Foxc1a调控斑马鱼胚胎心脏发生的分子机制研究

Foxc1 is a highly conserved transcription factor during evolution. It plays dual roles by acting as a transcriptional activator or transcriptional repressor. Foxc1 is involved in regulating mouse cardiogenesis. The mutated FOXC1 is associated with the congenital heart diseases in human. However, its functions and the molecular mechanisms underlying the vertebrate cardiogenesis remain largely unknown. Previously, we demonstrated that zebrafish embryos carrying null alleles of foxc1a, an orthologue gene of human FOXC1, exhibited severe defects in cardiogenesis. And our preliminary data from transcriptomic analysis further revealed that the expressions of nkx2.5 (a marker gene for the cardiac progenitor cells), 3 genes in Wnt pathway and 3 genes in retinoic acid (RA) signaling pathway，and 7 pri-miRNA, were greatly changed in zebrafish embryos though the formation of the anterior lateral plate mesoderm at somitogenesis stage and three germ layers at gastrula were normally formed. In this study, we will investigate whether foxc1a play essential roles in zebrafish cardiogenesis through directly activating or repressing the expressions of nkx2.5, the 3 genes in Wnt and the 3 ones in RA signaling pathways, and the 7 pri-miRNA in embryos by using genetic methods of creating knockout, conditional knockout and knock-in zebrafish mutants. The results from zebrafish, an excellent model for the research on vertebrate heart development, will help to reveal the molecular and genetic mechanisms of vertebrate cardiogenesis and provide clues to understand the etiology of the congenital heart diseases in human.

Foxc1是进化上高度保守的转录因子，具转录激活或抑制的双向调控作用。Foxc1参与调控小鼠心脏发生，FOXC1突变与人先天性心脏病发生相关。但有关Foxc1是以何种机制调控心脏发生尚未有系统研究。我们前期工作基础表明，在斑马鱼中敲除FOXC1的直系同源基因foxc1a致心脏发生异常；基于转录组学数据的研究显示突变胚胎体节期的前部侧板中胚层和原肠期的三个胚层都正常形成，但心脏前体细胞特化标识基因nkx2.5、3个Wnt信号通路基因和3个视黄酸信号通路基因、7个pri-miRNA分别表达异常。本申请将在已有工作基础上，采用基因敲除、条件性敲除和敲入等遗传学手段，研究Foxc1a是否通过直接激活或抑制nkx2.5、3个Wnt和3个视黄酸信号通路基因、7个pri-miRNA等的表达，决定模式动物斑马鱼心脏发生。研究结果将有助于深入揭示脊椎动物心脏发生的分子机制，为了解先天性心脏病的病因提供参考。

FOXC1作为进化中高度保守的转录因子，在脊椎动物心脏发生中发挥关键作用。我们利用斑马鱼为模型研究心脏发生的分子调控机制时发现，斑马鱼foxc1a纯合突变体具有严重的早期心脏缺陷，最早可追溯到前体细胞特化时期。.研究表明，斑马鱼foxc1a通过直接调控nkx2.5启动子上的两个富含T的非保守结合位点调控早期心脏发育。使用热启动蛋白启动子在10hpf热激启动nkx2.5 的过表达能够部分拯救50hpf的心室收缩和心室面积变小的表型以及108hpf的心包水肿指数。.在14hpf的foxc1a纯合突变体的转录组深度测序数据中显著下调的Wnt信号通路关键基因tmem88a通过qRT-PCR和原位杂交验证结果一致，条件性过表达tmem88a能够部分拯救108hpf的心包水肿指数，但是tmem88a的3Kbp启动子序列无法响应Foxc1a的调控，由此得出结论tmem88a并不能直接受到Foxc1a的调控。.RA信号通路关键基因aldh1a2在14hpffoxc1a纯合突变体的前部侧板中胚层后端异位表达。进一步研究发现异位上调的aldh1a2表达到了心肌前体细胞的区域，而条件性敲除foxc1a细胞中的aldh1a2的表达可以部分拯救心肌前体细胞标记基因nkx2.5的表达水平、50hpf的心室收缩能力、生存时长，但不能拯救纯合突变体的心包水肿表型。.人FOXC1能够以剂量依赖的方式结合人NKX2-5启动子序列。我们运用大鼠心肌细胞系和斑马鱼胚胎模型对FOXC1进行功能评价发现，ARS综合征家系中的不同突变形式的FOXC1对心脏前体细胞NKX2-5的响应不同，在斑马鱼中过表达不同突变形式的FOXC1对心室收缩能力的拯救效果也对应不同，相比之下斑马鱼模型更具有整体水平的优势。散发的先天性心脏病患者中的FOXC1突变对NKX2-5的表达水平没有影响，很可能是因为FOXC1的多态性形式。.研究还发现了HOXC11作为大鼠心肌细胞系中与FOXC1相互作用的转录因子，可以单独激活NKX2-5启动子的转录活性，但在斑马鱼胚胎中敲除其直系同源基因hoxc11a和hoxc11b不能影响心脏的发育和功能。