转录因子Msx1与哺乳动物上腭发育的前-后区域化

Cleft palate belongs to the most occurred birth defect in humans. The palatogenesis of mammals is coordinatedly and precisely regulated at the level of gene expression. Previous studies have indicated there is the heterogeneity on the gene expression along anterior and posterior axis of palatal shelf during the development. However, the molecular mechansim underlying the regional regulation still remains elusive. Both human MSX1 mutation and mouse Msx1 knockout result in the cleft palate. Our preliminary studies show that the expression pattern change on the mouse Msx1 by genetic modification led to the cleft secondary palate. In this grant proposal we plan to study the Msx1 regulatory mechanism of mouse palatogenesis along the anterior and posterior palatal axis using transgenic Msx1 gain of function mouse model. We plan to define the developmental role of Msx1 in the regional establishment of palatal shelf by characterizing the defect phenotype of palatogenesis using histology analyses, in vitro organ culture, in vitro gene knock down system. qRT-PCR and extensive RNA in situ hybridization will be performed to analyse the differential expression data provided by RNA-Seq to unbderstand the Msx1 regulation of those marker genes for the anterior or posterior palatal shelf; We also want to determine the potential target gene(s) and binding site(s) of Msx1 in the mesenchymal cells of the palatal shelf by means of chromatin immunoprecipitation assay (ChIP assay) and dual luciferase reporter system. The expression pattern of those potential Msx1 target genes during palatogenesis will be analysed by both in situ hybridization and quantitative analysis. Together, the role of Msx1-dependent regulation circuit/network in the control of regionalization along the anterior and posterior axis of palatal shelf will be elucidated through this study.

上腭裂是人类最常见的出生缺陷病。哺乳动物上腭发育过程涉及严密的转录水平的调控。过去的研究提示：上腭发育中存在沿胚胎上腭前-后轴在基因表达水平的区域特异性。但是，相关分子及遗传调控机理仍然不清。人类MSX1突变及模式小鼠敲除Msx1基因都可导致腭裂。我们前期初步发现，利用遗传修饰改变Msx1在上腭发育中的表达模式会造成上腭裂。项目利用小鼠Msx1基因修饰模型研究转录因子Msx1在小鼠上腭发育中在前-后轴性建立中的转录调控机理。通过对Msx1过表达小鼠形成上腭裂的分子发育生物学分析及对腭板组织RNA-seq数据中差异表达基因的原位杂交分析解析Msx1在控制腭板前后区域化建立中对特定基因表达的影响；利用染色质免疫沉淀和萤光素酶基因报告等手段确定上腭架细胞中Msx1的潜在靶点基因及结合位点；并通过对这些潜在靶分子表达谱的研究解析Msx1作为主要调控分子在上腭前后轴性建立中的作用机理。

上腭裂是人类最常见的出生缺陷病。以前的研究发现，上腭发育中存在沿胚胎上腭前-后轴在基因表达水平的区域特异性。然而，相关分子及遗传调控机理仍然不清。Msx1是一个在腭板发育过程中起重要作用的转录因子，人类MSX1突变及模式小鼠敲除Msx1基因都可导致腭裂。本项目使用Msx1过表达小鼠和敲除小鼠相结合进行研究，旨在揭示Msx1在上腭发育中的作用。我们发现使用Wnt1-Cre在小鼠神经嵴来源的腭板间充质细胞中过表达Msx1会导致腭裂，并且伴随腭板细胞的增殖缺陷以及异常细胞死亡。Msx1过表达小鼠的硬腭骨形成具有严重缺陷，腭骨形成严重受损并且骨发育关键调控因子Sp7的表达大量减少。我们使用RNA测序分析发现，一些和腭板发育相关的关键基因在Msx1过表达和敲除腭板中的表达有显著变化。其中，腭板前段特异性表达的Alx1的表达受Msx1促进，而中后段特异性表达的Barx1、Meox2和Tbx22的表达则受Msx1抑制。此外，我们使用染色体免疫共沉淀和双荧光素酶报告基因实验发现Tbx22的转录直接受Msx1抑制，Tbx22是Msx1的靶基因。我们进一步发现，Tbx22对Sp7有转录促进作用，并且过表达Tbx22可以挽救Msx1过表达小鼠中Sp7的减少。以上结果说明Msx1作为主要调控分子在上腭前后轴性建立中对腭板前后端特异性表达基因具有重要的调控功能。