高度表达。我们建议在小鼠胚胎干细胞中错误表达G1或ZNF 500以及神经元诱导剂，以确定其是否可以激活感光器特异性基因的表达。在第三个目标中，我们将使用RNAi策略来研究每个鉴定的Gl激活的直接靶标在光感受器发育中的作用。由于这些基因中的许多仍然是未知的，我们希望确定新的因素，有助于感光细胞的招聘，分化，生存或轴突靶向。

 DESCRIPTION (provided by applicant): Cell fate specification is imposed by transcriptional regulators that activate cell type specific gene expression patterns. Many of these master regulators are conserved from invertebrates to vertebrates, making the Drosophila eye an excellent model system in which to address this process. This proposal will investigate the Drosophila zinc finger transcription factor Glass (Gl), which appears to act as a key determinant in controlling the developmental switch from neuronal precursors to photoreceptor cell types. In gl mutants, eye progenitor cells initiate neuronal differentiation as detected by the presence of neuronal markers, but fail to express photoreceptor-specific genes, extend axons aberrantly into the brain, and die before reaching adulthood. We hypothesize that characterizing the role of Gl in photoreceptor differentiation will reveal novel molecular features underlying eye development. These findings will have translational implications in developing stem cell-based therapies for patients suffering from retinal degenerative diseases. The first aim is to identify the specific stages of photoreceptor development that require Gl function. Premature cell death has prevented a careful analysis of this requirement and it is presently not known if Gl is required at later stages for the differentiation and maintenance of photoreceptors or if its only role is to ac as an early determinant that activates the photoreceptor transcriptional network. To address this question we will inactivate gl expression at late stages, and examine the fate of cells incapable of inducing cell death in a gl mutant background. The second aim will determine if Gl is sufficient to induce photoreceptor development in Drosophila or mammalian cells. Using genetic tools we will misexpress Gl in fly tissues and determine by phenotypic analysis, immunohistochemistry and gene expression profiling if it can induce photoreceptor-specific genes or traits. Gl was shown to bind to vertebrate rhodopsin promoters making it a potentially powerful tool with which to induce gene expression changes in mammalian cells. Gl shares homology with an uncharacterized human zinc finger protein ZNF500, which is also highly expressed in the retina. We propose to misexpress Gl or ZNF500 together with neuronal inducers in mouse embryonic stem cells to determine whether it can activate the expression of photoreceptor-specific genes. In the third aim we will use an RNAi strategy to investigate the role of each of the identified direct targets of Gl activation in photoreceptor development. As many of these genes remain uncharacterized, we expect to identify novel factors that contribute to photoreceptor recruitment, differentiation, survival or axon targeting.