ZIC3 IS AN ESSENTIAL REGULATOR OF CONE PHOTORECEPTOR DEVELOPMENT
Johns Hopkins University
13-line ground squirrel(13-LGS) is one of the rare diurnal rodents with cone-dominant retinas. A comprehensive large-scale single-cell comparative genomics study previously conducted in our lab revealed significant differences in cone development patterns between the 13-LGS and the rod-dominant mouse, characterized by an extended period of cone generation into the postnatal period in 13-LGS. A list of candidate genes was selected based on their differential expression patterns between 13-LGS and mice, among which Zic3 was our top candidate for further investigation. While Zic3 expression is strictly restricted to early-stage retinal progenitors (RPCs) in mice, it is also robustly expressed in late-stage RPCs and cone cells in 13-LGS, indicating a potential role for Zic3 in promoting cone photoreceptor specification and differentiation. To investigate this potential role of Zic3 in regulating cone photoreceptor development, Zic3 conditional knockout and overexpression models were generated, then analyzed with immunostaining and single-cell RNA sequencing. Selective disruption of Zic3 expression during early retinal development resulted in a reduction of cone cell density, while in vivo overexpression of Zic3 in late-stage RPCs that do not normally generate cones induced photoreceptor to translocate towards the apical retina, mirroring the normal location of cone photoreceptor nuclei. Co-electroporation of Zic3 and Pou2f1, which also promotes cone photoreceptor specification and shows expanded expression in developing 13-LGS retina, in ex vivo retinal explants resulted in a synergistic increase in generation of early-born retinal cell types, including cone photoreceptors, and suppression of rod-specific genes. Ex vivo overexpression of the transcription factor Onecut1, which also shows expanded expression in late-stage RPCs in 13-LGS, also strongly inhibited expression of rod-specific genes such as Nr2e3. These findings validated the role of transcription factor Zic3 in promoting cone specification during retinal development and potentially provided a new insight for the realization of directed induction of cone cell differentiation in stem cell therapy.
Zic3, cone fate specification, cone development, retina development. single-cell RNA sequencing, immunohistochemistry, 13-LGS, Onecut1, Pou2f1