Identification of TWIST1 transcriptional targets in the cranial mesoderm [E8_5]

TWIST1, a basic helix-loop-helix transcription factor is essential for the development of cranial mesoderm and cranial neural crest-derived craniofacial structures. Our previous work showed that, in the absence of TWIST1, some cells within the cranial mesoderm adopt an abnormal epithelial configuration. Here, we show by transcriptome analysis that loss of TWIST1 in the cranial mesoderm is accompanied by a reduction in the expression of genes that are associated with cell-extracellular matrix interactions and the acquisition of mesenchymal characteristics. By comparing the transcriptional profiles of cranial mesoderm-specific Twist1 loss-of-function mutant and control mouse embryos, we identified a set of genes that are both TWIST1-dependent and predominantly expressed in the mesoderm. By ChIP-seq in a cell line model of a TWIST1-dependent mesenchymal state, we identified, among the downstream genes, three direct transcriptional targets of TWIST1: Ddr2, Pcolce and Tgfbi. Our findings show that the mesenchymal properties of the cranial mesoderm is likely to be regulated by a network of TWIST1 targets genes that influence the extracellular matrix and cell-matrix interactions, and collectively they are required for the morphogenesis of the craniofacial structures. For microarray analysis of CM-CKO embryos, embryo heads of four genotypes were collected at E8.5 (5-7 somites) and E9.5 (18- 20 somites): CM-CKO (Twist1flox/del; Mesp1Cre/+), CM-Het (Twist1flox/wt; Mesp1Cre/+), Het (Twist1flox/del; Mesp1+/+) and Control (Twist1flox/wt; Mesp1+/+). Sample sizes for E8.5 embryos were as follows: Control, n=4 CM-CKO, n=4; Het, n=3; CM-Hets, n=3).TWIST1是一种碱性螺旋-环-螺旋（basic helix-loop-helix）转录因子，对颅中胚层及颅神经嵴来源的颅面结构发育具有关键调控作用。本团队既往研究证实，在TWIST1缺失的背景下，颅中胚层内的部分细胞会呈现异常的上皮细胞构型。本研究通过转录组分析发现，颅中胚层中TWIST1的缺失会伴随与细胞-细胞外基质相互作用及间充质特性获得相关的基因表达水平下调。通过对比颅中胚层特异性Twist1功能缺失突变小鼠胚胎与对照胚胎的转录谱，我们鉴定出了一组同时受TWIST1调控且主要在间充质中表达的基因。随后，在TWIST1依赖的间充质状态细胞系模型中开展染色质免疫共沉淀测序（ChIP-seq），我们在下游靶基因中筛选得到三个TWIST1的直接转录靶点：Ddr2、Pcolce与Tgfbi。本研究结果表明，颅中胚层的间充质特性很可能受一系列TWIST1靶基因组成的调控网络所调控，这类靶基因可通过影响细胞外基质及细胞-基质相互作用，共同参与颅面结构的形态发生过程。针对颅中胚层特异性敲除（CM-CKO）胚胎的微阵列分析中，我们于E8.5（5-7体节）及E9.5（18-20体节）阶段收集了四种基因型的胚胎头部：CM-CKO（Twist1flox/del; Mesp1Cre/+）、CM-Het（Twist1flox/wt; Mesp1Cre/+）、Het（Twist1flox/del; Mesp1+/+）以及对照（Twist1flox/wt; Mesp1+/+）。E8.5阶段胚胎的样本量设置如下：对照组n=4、CM-CKO组n=4；Het组n=3；CM-Het组n=3。