Identifying gene regulatory networks controlling photoreceptor specification by transcriptomic and epigenomic analysis of retinal development in cone-dominant retina

项目摘要 虽然锥状光感受器的退化是光感受器营养不良中失明的最终原因， 老鼠和人类的视网膜都是视杆细胞感光器占主导地位，这一事实阻碍了对视网膜的理解 如何指定圆锥体。对这一问题的新见解可能来自于研究分子 具有天然视锥细胞优势视网膜的哺乳动物物种的感光细胞发育机制。一 这种物种是13线地松鼠（13-LGS）Ictidomys tridecemlineatus，这是特有的 美国中西部。13-LGS的基因组被完全测序，它们的发育阶段很好- 表征并容易与小鼠比较。我们建议全面分析基因表达， 使用单细胞RNA和ATAC-Seq在13-LGS中视网膜神经发生期间的染色质可及性我们将 然后使用计算方法来识别预测控制锥状细胞的基因调控网络， 光感受器的发展，并比较这些结果从小鼠和人类 视网膜，以确定在基因表达和调节的物种特异性差异，这些差异是在视网膜中的差异的基础。 杆锥比我们还将测试Nrl基因的13-LGS直向同源物，Nrl基因的主要调节因子，是否与Nrl基因的13-LGS同源。 光感受器规格，显示相对于 老鼠的对应物。我们希望这些研究将确定多个以前未被识别的基因， 强候选人的积极和消极的调节锥发展，并可以在未来的功能测试 问题研究最终，这可能有助于改进用于定向区分视锥光感受器和视神经细胞的方案。 用于治疗性移植干细胞和祖细胞。

Project Summary While degeneration of cone photoreceptors is the ultimate cause of blindness in photoreceptor dystrophies, mouse and human retinas are rod photoreceptor-dominant, a fact that has hindered progress in understanding how cones are specified. New insight into this problem can potentially come from studying the molecular mechanisms of photoreceptor development in mammalian species with naturally cone-dominant retinas. One such species is the 13-lined ground squirrel (13-LGS) Ictidomys tridecemlineatus, which is endemic to the American Midwest. The genome of 13-LGS is fully sequenced, and their developmental staging is well- characterized and readily comparable to mouse. We propose to comprehensively profile gene expression and chromatin accessibility during retinal neurogenesis in 13-LGS using single-cell RNA- and ATAC-Seq. We will then use computational approaches to identify gene regulatory networks predicted to control cone photoreceptor development in 13-LGS, and compare these with results obtained from mouse and human retinas to identify species-specific differences in gene expression and regulation that underlie differences in the rod:cone ratio. We will also test whether the 13-LGS orthologue of the Nrl gene, a master regulator of photoreceptor specification, shows reduced ability to promote rod, and repress cone, development relative to its mouse counterpart. We expect these studies will identify multiple previously unidentified genes that are strong candidate positive and negative regulators of cone development, and can be functionally tested in future studies. Ultimately, this may help improve protocols for directed differentiation of cone photoreceptors from stem and progenitor cells for use in therapeutic transplantation.