Transforming growth factor β2 is negatively regulated by endogenous retinoic acid during early heart morphogenesis

Vitamin A-deficient (VAD) quail embryos lack the vitamin A-active form, retinoic acid (RA) and are characterized by a phenotype that includes a grossly abnormal cardiovascular system that can be rescued by RA. Here we report that the transforming growth factor, TGF beta 2 is involved in RA-regulated cardiovascular development. In VAD embryos TGF beta 2 mRNA and protein expression are greatly elevated. The expression of TGF beta receptor II is also elevated in VAD embryos but is normalized by treatment with TGF beta 2-specific antisense oligonucleotides (AS). Administration of this AS or an antibody specific for TGF beta 2 to VAD embryos normalizes posterior heart development and vascularization, while the administration of exogenous active TGF beta 2 protein to normal quail embryos mimics the excessive TGF beta 2 status of VAD embryos and induces VAD cardiovascular phenotype. In VAD embryos pSmad2/3 and pErk1 are not activated, while pErk2 and pcRaf are elevated and pSmad1/5/8 is diminished. We conclude that in the early avian embryo TGF beta 2 has a major role in the retinoic acid-regulated posterior heart morphogenesis for which it does not use Smad2/3 pathways, but may use other signaling pathways. Importantly, we conclude that retinoic acid is a critical negative physiological regulator of the magnitude of TGF beta 2 signals during vertebrate heart formation.