Ferrena Alexander, Zhang Xusheng, Shrestha Rupendra, Zheng Deyou, Liu Wei
bioRxiv. 2023 May 3:2023.05.03.539288. doi: 10.1101/2023.05.03.539288.
Formation, maintenance, and differentiation of tissue-specific progenitor cells are fundamental tasks during organogenesis. Retinal development is an excellent model for dissecting these processes; mechanisms of retinal differentiation can be harnessed for retinal regeneration toward curing blindness. Using single-cell RNA sequencing of embryonic mouse eye cups in which transcription factor Six3 was conditionally inactivated in peripheral retinas on top of germline deletion of its close paralog Six6 ("DKO"), we identified cell clusters and then inferred developmental trajectories in the integrated dataset. In control retinas, naïve retinal progenitor cells had two major trajectories leading to ciliary margin cells and retinal neurons, respectively. The ciliary margin trajectory was directly from naïve retinal progenitor cells at G1 phase, and the retinal neuron trajectory was through a neurogenic state marked by expression. Upon and dual deficiency, both naïve and neurogenic retinal progenitor cells were defective. Ciliary margin differentiation was enhanced, and multi-lineage retinal differentiation was disrupted. An ectopic neuronal trajectory lacking the Atoh7+ state led to ectopic neurons. Differential expression analysis not only confirmed previous phenotype studies but also identified novel candidate genes regulated by . Six3 and Six6 were jointly required for balancing the opposing gradients of the Fgf and Wnt signaling in the central-peripheral patterning of the eye cups. Taken together, we identify transcriptomes and developmental trajectories jointly regulated by Six3 and Six6, providing deeper insight into molecular mechanisms underlying early retinal differentiation.
组织特异性祖细胞的形成、维持和分化是器官发生过程中的基本任务。视网膜发育是剖析这些过程的绝佳模型；视网膜分化机制可用于促进视网膜再生以治愈失明。通过对胚胎小鼠眼杯进行单细胞RNA测序，在其近亲同源基因Six6的种系缺失基础上，转录因子Six3在外周视网膜中被条件性失活（“双敲除”），我们识别出细胞簇，然后在整合数据集中推断发育轨迹。在对照视网膜中，幼稚视网膜祖细胞有两条主要轨迹，分别通向睫状缘细胞和视网膜神经元。睫状缘轨迹直接来自处于G1期的幼稚视网膜祖细胞，而视网膜神经元轨迹则通过以 表达为特征的神经源性状态。在Six3和Six6双缺失时，幼稚和神经源性视网膜祖细胞均有缺陷。睫状缘分化增强，多谱系视网膜分化受到破坏。一条缺乏Atoh7 + 状态的异位神经元轨迹导致异位神经元。差异表达分析不仅证实了先前的表型研究，还鉴定了受 调控的新候选基因。Six3和Six6共同作用，以平衡眼杯中央 - 外周模式中Fgf和Wnt信号的相反梯度。综上所述，我们鉴定了由Six3和Six6共同调控的转录组和发育轨迹，为早期视网膜分化的分子机制提供了更深入的见解。
