Cdx/Hox通路在哺乳动物心脏发育中的作用及分子机理研究

Cdx family members are caudal-related transcription factors, which are involved in anterior-posterior body patterning through Hox gene regulation. Their functions and molecular mechanism in cardiac development remain to be elucidated. Previous reports demonstrate that several signal molecules, including Wnt pathway, regulate cardiac development. In this study, our preliminary data revealed that Wnt can regulate Cdx gene expression in cardiac cells. In addition, gain or loss of function of Cdx family members led to cardiac malformation with disrupted Hox gene expression pattern during embryonic development, thereby suggesting a Wnt-Cdx regulated "Hox code" may exist in cardiac development. In addition, abnormal Cdx and Hox gene expression patterns were observed in a mouse model with cardiac hypertrophy caused by transverse aortic constriction, indicating Cdx/Hox pathway may also play an important role in cardiac disease formation. In order to further elucidate the molecular mechanism of Cdx/Hox involved in cardiac development and heart diseases, embryonic stem cell (ESC) lines to conditionally induce Cdx or Hox gene expression, or ESC lines with single or compound Cdx knockdown or knockout were established. In this proposal, we first sought to confirm the biological function of Cdx/Hox in cardiac development using ESC in vitro differentiation system together with transgenic mouse models. In addition, we also plan to explore the molecular mechanism of Cdx/Hox pathway in cardiac development and heart disease formation, particularly to identify the target genes and interaction partners of Cdx and certain Hox genes in cardiomyocytes. Further, we will investigate the potential role of Cdx/Hox pathway in cardiac disease formation, including pathlogical cardiac hypertrophy development. Our study will shed light on our understanding torward network interaction of trancriptional regulation in cardiac development and heart diseases, but also provide invaluable information on the cardiac differentiation from pluripotent stem cells, and on new drug targets in clinical therapy of cardiac diseases.

Cdx/Hox是一类具有Homeodomain的转录因子，在心脏发育及疾病中的分子作用机制尚无报道。已知哺乳动物心脏发育受Wnt等多种信号的调控，而我们的初步实验结果发现Cdx基因的表达异常影响心肌的发育，且受Wnt信号调控，调节Hox基因的表达，意味着Cdx可能介导Wnt等信号形成决定性的"Hox code"，影响心肌的形成。另外，病理性心肌肥大伴随着Cdx及Hox基因表达异常，表明Cdx/Hox可能与心脏疾病发生有关。为了进一步揭示Cdx/Hox通路在心脏发育及疾病发生中的分子机制，我们已建立了条件诱导Cdx表达或缺失的胚胎干细胞株及转基因小鼠，Hox基因过表达载体等，拟利用胚胎干细胞体外分化体系，结合转基因小鼠病理模型，进一步确定Cdx/Hox在胚胎干细胞心肌特化及心脏疾病方面的生物学意义，寻找Cdx/Hox通路在心肌细胞中的靶基因及作用伴侣，为心脏疾病的基因治疗及药物开发提供新靶点

在胚胎发育的E9.5-E11.5，位于心管和肝脏附近的一团原心外膜细胞迁移至心管，经粘附和增殖形成心外膜上皮细胞，从胚胎E11.5开始，心外膜上皮细胞增殖并且发生上皮-间质转化形成心外膜衍生的间充质细胞（Epicardial derived cells, EPDCs），之后EPDC发生迁移分化，对于心脏中血管平滑肌、内皮细胞、心脏成纤维细胞和小部分心肌细胞的发育都有作用，尤其是冠状血管的形成意义重大。之前尚未有Cdx基因与心脏外膜发育相关的报道。我们发现Cdx1表达于心外膜细胞及心肌细胞中，在心脏内部的非心肌细胞中不表达。我们因而建立了四环素诱导表达的Cdx1转基因胚胎干细胞及小鼠模型。发现Cdx1诱导心脏外层细胞加厚，心外膜细胞的标志性基因WT1表达消失，而胚胎发育早期的Cdx1过表达导致冠状动脉形成障碍，并进一步Cdx1的过表达促进了心外膜细胞的上皮-间质转化，并从体内和体外的实验中证实了Cdx1的持续性高表达抑制了间充质细胞向心肌层的迁移，影响血管平滑肌的形成，因而导致冠状动脉形成异常。RNA-seq的结果表明Cdx1的过表达促进神经特异性基因的表达，抑制心血管发育相关基因的转录水平。综上所述，我们的研究揭示了Cdx1在心脏发育中的一个新功能及其可能的分子作用机制。