A cis-regulatory module underlies retinal ganglion cell genesis and axonogenesis [Set3.Bulk RNA-Seq retina E14.5 WT and Atoh7 Remote Enhancer KO]. A cis-regulatory module underlies retinal ganglion cell genesis and axonogenesis [Set3.Bulk RNA-Seq retina E14.5 WT and Atoh7 Remote Enhancer KO]

During retinogenesis, the proneural bHLH transcription factor Atoh7 is transiently expressed in retinal progenitor cells (RPCs) and is required for retinal ganglion cell (RGC) differentiation. In humans, a deletion in a distal non-coding regulatory region upstream of ATOH7 is associated with nonsyndromic congenital retinal nonattachment (NCRNA) disorder, characterized by optic nerve atrophy and complete blindness. Here we functionally interrogate the significance of the Atoh7 enhancer landscape to retinogenesis. We demonstrate that deletion of the enhancer structure upstream of Atoh7 in mice leads to RGC deficiency, optic nerve hypoplasia and blood vascular abnormalities, phenocopying inactivation of Atoh7 and recapitulating key features of NCRNA. We further provide evidence that the loss of the Atoh7 remote enhancer impacts ipsilaterally-projecting RGCs and disrupts proper axonal projections to the brain targets. Transcriptionally, deletion of the Atoh7 remote enhancer is associated with dysregulation of axonogenesis genes, including the derepression of the axon repulsive cue Robo3 which is normally epigenetically silenced in the developing retina. Our data provide novel insights into how Atoh7 enhancer elements function to promote RGC development and optic nerve formation and uncover a key role of Atoh7 in the transcriptional control of axon guidance molecules possibly via epigenetic mechanisms. Overall design: Bulk RNA-Seq retina E14.5 WT and Atoh7 Remote Enhancer KO: three independent replicates from WT or Atoh7 REN KO retinae at E14.5 were processed for RNA extraction and submitted for RNA-Seq

在视网膜发生（retinogenesis）过程中，前神经元碱性螺旋-环-螺旋（basic helix-loop-helix, bHLH）转录因子Atoh7会在视网膜祖细胞（retinal progenitor cells, RPCs）中瞬时表达，且为视网膜神经节细胞（retinal ganglion cell, RGC）分化所必需。在人类中，ATOH7上游远端非编码调控区域的缺失与非综合征性先天性视网膜脱离（nonsyndromic congenital retinal nonattachment, NCRNA）相关，该疾病以视神经萎缩和完全失明为典型特征。本研究对Atoh7增强子调控图谱在视网膜发生中的功能意义展开了探究。实验证实，小鼠体内Atoh7上游增强子结构的缺失会引发RGC缺陷、视神经发育不全及血管异常，其表型与Atoh7功能失活一致，并重现了NCRNA的关键病理特征。本研究进一步发现，Atoh7远端增强子的缺失会影响同侧投射型RGC，并破坏其向脑内靶区的正常轴突投射。转录层面分析显示，Atoh7远端增强子的缺失与轴突发生相关基因的表达失调密切相关，包括通常在发育视网膜中表观遗传沉默的轴突排斥性信号分子Robo3的去抑制。本研究数据为Atoh7增强子元件如何促进RGC发育与视神经形成提供了全新见解，并揭示了Atoh7可能通过表观遗传机制参与轴突导向分子转录调控的关键作用。总体实验设计：针对E14.5胎鼠视网膜的批量RNA测序（Bulk RNA-Seq）：野生型（wild type, WT）与Atoh7远端增强子敲除（Atoh7 Remote Enhancer KO, REN KO）组各设置3次独立生物学重复，提取总RNA后进行RNA测序。