Six3 and Six6 jointly regulate the identities and developmental trajectories of multipotent retinal progenitor cells in the embryonic mouse retina

Regulation of retinal differentiation in mammals is not adequately understood. Using single-cell RNA sequencing of control and Six3 and Six6 compound-mutant mouse embryonic eye-cups, we identified cell clusters and developmental trajectories jointly regulated by transcription factor Six3 and its close paralog Six6. In control retinas, nave retinal progenitor cells had two major trajectories leading to ciliary margin cells and retinal neurons, respectively. The ciliary margin trajectory was directly from nave retinal progenitor cells at G1 phase whereas the neuronal trajectory was through a neurogenic state marked by Atoh7 expression. Upon Six3 and Six6 dual deficiencies, both nave and neurogenic retinal progenitors were defective, ciliary margin differentiation was enhanced, and multi-lineage neuronal differentiation was disrupted. An ectopic neuronal trajectory lacking the Atoh7+ state led to ectopic neurons. Additionally, opposing gradients of Wnt and Fgf signaling were perturbed. Our findings provide deeper insight into molecular mechanisms underlying early retinal differentiation in mammals. Mouse embryos from the mating between Six3F/F;Six6-/-;a-Cre male and Six3F/F;Six6+/- female were harvested at E13.5. Intact eye cups containing the neural retina and lens were dissected out from neighboring tissues. Eye cups from an embryo with the genotype of Six3F/F;Six6-/-;a-Cre were identified based on GFP+ rosettes in the retinas under a stereo fluorescence microscope. The correlation between GFP+ rosettes and the genotype of Six3F/F;Six6-/-;a-Cre was previously established in pilot studies. Eye cups from an embryo without GFP were used as a control. Tails of the selected embryos were collected for genotyping. Then, eye cups were dissociated into single cells using activated Papain (Worthington Biochemical) for cell capture using the 10x Chromium fluid device, targeting 10,000 cells for each sample. Genotyping of the embryos confirmed the genotype of Six3F/F;Six6-/-;a-Cre for DKO retinas and the genotype of Six3+/-;Six6+/- for the littermate control. Previous phenotype analysis indicates that embryos with the genotype of Six3+/-;Six6+/- are nearly indistinguishable from wildtype embryos (Diacou et al., 2018). Captured cells were used for library preparation using the 10x Genomics Single Cell 3 kit (version 3).哺乳动物视网膜分化的调控机制尚未得到充分阐明。本研究通过对对照组以及Six3与Six6复合突变的小鼠胚胎视杯开展单细胞RNA测序（single-cell RNA sequencing），鉴定出受转录因子（transcription factor）Six3及其近缘旁系同源基因（paralog）Six6共同调控的细胞簇与发育轨迹。在对照组视网膜中，幼稚视网膜祖细胞（retinal progenitor cells）存在两条主要发育轨迹，分别朝向睫状缘（ciliary margin）细胞与视网膜神经元分化。其中睫状缘发育轨迹直接源自处于G1期的幼稚视网膜祖细胞，而神经元发育轨迹则需经过以Atoh7表达为特征的神经发生状态（neurogenic state）。当Six3与Six6双缺失时，幼稚视网膜祖细胞与神经发生状态的视网膜祖细胞均出现功能缺陷，睫状缘分化被增强，而多谱系神经元分化则受到扰乱。一条缺失Atoh7+细胞状态的异位（ectopic）神经元发育轨迹可产生异位神经元。此外，Wnt信号通路（Wnt signaling）与成纤维细胞生长因子（fibroblast growth factor, Fgf）信号的反向浓度梯度受到扰动。本研究结果为哺乳动物早期视网膜分化的分子机制提供了更深入的认知。本研究通过将Six3F/F;Six6-/-;a-Cre雄性小鼠与Six3F/F;Six6+/-雌性小鼠交配，在胚胎发育第13.5天（E13.5）收集胚胎。从周围组织中分离出包含神经视网膜与晶状体的完整视杯。通过体式荧光显微镜（stereo fluorescence microscope）观察视网膜中的绿色荧光蛋白（green fluorescent protein, GFP）阳性玫瑰结样结构，鉴定出基因型为Six3F/F;Six6-/-;a-Cre的胚胎视杯。此前的预实验已验证GFP+玫瑰结样结构与Six3F/F;Six6-/-;a-Cre基因型之间的对应关系。无GFP信号的胚胎视杯则作为对照组。收集所选胚胎的尾部组织用于基因型鉴定。随后，使用活化的木瓜蛋白酶（Papain, Worthington Biochemical）将视杯解离为单细胞，通过10x Chromium微流控设备（10x Chromium fluid device）进行细胞捕获，每个样本目标捕获10000个细胞。胚胎基因型鉴定结果确认，双敲除（DKO）视网膜的基因型为Six3F/F;Six6-/-;a-Cre，同窝对照组的基因型为Six3+/-;Six6+/-。此前的表型分析表明，基因型为Six3+/-;Six6+/-的胚胎与野生型胚胎几乎无差异（Diacou et al., 2018）。捕获的细胞使用10x Genomics单细胞3'转录组建库试剂盒（10x Genomics Single Cell 3 kit, version 3）进行文库制备。