Abstract:
The proneural transcription factor atonal basic helix-loop-helix transcription factor 7 (ATOH7) is expressed in early progenitors in the developing neuroretina. In vertebrates, this is crucial for the development of retinal ganglion cells (RGCs), as mutant animals show an almost complete absence of RGCs, underdeveloped optic nerves, and aberrations in retinal vessel development. Human mutations are rare and result in autosomal recessive optic nerve hypoplasia (ONH) or severe vascular changes, diagnosed as autosomal recessive persistent hyperplasia of the primary vitreous (PHPVAR). To better understand the role of ATOH7 in neuroretinal development, we created ATOH7 knockout and eGFP-expressing ATOH7 reporter human induced pluripotent stem cells (hiPSCs), which were differentiated into early-stage retinal organoids. Target loci regulated by ATOH7 were identified by Cleavage Under Targets and Release Using Nuclease with sequencing (CUT&RUN-seq) and differential expression by RNA sequencing (RNA-seq) of wildtype and mutant organoid-derived reporter cells. Additionally, single-cell RNA sequencing (scRNA-seq) was performed on whole organoids to identify cell type-specific genes. Mutant organoids displayed substantial deficiency in axon sprouting, reduction in RGCs, and an increase in other cell types. We identified 469 differentially expressed target genes, with an overrepresentation of genes belonging to axon development/guidance and Notch signaling. Taken together, we consolidate the function of human ATOH7 in guiding progenitor competence by inducing RGC-specific genes while inhibiting other cell fates. Furthermore, we highlight candidate genes responsible for ATOH7-associated optic nerve and retinovascular anomalies, which sheds light to potential future therapy targets for related disorders.
摘要:
前神经转录因子无碱性螺旋-环-螺旋转录因子7(ATOH7)在发育中的神经视网膜的早期祖细胞中表达。在脊椎动物中,这对视网膜神经节细胞(RGC)的发育至关重要,由于突变动物几乎完全没有RGC,视神经发育不全,和视网膜血管发育的像差。人类突变是罕见的,并导致常染色体隐性视神经发育不全(ONH)或严重的血管变化,诊断为原发性玻璃体常染色体隐性持续增生(PHPVAR)。为了更好地理解ATOH7在神经视网膜发育中的作用,我们创建了ATOH7敲除和表达eGFP的ATOH7报告人诱导多能干细胞(hiPSCs),分化为早期视网膜类器官。由ATOH7调节的靶基因座通过在靶下切割和使用核酸酶的释放进行测序(CUT&RUN-seq)和通过野生型和突变型类器官衍生的报告细胞的RNA测序(RNA-seq)的差异表达来鉴定。此外,对整个类器官进行单细胞RNA测序(scRNA-seq)以鉴定细胞类型特异性基因.突变的类器官在轴突发芽中表现出实质性的缺陷,RGC的减少,以及其他细胞类型的增加。我们确定了469个差异表达的靶基因,属于轴突发育/指导和Notch信号传导的基因过度表达。一起来看,我们通过诱导RGC特异性基因,同时抑制其他细胞命运,巩固了人类ATOH7在指导祖细胞能力方面的功能。此外,我们强调了负责ATOH7相关视神经和视网膜血管异常的候选基因,这揭示了相关疾病的潜在未来治疗目标。