Heterochronic transcription factor expression drives cone-dominant retina development in 13-lined ground squirrels. [scATAC-Seq]

Evolutionary adaptation to diurnal vision in ground squirrels has led to the development of a cone-dominant retina, in stark contrast to the rod-dominant retinas of most mammals. The molecular mechanisms driving this shift remain largely unexplored. Here, we perform single-cell RNA sequencing (scRNA-Seq) and chromatin accessibility profiling (scATAC-Seq) across developmental retinal neurogenesis in the 13-lined ground squirrel (13LGS) to uncover the regulatory basis of this adaptation. We find that 13LGS cone photoreceptors arise not only from early-stage neurogenic progenitors, as seen in rod-dominant species like mice, but also from late-stage neurogenic progenitors. This extended period of cone generation is driven by a heterochronic shift in transcription factor expression, with cone-promoting factors such as Onecut2, Pou2f1, and Zic3 remaining active in late-stage progenitors, while cone-differentiation factors Thrb, Rxrg, and Mef2c are expressed precociously in cone specification. Functional analyses reveal that Zic3 and Mef2c promote cone specification, acting through species-specific regulatory elements that drive their expression in late-stage progenitors. These results demonstrate that evolutionary modifications to gene regulatory networks underlie the development of cone-dominant retinas, providing insight into mechanisms of sensory adaptation and potential strategies for cone photoreceptor regeneration in vision disorders. In 13LGS, retinal neurogenesis begins around embryonic day 18 (E18), with birth occurring at E27 (blue-dotted line in Fig. 1A), and concludes by postnatal day 17 (P17)27. To comprehensively profile cellular-level changes in gene expression and chromatin accessibility, we conducted scRNA-Seq and scATAC-Seq analyses across ten developmental time points, spanning the full course of neurogenesis and the immediate post-neurogenesis period (E18–P21).