Analysis of cardiovascular development and pathophysiology by gene engineering of the endothelin system in mice

关于内皮素(ET)系统参与心血管和颅面发育以及病理生理学的分子机制，我们取得了以下结果。结论：1.建立了Cre-重组酶介导的小鼠ES细胞基因敲入系统，系统地用外源基因替代ET-A受体基因。利用这个系统，我们敲入了编码ET-B受体(ETBR)和ETAR-ETBR嵌合受体的cDNA。这些实验表明：1)STAR亚型选择性和非选择性信号都参与了颅面发育；II)Dlx5/Dlx6同源盒基因的诱导和随后的下颌识别是由STAR亚型选择性、Gq/G11介导的信号通路介导的。LacZ基因的敲入揭示了一种可能起源于心脏新月的新的细胞谱系，并对早期心血管发育做出了贡献。绿色荧光蛋白的敲入使我们能够在小鼠体内原位显示STAR阳性细胞，这对于分析STAR阳性细胞在胚胎血管形成过程中的动态以及在生理和病理生理过程中的作用是有用的。我们已经确定Calain-6是ET-1/STAR信号通路在颅面发育过程中的靶分子。我们发现了Calain-6在稳定微管和参与细胞形态和运动的肌动蛋白组织中的新功能。对Calain-6发育作用的研究始于最近建立的其基因敲除小鼠，这些成果有助于了解心血管和颅面发育的机制，并为开发适用于涉及ET系统的(病理)生理学研究的实验系统铺平了道路。

We have achieved the following results in regard to the molecular mechanisms underlying the involvement of the endothelin (ET) system in cardiovascular and craniofacial development and pathophysiology.1. We have established the Cre-recombinase-mediated gene knock-in system in mouse ES cells, in which we can systematically replace the ET-A receptor (STAR) gene with exogenous genes.2. By using this system, we knocked-in cDNAs encoding the ET-B receptor (ETBR) and ETAR-ETBR chimeric receptors. These experiments have revealed that i) both STAR subtype-selective and nonselective signaling are involved in the craniofacial development and ii) the induction of Dlx5/Dlx6 homeobox genes and subsequent specification of the mandibular identity is mediated by the STAR subtype-selective, Gq/G11-mediated signaling pathway.3. Knock-in of the lacZ gene revealed a possible novel cell lineage originating within the cardiac crescent and contributing to the early cardiovascular development.4. Knock-in of EGFP enabled us to visualize the STAR-positive cells in situ in mice, which is useful for the analysis of the dynamics of STAR-positive cells (e.g. smooth muscle cells) in embryonic vascular formation and in the physiological and pathophysiological processes.5. We have identified Calpain-6 as a target molecule of the ET-1/STAR signaling pathway in craniofacial development. We have discovered the novel function of Calpain-6 in the stabilization of microtubules and actin organization involved in cellular morphology and motility. The studies on the developmental role of Calpain-6 are starting with its knockout mice established recently.These achievements contribute to the understanding of the mechanisms of the cardiovascular and craniofacial development and pave a way to the development of experimental systems applicable to the studies on (patho)physiology involving the ET system.