Abstract 800: Zebrafish BMPR2 is Required for Normal Vasculogenesis and Vessel Patterning during Angiogenesis in vivo
Mutations in the BMP type 2 receptor (BMPR2) cause pulmonary hypertension, but the disease mechanisms remain incompletely understood. To define the in vivo effects of perturbed BMPR2 signaling, we studied zebrafish (zf) embryos in which the BMPR2 gene had been knocked down with antisense morpholino oligos (MO). ZfBMPR2 was cloned and MOs targeting splice-junctions were designed. MO injected embryos were morphologically staged and cardiovascular phenotyping was performed. Phospho (p)Smad-1/5/8 signaling was assessed using an in vivo id1 promoter/reporter system: a plasmid with the id1 promoter controlling firefly luciferase (luc) was coinjected with a control plasmid (HSV TK promoter directing renilla luc) enabling measurement of pSmad-responsive luc activity in embryo lysates. Zf express a single 3.6 kbp BMPR2 cDNA predicted to encode a polypeptide with 56% identity and 62% similarity to human BMPR2. In situ hybridization with an antisense BMPR2 riboprobe showed widespread expression throughout development. MO-mediated BMPR2 knockdown in flk1::GFP transgenic embryos (with endothelial gfp) revealed consistent regional vascular patterning defects. In MO-treated embryos, impaired vasculogenesis results in proximal aorto-venous fusion, premature transit of arterial to venous circulation, and a single distal tubular vascular chamber in the tail. In contrast to controls where angiogenesis occurs in a regular, ordered fashion, MO treated embryos showed mispatterned, disorganized intersomitic vessels. In situ immunohistochemical analyses with anti pSmad-1/5/8 demonstrates nuclear staining in control zebrafish embryos, which was increased in intensity in MO treated embryos. This was confirmed by id1:luc analysis which showed a significant increase (3- and 2.5-fold increase at 24 and 48 h post fertilization respectively) in pSmad-1/5/8 signaling in MO treated embryos (p < 0.001, both time points). Together these data demonstrate that BMPR2 is required for normal vessel development and patterning during vasculogenesis and angiogenesis, and suggest that these effects may be mediated through pSmad-1/5/8 signaling. The fundamental mechanisms of BMPR2-related cardiovascular disease are approachable in vivo using this tractable zf model.