Abstract 3940: Paradoxical Augmentation Of BMP-signaling In Vivo Following Loss Of BMPR2 In Zebrafish Is Mediated Through Recruitment Of An Alternative Type 2 Receptor
Defective vasculogenesis and angiogenesis result from knockdown of the type 2 BMP receptor (BMPR2) in developing zebrafish (ZF). The paradoxical increase in BMP-mediated Smad-1/5/8 phosphorylation following BMPR2 knockdown plays a key role in vascular defects in vivo and may represent a central molecular link between mutations in BMPR2 and heritable forms of pulmonary hypertension or vascular disease. In vitro studies (Yu PB et al. J Biol Chem 2005; 280:24443) have demonstrated ligand specific gain of BMP signaling following deletion of BMPR2 through functional substitution with ActRIIa. We therefore explored in ZF whether recruitment of alternative type 2 BMP receptors in vivo explains the signaling defects, and consequently the vasculogenic and angiogenic defects, we have identified following loss of BMPR2. Fertilized ZF oocytes were co-injected with morpholino oligos (MOs) targeting BMPR2 and either ACVR2A/ActRIIa or ACVR2B/ActRIIb. Embryos were collected at 24 hours post fertilization. RNA was harvested for expression analysis and total embryo lysates analyzed by western blot. ZF injected with MO targeting BMPR2 show increased pSMAD-1/5/8 phosphorylation and embryos co-injected with MOs targeting both BMPR2 and ACVR2B did not exhibit significantly different pSMAD-1/5/8 levels. For ZF co-injected with BMPR2 and ACVR2A MO, pSMAD-1/5/8 levels decreased by 45%. Expression studies demonstrate no compensatory changes in type 2 receptors or BMP ligands. These studies demonstrate that the signaling abnormalities in ZF following loss of BMPR2 do not result from compensatory changes in the expression of BMP family members. Instead, we conclude that ACVR2A plays a key role in the gain of BMP-mediated SMAD signaling, based on double MO knockdown experiments showing that Smad-1/5/8 phosphorylation is attenuated only with concurrent knockdown of both BMPR2 and ACVR2A. Translational studies of this model in other species will establish whether orthologous mechanisms underlie mammalian development and human disease.
This research has received full or partial funding support from the American Heart Association, AHA National Center.