Retinoic acid-regulated epigenetic marks identify Alx1 as a direct target gene required for optic cup formation [RNA-seq]

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 identity genes that are directly regulated by RA. Here, we combined ChIP-seq for epigenetic marks with RNA-seq on eye tissue from wild-type embryos and Rdh10-/- embryos that exhibit failure in optic cup formation. We identified a small number of genes with decreased expression when RA is absent that also have 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 strong evidence that RA directly activates Alx1. In situ hybridization studies showed that Rdh10-/- embryos exhibit a large decrease in eye Alx1 expression. CRISPR/Cas9 knockout of Alx1 resulted in a failure in optic cup formation, thus demonstrating that RA directly activates Alx1 in order to stimulate this stage of eye development. Gene expression comparison of Rdh10 KO and WT eye in mice using RNA-seq.