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

项目摘要 虽然视锥感光细胞退化是感光细胞营养不良失明的最终原因， 小鼠和人类的视网膜是杆状感光感受器占优势的，这一事实阻碍了对这一认识的进展 如何指定圆锥体。对这个问题的新见解可能来自于对分子的研究 具有自然视锥优势视网膜的哺乳动物物种中光感受器发育的机制。一 这些物种是13线地松鼠(13-lgs)Ictidomys tridecemlineatus，是当地特有的 美国中西部。13-LGS的基因组已完全测序，其发育阶段也很好- 具有老鼠的特征，很容易与老鼠相提并论。我们建议全面分析基因表达和 应用单细胞RNA和ATAC-Seq研究13-LGS视网膜神经发生过程中染色质的可及性。我们会 然后使用计算方法识别预测控制锥体的基因调控网络 13-LGS中光感受器的发育，并与小鼠和人的结果进行比较 视网膜以确定基因表达和调控的物种特定差异，这些差异是 杆：锥比。我们还将测试NRL基因的13-LGS同源基因是否是 光感受器规格，显示促进视杆细胞发育和抑制视锥细胞发育的能力降低 它的鼠标对应物。我们预计这些研究将识别出多个以前未识别的基因 锥体发育的强有力的候选正负调节器，并可在未来进行功能测试 学习。最终，这可能有助于改进视锥细胞光感受器与 用于治疗性移植的干细胞和祖细胞。

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.