Six3 and Six6 jointly control diverse target genes in multiple cell populations over developmental trajectories of mouse embryonic retinal progenitor cells.

How tissue-specific progenitor cells generate adult tissues is a puzzle in organogenesis. Using single-cell RNA sequencing of control and Six3 and Six6 compound-mutant mouse embryonic eyecups, we demonstrated that these two closely related transcription factors jointly control diverse target genes in multiple cell populations over the developmental trajectories of mouse embryonic retinal progenitor cells. In the Uniform Manifold Approximation and Projection for Dimension Reduction (UMAP) graph of control retinas, naïve retinal progenitor cells had two major trajectories leading to ciliary margin cells and retinal neurons, respectively. The ciliary margin trajectory was from naïve retinal progenitor cells in the G1 phase directly to ciliary margin cells, whereas the neuronal trajectory went through an intermediate neurogenic state marked by Atoh7 expression. Neurogenic retinal progenitor cells (Atoh7+) were still proliferative; early retinal neurons branched out from Atoh7+ retina progenitor cells in the G1 phase. Upon Six3 and Six6 dual deficiency, both naïve and neurogenic retinal progenitor cells 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, Wnt signaling was upregulated, whereas FGF signaling was downregulated. Notably, Six3 and Six6 proteins occupied the loci of diverse genes that were differentially expressed in distinct cell populations, and expression of these genes was significantly altered upon Six3 and Six6 dual deficiency. Our findings provide deeper insight into the molecular mechanisms underlying early retinal differentiation in mammals.