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 Overall design: 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-/-小鼠发生唇腭裂（Cleft Lip/Palate, CL/P）的分子机制，我们分别采集了对照及Esrp1-/-胚胎的上皮细胞与间充质细胞，并开展RNA测序（RNA-Seq）分析。我们采用已报道的方法，在胚胎发育第12.0天（E12.0，此时唇部融合正处于进行阶段）从面部突起中分离面部外胚层与间充质（Li及Williams, 2013）。我们收集混合配对的外胚层与间充质样本，以获取足够的实验材料，用于野生型（Wild Type, WT）及Esrp1-/-胚胎的外胚层与间充质组分各4次生物学重复，并制备总RNA用于RNA测序（RNA-Seq）。我们采用双端测序（paired end sequencing）技术，获得了充足的测序覆盖度，每个生物学重复的平均读对数量达1亿条。我们对野生型对照上皮与间充质样本的RNA测序数据中的每百万转录本（TPM）值进行初步分析，通过一组包含Esrp1在内的标准上皮及间充质细胞类型特异性标志物组合，验证了这些样本分别来源于相对纯净的对应细胞群。实验设计概述：对野生型及Esrp1基因敲除（Esrp1 KO）小鼠胚胎在E12.0时面部突起的外胚层与间充质中的转录组变化进行RNA测序分析。