Cleft lip and cleft palate (CL/P) in Esrp1 KO mice is associated with alterations in Wnt signaling and epithelial-mesenchymal crosstalk

To investigate the molecular mechanisms that lead to CL/P in Esrp1-/- mice, we performed RNA-Seq using RNAs collected from both epithelial cells and mesenchymal cells from control and Esrp1-/- embryos. We used a previously described method to separate facial ectoderm and mesenchyme from facial prominences at E12.0, a stage at which lip fusion is underway (Li and Williams, 2013). We collected pooled paired ectoderm and mesenchyme samples to obtain sufficient material for four replicates each of ectoderm and mesenchyme fractions from WT and Esrp1-/- embryos and prepared total RNA for RNA-Seq. We used paired end sequencing and obtained deep coverage with an average of 100 million read pairs per replicate. Preliminary analysis of transcripts per million (TPM) values in the RNA-Seq analysis from epithelial and mesenchymal control samples validated that they were derived from relatively pure populations of each cell type using a panel of standard epithelial and mesenchymal cell type-specific markers, including Esrp1 RNA-Seq analysis of transcriptomic changes in ectoderm and mesenchyme of the facial prominences at E12 in wild-type and Esrp1 KO mouse embryos.为探究Esrp1基因敲除纯合子（Esrp1-/-）小鼠发生唇腭裂（CL/P）的分子机制，本研究利用从野生型对照及Esrp1-/-胚胎的上皮细胞（epithelial cells）与间充质细胞（mesenchymal cells）中提取的RNA开展RNA测序（RNA-Seq）分析。本研究采用已发表的方法，于胚胎发育第12.0天（E12.0，该时期正值唇融合进程）从面部突起中分离面部外胚层与间充质组织（Li及Williams，2013）。为获取足够的实验材料，我们将配对的外胚层与间充质样本混合，分别为野生型（WT，wild type）及Esrp1-/-胚胎的外胚层组分、间充质组分各设置4个生物学重复，并提取总RNA用于RNA测序。本研究采用双端测序（paired end sequencing），获得了较高的测序深度，每个重复的平均读对（read pairs）数量达1亿条。通过使用一组标准的上皮细胞与间充质细胞特异性标志物（包括Esrp1）对野生型对照样本的RNA测序数据进行每百万转录本（TPM，transcripts per million）值的初步分析，验证了所收集的细胞群体具有较高的纯度。本研究的RNA测序分析聚焦于野生型及Esrp1敲除（KO，knockout）小鼠胚胎在E12时期的面部突起外胚层与间充质的转录组变化。