Retinoic Acid-Regulated Epigenetic Marks Identify Alx1 as a Direct Target Gene Required for Optic Cup Formation

Background/Objectives: Retinoic acid (RA) is a transcriptional control agent that regulates several aspects of eye development including invagination of the optic vesicle to form the optic cup, although a target gene for this role has not been previously identified. As loss of RA synthesis in Rdh10 knockout embryos affects the expression levels of thousands of genes, a different approach is needed to identify genes that are directly regulated by RA. Methods: Here, we combined ChIP-seq for the H3K27ac epigenetic mark with RNA-seq on optic field tissue from E10 wild-type and Rdh10−/− embryos that exhibit failure in optic cup formation. Results: We identified a small number of genes with decreased expression when RA is absent that also have a decreased presence of a nearby epigenetic gene activation mark (H3K27ac). One such gene was Alx1 that also has an RA response element (RARE) located near the RA-regulated H3K27ac mark, providing evidence that RA directly activates Alx1. In situ hybridization studies showed that Rdh10−/− embryos exhibit a large decrease of Alx1 expression in the optic field. CRISPR/Cas9 knockout of Alx1 resulted in a defect in optic cup formation due to a failure of perioptic mesenchyme to migrate and separate the optic cup epithelium from the forebrain neuroepithelium. Conclusions: Our studies support a model in which RA functions to directly activate Alx1 in perioptic mesenchyme to stimulate an early stage of eye development during which the optic vesicle folds into an optic cup that forms the retina.