Genome-wide enhancer profiling identifies novel Shh regulated gene targets in the developing cochlear duct

The development of the mammalian inner ears is a highly dynamic process that is dependent on the interplay of multiple signaling molecules. Previous studies demonstrated the role of Shh in the initiation and outgrowth of the cochlea. However, the gene targets of Shh in the cochlea largely remain unknown. We show in this study, Shh responsive genes that are loss in the cochlea of Smoecko mutant (Smo null) and upregulated in Shh-P1 mutant embryos through comparative RNA-seq profiling. We performed a genome-wide cis-regulatory element (cRE) study (via ATAC-seq and Gli2 ChIP-seq) and identified potential cREs that are targets of the Shh signaling pathway in the cochlea. Our data unravel novel Shh signaling regulated gene targets that have important functional roles in the development of the cochlea. 1) Examination of genome wide gene expression in 2 different inner ear mutants at E11.5, 2) Examination genome wide of open chromatin regions using ATAC-seq in E11.5 inner ear, 3) Examination of genome wide Gli2 binding site in the E10.5 embryonic head哺乳动物内耳发育是一个高度动态的过程，依赖于多种信号分子的相互作用。既往研究已证实音猬因子（Sonic Hedgehog, Shh）在耳蜗起始与生长过程中的作用，但目前对于耳蜗中Shh的基因靶标仍知之甚少。本研究通过比较RNA测序（RNA-seq）分析，鉴定出在Smoecko突变体（平滑蛋白Smoothened, Smo功能缺失型，Smo null）耳蜗中表达缺失、且在Shh-P1突变胚胎中上调的Shh应答基因。我们通过转座酶可及性测序（Assay for Transposase-Accessible Chromatin using sequencing, ATAC-seq）与Gli2染色质免疫共沉淀测序（Chromatin Immunoprecipitation sequencing, ChIP-seq）开展了全基因组顺式调控元件（cis-regulatory element, cRE）分析，鉴定出耳蜗中Shh信号通路潜在靶标的顺式调控元件。本研究数据揭示了一批全新的Shh信号通路调控基因靶标，这些靶标在耳蜗发育过程中发挥重要功能。具体实验内容包括：1. 对E11.5时期的2种不同内耳突变体开展全基因组基因表达检测；2. 利用ATAC-seq对E11.5时期内耳的全基因组开放染色质区域进行检测；3. 对E10.5时期胚胎头部开展全基因组Gli2结合位点检测。