Onecut1 and Otx2 directly regulate multiple genes expressed early in cone and horizontal cell development [scRNA-seq]

The development of the retina into a highly organized structure occurs via the production of over 100 cell types from a pool of retinal progenitor cells (RPCs). While some RPCs are capable of making many types of retinal cell types, some terminally dividing RPCs are restricted to the production of specific types of daughter cells. Notably, although such restrictions can be limited to the production of a very specific single cell type, in other cases, production of two very different cell types, such as a photoreceptor and an interneuron, occurs. How specific types of RPCs make specific types of neuron is not fully understood. Combining RNA-seq and ATAC-seq, we compared the transcriptome and chromatin profiles between one type of biased RPC and other types of RPCs. The biased RPC that was studied is one that we previously defined by its expression of a cis-regulatory module (CRM) for the thyroid hormone receptor beta gene (Thrb), the earliest known marker of cone photoreceptors. In this earlier study, we found that Otx2 and Onecut1 drove expression of this Thrb CRM, as well as regulated expression of the Thrb gene. Here we explored the roles of these Otx2 and Oc1 further. We discovered that their roles extend well beyond regulating Thrb. They collaborate to regulate multiple genes important in cone development and/or function, including Rxrg, a known partner of Thrb. We also identified several new enhancers of genes active in developing cones. These data expand our understanding of the gene regulatory network (GRN) for cone development and contribute new CRMs for vectors that are expressed only in cones, as well as enable the genesis of cones from stem cells, for their use in retinal disease cell and gene therapies. RNA-seq for ThrbPos and ThrbNeg cells in chick

视网膜发育为高度有序结构的过程，源自视网膜祖细胞（retinal progenitor cells, RPCs）池的分化，最终产生超过100种细胞类型。部分视网膜祖细胞可生成多种视网膜细胞类型，而部分终末分裂的视网膜祖细胞则仅能定向产生特定类型的子代细胞。值得注意的是，这类定向分化限制有时可局限于单一特异性细胞类型的生成，而在其他情况下，则可产生两种截然不同的细胞类型，例如视锥感光细胞与中间神经元。目前，特异性视网膜祖细胞如何产生特异性神经元的分子机制尚未完全阐明。
本研究结合RNA测序（RNA-seq）与转座酶可及性测序（ATAC-seq），对一类偏分化倾向的视网膜祖细胞与其他类型视网膜祖细胞的转录组及染色质图谱进行了对比分析。本次研究的偏分化祖细胞，是我们此前通过甲状腺激素受体β基因（thyroid hormone receptor beta gene, Thrb）的顺式调控模块（cis-regulatory module, CRM）的表达所定义的，该模块是目前已知最早的视锥感光细胞标记物。在先前的研究中，我们发现Otx2与Onecut1可调控该Thrb顺式调控模块的活性，同时也可直接调控Thrb基因的表达。
本研究进一步探究了Otx2与Onecut1（亦可简称为Oc1）的调控功能，发现其作用远不止于调控Thrb基因。二者协同调控了多个对视锥细胞发育和/或功能至关重要的基因，包括已知的Thrb互作搭档Rxrg。此外，我们还鉴定出多个在发育中的视锥细胞中活跃的基因的新型增强子。
上述数据拓展了我们对视锥细胞发育相关基因调控网络（gene regulatory network, GRN）的认知，同时为仅在视锥细胞中特异性表达的载体提供了全新的顺式调控模块，也为通过干细胞定向诱导生成视锥细胞以应用于视网膜疾病的细胞与基因治疗提供了新的可能。本数据集包含鸡胚中Thrb阳性（ThrbPos）与Thrb阴性（ThrbNeg）细胞的RNA测序数据。