Shox2调控上腭间充质细胞成骨分化的分子机制

The morphogenetic pattern and gene expression of palate was similar between mouse and human, thus, mouse is an excellent model for studying palate development. Gene knockout or mutant mouse show the significantly reduced bone formation phenotype during palate development. However, the molecular mechanisms underlying palatal bone formation remain elusive. The homeobox gene Shox2 is expressed specifically in the anterior palatal mesenchyme, overlapping the future bony hard palate domain. Inactivation of Shox2 in the cranial neural crest-derived palatal mesenchymal cells leads to significantly reduced bone formation in the hard palate. In addition, our preliminary studies using Shox2 ChIP-Seq on developing palate reveal predominant binding of Shox2 to the distal acting enhancers of osteogenic genes, further indicating an essential role for Shox2 in osteogenesis. Since Shox2 is also expressed in non-osteogenic cell lineages in the palatal mesenchyme, in this proposal, we will determine the cell autonomous function of Shox2 in osteogenic lineage using the 3.6k-Col1a1-Cre allele that has been shown to become activated in osteogenic precursor cells. We will further conduct RNA-Seq and ChIP-Seq to reveal molecular function of Shox2 at the genomic level. The results gained from the proposed research will greatly enhance our understanding of molecular mechanisms underlying palate development and osteogenesis.

小鼠的上腭发育与基因调控模式与人类相同，为研究上腭器官发育绝佳的动物模型。一些基因突变的小鼠表现出上腭板的骨头发育不全的表型，但对其中上腭间充质细胞成骨分化的分子机制解析甚少。Shox2作为一个重要的转录因子，在上腭前端特异性表达，与上腭发生成骨分化区域重合。前期研究表明上腭间充质中敲除Shox2会导致罕见的前腭裂并伴随骨量大幅减少。而本项目综合RNA-Seq和ChIP-Seq高通量测序与生物信息学综合分析后证实Shox2异常会导致上腭骨相关基因的表达异常，并可能是通过结合增强子调控成骨分化，因此Shox2对上腭的骨发育至关重要。本项目将利用成骨前体细胞中特异敲除Shox2的遗传小鼠模型，在细胞特异性谱系发育层面上，研究Shox2在间充质细胞成骨分化中的自主性调控功能，并从基因组学水平上进一步阐明Shox2调控上腭间充质细胞成骨分化的分子机制，为探究上腭骨发育畸形的机理提供理论

小鼠的上腭发育与基因调控模式与人类相同，为研究上腭器官发育绝佳的动物模型。一些基因突变的小鼠表现出上腭板的骨头发育不全的表型，但对其中上腭间充质细胞成骨分化的分子机制解析甚少。Shox2作为一个重要的转录因子，在上腭前端特异性表达，并且与上腭发生成骨分化区域重合。本项目首先利用3.6k-Col1a1-Cre（成骨细胞特异性Cre）小鼠与Shox2条件性敲除小鼠交配，发现在成骨细胞谱系中敲除Shox2导致了上腭板中成骨细胞大量减少，同时成骨分化基因Runx2和Sp7表达下调，证明了Shox2是在Shox2以细胞自主的方式调节上腭间充质的成骨分化。其次，通过流式细胞分选出成骨细胞谱系中Shox2敲除的细胞，进行转录组分析，筛选出一批Shox2所调控的成骨分化相关基因，并进行了表达验证。最后，通过ChIP-Seq分析，发现Shox2作为转录因子，结合到成骨相关基因的远端增强子上调控基因的表达。本项目解析了Shox2作为转录因子调控上腭板间充质细胞成骨分化的机制，筛选出了一批Shox2结合的并与成骨分化相关的增强子序列，这些序列将为上腭板间充细胞成骨分化机制的解析，以及人类相关非编码区的疾病SNP挖掘提供理论基础。