Abstract
Purpose :
Vsx2 is an important and highly conserved transcription factor during retinal development process and its mutations lead to microphthalmia in humans and mice. During early development, Vsx2 is expressed in retinal progenitor cells (RPCs) while later it is exclusively expressed in bipolar neurons and at low levels in Müller glia. How is this precise temporal and spatial pattern of expression achieved during retinal development remains poorly understood. We hypothesized that enhancers should playing an important role in achieving the precise expression of Vsx2.
Methods :
Enhancers were tested for their ability to activate expression of reporter genes in developing mouse retinas, using electroporation into retinal explants. We deleted one promising enhancer in mouse using CRISPR/CAS9 technology to examine the functionality of it. Retinas at E14.5 and adult stages were then collected for immunostaining or RNA-seq study.
Results :
We identified Vsx2 elements that drive robust reporter expression within the developing retina. Genetic deletion of a Vsx2 enhancer element leads to microphthalmia, recapitulating retinal defects observed in Vsx2-null mutant mice. However, different from Vsx2-null mutant mice, these knock out mice still have bipolar cells. RNA-seq results from E14.5 samples showed that cell proliferation process was affected in the knock-out mice. And these results were further confirmed by immunostaining of EdU and Ki67.
Conclusions :
Taken together, our results revealed a cell specific cis-regulatory element which is responsible for the expression of Vsx2 and proliferation of retinal progenitor cells in retinal development. Thus, providing insights onto gene regulatory networks that govern Vsx2 expression during retinal development.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.