The role of endothelin-1/endothelin-A signaling in pharyngeal arch development and teratogenicity

Endothelin-1 (ET-1) activation of its endothelin-A (ET_A) receptor is an evolutionarily conserved pathway that is critical to development of the pharyngeal (or branchial) arches. In the absence of ET-1/ET_A signaling, embryos develop particularly severe defects of the lower jaw, a first arch derivative. The studies presented here were designed to elucidate how the ET-1/ET_A pathway influences mandibular development in the mouse embryo. In these experiments, endothelin receptor antagonists were used to transiently disrupt receptor activity in utero or in embryo culture. Cultured CD-1 mouse embryos were exposed to BQ-123, an ET_A receptor selective antagonist, resulting in dysmorphology of the mandibular processes of the first arch similar to that observed in ET-1 or ET_A knockout mice. Experiments carried out in vitro established that embryos were more sensitive to antagonism on gestation day (gd) 8 than on gd 9. Pregnant CD-1 mice were dosed with the orally available endothelin mixed receptor antagonist L-754,142 on gd 8 based on in vitro sensitive window data. Fetuses from exposed dams appeared to have an inverted, duplicate maxilla in place of the mandible, and analysis of morphology at various stages of development indicated that the mirror-image duplicate maxilla developed in parallel with the normal upper jaw. While this finding was not inconsistent with observations in both ET-1 and ET_A null mice, the concept of the mandible being transformed to a maxilla was not previously discussed. Analysis of pathogenesis in embryos treated in vitro with BQ-123 revealed increases in cell death in the proximal first and second arches at 24 hours in culture, a localized decrease in cell proliferation in the proximal mandibular process at 48 hours, but no effects on migration or distribution of neural crest cells. From these results, we conclude that not only is ET-1/ET_A signaling required for mandibular development, but it is also involved in specifying mandibular phenotype during early (gd 8) arch patterning. Furthermore, the pathogenesis that follows this mispatterning of the arch includes increases in cell death and localized decreases in proliferation.