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, 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 whereas the neuronal trajectory was through a neurogenic state marked by Atoh7 expression. Upon Six3 and Six6 dual deficiencies, both naïve 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. Overall design: 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).