The endothelin (ET) system consists of three isopeptides (ET-1, 2, and 3) sharing two distinct G protein-coupled receptors (ET-A and ET-B) with different affinities and is thought to participate in various biological events including the regulation of cardiovascular homeostasis. To clarify the physiological and pathological role of ET-1, we disrupted the murine ET-1 gene as reported previously (Kurihara Y, et al. Nature 368: 703-710, 1994) . ET-1 knockout mice are characterized by morphological abnormalities in pharyngeal arch-derived tissues and organs. In addition, ET-1 knockout mice demonstrate cardiovascular defects including aortic arch interruption or hypoplasia and ventricular septal defect with outflow malalignment, which seem to be due to maldevelopment of pharyngeal arch arteries and outflow tract. These abnormalities quite resemble the outcome of neural crest ablation experiments and a human disease, CATCH22/DiGeorge complex, which is regarded as a neural crest disease. This analogy and anatomical analysis of ET-1 knockout mice lead us to hypothesize that ET-1 is an essential factor in the development of cranial and cardiac neural crest cell lineages. To further investigate the role of ET-1 in embryonic development, we examined ET-1 gene expression in embryos by in situ hybridization. At an earlier embryonic stage, formation of pharyngeal arch arteries and endocardial cushion is disturbed in ET-1 knockout homozygotes. Correspondingly, in situ hybridization of embryos demonstrated that ET-1 is expressed in the endothelium of the arch arteries and outflow tract of the heart as well as in the epithelium of the pharyngeal arches, whereas ET-A and ET-B receptors are expressed in the neural crest derivatives. Recent observation by Yanagisawa et al. that ET-A receptor knockout mice demonstrate the same phenotype as ET-1 knockout mice suggests the importance of the ET-1-ET-A receptor axis in cranial/cardiac neural crest development. To determine the molecular mechanism how ET-1 is involved in neural crest development, we examined the gene expression of several nuclear factors in ET-1 knockout mice by whole mount in situ hybridization. In 9-11 days post coitum wild type mouse embryos, cranial/cardiac neural crest cell-derived ectomesenchymal cells highly expressed dHAND and eHAND, novel bHLH genes involved in neural crest and heart development. The expression of dHAND and eHAND was diminished in the pharyngeal arches and was relatively low in the great vessels of ET-1 knockout mice. The dHAND expression in the limb bud was not different between wild type and ET-1 knockout mice, suggesting that the suppression of HAND gene expression is specific for the cardiovascular and pharyngeal region. These results suggest that ET-1 may act as a mediator of epithelial/mesenchymal interaction and regulate the expression of the HAND genes in cranial/cardiac neural crest-derived cell lineages, leading to normal craniofacial and cardiovascular morphogenesis.

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