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

Abstract

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, TGFb2 is involved in RA-regulated cardiovascular development. In VAD embryos TGFb2 mRNA and protein expression are greatly elevated. The expression of TGFb receptor II is also elevated in VAD embryos but is normalized by treatment with TGFb2-specific antisense oligonucleotides (AS). Administration of this AS or an antibody specific for TGFb2 to VAD embryos normalizes posterior heart development and vascularization, while the administration of exogenous active TGFb2 protein to normal quail embryos mimics the excessive TGFb2 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 TGFb2 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 TGFb2 signals during vertebrate heart formation.

Retinoids and Cardiac Development

“…More recently, N-cadherin and TGFβ2 expression have been shown to be upregulated in VAD quail embryos [60,61]. Studies conducted by blocking the function of those over-expressed molecules or in the case of TGFβ2, treatment of normal embryos with the growth factor, have led to the conclusion that their deregulated activities contribute to the cardiac phenotype of the VAD quail embryo [60,61]. Interestingly, a recent transcriptomic analysis aiming to identify genes deregulated in the early Raldh2 -/-mutant embryos (4 somite stage)…”

“…Furthermore, addition of exogenous Bmp2 can function additively with forced expression of Gata4 to overcome the loss of retinoid signaling, indicating the presence of an active RA-BMP-GATA network for posterior heart tube development. More recently, N-cadherin and TGFβ2 expression have been shown to be upregulated in VAD quail embryos [60,61]. Studies conducted by blocking the function of those over-expressed molecules or in the case of TGFβ2, treatment of normal embryos with the growth factor, have led to the conclusion that their deregulated activities contribute to the cardiac phenotype of the VAD quail embryo [60,61].…”

Vitamin A–Not for Your Eyes Only: Requirement for Heart Formation Begins Early in Embryogenesis

“…It is important to note that severe cardiac malformations are observed in an estimated 10% of early miscarriages [73]; some of them may be linked to disturbances in vitamin A function during the early critical time of heart formation; c) . The identification of a cluster of developmental genes that are regulated by RA during early heart formation; they include the cardiac transcription factor GATA4 [68], the heart asymmetry genes Nodal, Snail and Pitx2 [66], the global growth factor Bmp2 [81], the adhesion molecule N-cadherin [82] and the global growth regulator TGFβ2 [83]; and d) . The discovery that all retinoid receptors are expressed in the early avian embryo (67, 84-86), but that during the critical RA-requiring developmental window RARα2, RARγ and RXRα are the active transcription factors transducing the RA signal to target genes [84,85].…”

“…We have identified the following cluster of molecules that are regulated by RA during IFT formation: a) The retinoid receptors, which are also transcription factors, are expressed in the heart forming areas of the early quail embryo [84], but in the VAD embryos the expression of the RA receptor RARα2 is severely decreased in these regions [84]; blocking the expression of this receptor in normal embryos interferes specifically with the formation of IFTs [85] providing evidence for a specific role of RARα2 in regulating heart IFT morphogenesis; b) The cardiogenic transcription factor GATA4 may be a contributor to RA-regulated IFT formation, as normally GATA4 transcripts are particularly high in the posterior heart region [94], but in the VAD quail embryos they are severely diminished at these sites [68]; and c) .The overexpression of N-cadherin [82] and TGFβ2 [83] observed in VAD quail embryos, is linked to the abnormal heart IFT formation; this is discussed in the following sections. …”

“…Our most recent studies have focused on TGFβ2 as a major regulatory target of RA during early embryonic development [83]. It is well known that TGFβs and RA share global growth regulatory characteristics, e.g., modulation of cell proliferation, apoptosis and differentiation [4,6].…”