Kempf H, Linares C, Corvol P, Gasc J M
Collège de France, INSERM U36, 75005 Paris, France.
Development. 1998 Dec;125(24):4931-41. doi: 10.1242/dev.125.24.4931.
In the present study, we have applied an antagonist treatment to the chick embryo in ovo in order to demonstrate and dissect the essential roles of the endothelin type A (ETA) receptor in the embryonic development. We have cloned, sequenced and expressed the cDNA of the chick ETA receptor and shown that its affinity for endothelin antagonists is very similar to that shown by its mammalian counterparts. We have studied the spatio-temporal expression pattern of this receptor by in situ hybridization and shown that there is a high level of its mRNA within the mesenchyme of the branchial arches at E3-E5, in keeping with the direct effect of endothelin-1 (ET-1) on the fate of this region of the embryo. Unlike the endothelin type B (ETB) receptor mRNA, ETA mRNA is not expressed in neural crest cells during emigration from the neural tube, but is detected in neural crest-derived ectomesenchyme of the branchial arches. Finally, the functional involvement of this receptor in craniofacial and cardiovascular organogenesis was assessed by selectively inactivating the ETA receptor with specific antagonists applied during the time period corresponding to the expression of the ETA receptor and colonisation of the branchial arches. Embryos treated by these antagonists show a severe reduction and dysmorphogenesis of the hypobranchial skeleton, as well as heart and aortic arch derivative defects. This phenotype is very similar to that obtained in mice by gene inactivations of ET-1 and ETA. These results are observed with ETA antagonists but not with an ETB antagonist, and are dependent on the dose of the antagonists used and on the time of application to the embryo. Altogether, these data strongly show that the ET-1/ETA pathway, in chicken as in mammals, is a major factor involved directly and functionally in morphogenesis of the face and heart. This experimental model of pharmacological inactivation of a gene product described in this study offers a simple and rapid alternative to gene inactivation in mouse. This strategy can be applied to other ligand-receptor systems and extended to compounds of various chemical and functional natures.
在本研究中，我们对鸡胚进行了卵内拮抗剂处理，以证明和剖析A型内皮素（ETA）受体在胚胎发育中的重要作用。我们克隆、测序并表达了鸡ETA受体的cDNA，结果表明其对内皮素拮抗剂的亲和力与哺乳动物对应物非常相似。我们通过原位杂交研究了该受体的时空表达模式，结果显示在E3-E5期鳃弓间充质中有高水平的mRNA，这与内皮素-1（ET-1）对胚胎该区域命运的直接影响一致。与B型内皮素（ETB）受体mRNA不同，ETA mRNA在神经嵴细胞从神经管迁出期间不表达，但在鳃弓神经嵴衍生的外胚间充质中可检测到。最后，通过在与ETA受体表达及鳃弓定植相对应的时间段应用特异性拮抗剂选择性地使ETA受体失活，评估了该受体在颅面和心血管器官发生中的功能参与情况。用这些拮抗剂处理的胚胎显示鳃下骨骼严重减少和畸形发生，以及心脏和主动脉弓衍生物缺陷。该表型与通过ET-1和ETA基因失活在小鼠中获得的表型非常相似。这些结果在用ETA拮抗剂处理时出现，而用ETB拮抗剂处理时未出现，并且取决于所用拮抗剂的剂量和应用于胚胎的时间。总之，这些数据有力地表明，在鸡和哺乳动物中，ET-1/ETA途径是直接和功能性参与面部和心脏形态发生的主要因素。本研究中描述的这种基因产物药理学失活的实验模型为小鼠基因失活提供了一种简单快速的替代方法。该策略可应用于其他配体-受体系统，并扩展到各种化学和功能性质的化合物。
