Heterochronic transcription factor expression drives cone-dominant retina development in 13-lined ground squirrels. [CUT&RUN]

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. Nuclei were isolated from snap-frozen P5 13LGS and P2 mouse retinas for use in CUT&RUN-sequencing. Two biological replicates were assayed for each species, with each 13LGS replicate consisting of a pool of retinas from two individuals, and each mouse replicate consisting of a pool of retinas from 3 individuals.