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Fig. 5
Bmp10/Alk1 lies downstream of blood flow in regulation of pSmad1/5/9, cxcr4a and edn1. (A) BRE:luciferase activity in C2C12 cells transfected with human (h) ALK1, zebrafish (z) alk1, hALK1 kinase dead mutant (R411Q), hALK3 (which does not bind BMP10) or lipofectamine (lipo), and treated with rhBMP10. Data are normalized to pTK-Renilla luciferase activity. Values are mean±s.e.m., n=3 biological replicates, each representing 3 technical replicates. (B) pSmad1/5/9 (middle column) in endothelial cells (nuclei marked by fli1a:negfp transgene, left column) of the CaDI (caudal division of the internal carotid artery) of 36 hpf tnnt2a morphants in which endothelial cell alk1 expression [Tg(fli1a:alk1-myc)], BMP10 availability (rhBMP10; 2 nl of 10 μM rhBMP10 injected into base of CaDI at 28 hpf), or both, are restored. In merge (right column), EGFP-expressing endothelial cell nuclei are green, pSmad1/5/9 immunofluorescence is magenta. Yellow arrows indicate endothelial cells in the injected CaDI. 2D confocal projections of 50 μm frontal sections, dorsal upwards. Scale bar: 10 μm. See supplementary material Table S1 for fluorescence quantitation. (C) Whole-mount in situ hybridization for cxcr4a, edn1 and cdh5 (pan-endothelial control) in 36 hpf tnnt2a morphant;Tg(fli1a:alk1-myc) embryos injected at 28 hpf into the base of the left CaDI with 2 nl tracer with or without 10 μM rhBMP10. Yellow arrow indicates injected CaDI, red arrow indicates uninjected CaDI. Asterisk indicates injection site. Frontal views, dorsal upwards; lateral views, right (uninjected) side or left (injected) side. Scale bar: 50 μm. (D) Quantitation of CaDI endothelial cell number in 36 hpf Tg(fli1a:alk1-myc) embryos left uninjected (white bars) or injected at the one-cell stage with tnnt2a morpholino (gray, black bars). At 28 hpf, tnnt2a morphants were injected in the right CaDI with 2 nl tracer (gray bars) or 2 nl 10μM rhBMP10 (black bars). tnnt2a morphants injected intravascularly with tracer exhibited increased endothelial cell number throughout the CaDI/BCA, whereas intravascular injection of rhBMP10 normalized endothelial cell number in the injected CaDI and BCA (basal communicating artery). n=10-12 per group in two independent experiments. Values are mean±s.e.m. Student’s t-test: *P<0.05; **P<0.001; ns, not significant.