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.视黄酸（Retinoic acid, RA）是一类转录调控因子，可调控眼部发育的多个进程，其中包括视泡内陷形成视杯的过程，但此前尚未明确该功能对应的靶基因。由于Rdh10基因敲除胚胎中视黄酸合成缺失会导致数千个基因的表达水平发生改变，因此需要采用其他方法来鉴定直接受视黄酸调控的基因。本研究针对表现出视杯形成障碍的野生型胚胎及Rdh10敲除（Rdh10-/-）胚胎的眼部组织，联合采用染色质免疫共沉淀测序（ChIP-seq）检测表观遗传标记、RNA测序（RNA-seq）分析基因表达谱。我们筛选出了一类在视黄酸缺失时表达量下调，且其邻近区域的基因激活表观遗传标记——组蛋白H3赖氨酸27乙酰化（H3K27ac）——水平也同步降低的少量基因。其中Alx1基因不仅符合上述特征，其邻近视黄酸调控的H3K27ac标记区域还存在视黄酸应答元件（RA response element, RARE），这为视黄酸直接激活Alx1提供了强有力的证据。原位杂交（in situ hybridization）实验结果显示，Rdh10-/-胚胎眼部的Alx1表达量显著下调。通过CRISPR/Cas9技术敲除Alx1会导致视杯形成障碍，由此证实视黄酸可通过直接激活Alx1来促进眼部发育的该阶段进程。本研究通过RNA-seq对小鼠Rdh10敲除与野生型眼部组织开展了基因表达比较分析。