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

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 inRdh10knockout 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 andRdh10-/-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 wasAlx1that also has an RA response element (RARE) located near the RA-regulated H3K27ac mark, providing strong evidence that RA directly activatesAlx1. In situ hybridization studies showed thatRdh10-/-embryos exhibit a large decrease in eyeAlx1expression. CRISPR/Cas9 knockout ofAlx1resulted in a defect in optic cup formation, thus demonstrating that RA directly activatesAlx1in order to stimulate this stage of eye development.