A cis-regulatory module underlies retinal ganglion cell genesis and axonogenesis [Set2.Bulk RNA-Seq retina E14.5 WT and Atoh7 Large 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. Bulk RNA-Seq retina E14.5 WT and Atoh7 Large Enhancer KO: four independent replicates from WT or Atoh7 TEN 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，神经前体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）的分化所必需。在人类中，ATOH7基因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）病症相关，该病症以视神经萎缩和完全失明为典型特征。本研究通过功能实验探究了Atoh7增强子图谱对视网膜发生的重要意义。我们证实，在小鼠体内敲除Atoh7上游的增强子结构，会导致视网膜神经节细胞缺失、视神经发育不全及血管异常，该表型与Atoh7失活的表型一致，并重现了NCRNA的关键临床特征。我们进一步提供证据表明，Atoh7远端增强子的缺失会影响同侧投射型视网膜神经节细胞，并破坏其向大脑靶区的正常轴突投射。在转录调控层面，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被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视网膜神经节细胞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转录调控轴突导向分子的关键作用。本数据集包含小鼠胚胎期14.5天（E14.5）野生型（wild type, WT）与Atoh7大型增强子敲除（Atoh7 Large Enhancer KO）视网膜的批量RNA测序（Bulk RNA-Seq）数据：从E14.5天的野生型或Atoh7 TEN KO视网膜中获取4份独立重复样本，进行RNA提取后提交进行RNA测序分析。