Fig. 1

BMPR2 is required for CVP sprouting angiogenesis and EC filopodia formation, contributing to the endothelial tip cell phenotype.

a Zebrafish embryo expressing the endothelial Tg(kdrl-GFP) reporter transgene and maximum projection images of the caudal vein plexus (CVP) at 25 h post fertilization (hpf) from control or bmpr2b MO-treated embryos (upper) and control or bmpr2b crispant-treated embryos (lower) (arrows indicate individual tip cells). Scale bar: 50 µm. Right, close-up of regions (I and II) of interest depicted in bmpr2b MO-treated embryos (upper) and close-up of regions (III and IV) of interest depicted in bmpr2b crispant-treated embryos (lower) (arrows indicate individual filopodia at tip cell). b Quantification of the number of protruding cells at the CVP of control or bmpr2b MO-treated zebrafish and control or bmpr2b crispant-treated zebrafish at 25hpf. ***p < 0.001. c Representative images of optical sections showing part of the caudal vein plexus (CVP) in Tg(kdrl:GFP) endothelial reporter embryos. In control embryos, CD34 is present in many cells along the front of the CVP (yellow asterisks) whereas in bmpr2b-crispants most of the cells lack CD34 (indicated by white asterisks). Scale bar: 10 µm. Quantification of CD34⁺/ GFP⁺ cells in comparison to GFP⁺ cells in the CVP from Tg(kdrl:GFP) endothelial reporter embryos. ***p < 0.005. d Scheme describing formation of leader cells by gap closure assay using silicone-inserts. Cells are seeded in a silicone-insert on a glass coverslip. After cell-adherence, the silicone-insert is removed creating a cell-free gap towards which the first rows of cells polarize and migrate. Samples are then fixed and treated for immunofluorescence staining. e Immunofluorescence pictures of control and BMPR2^+/- ECs, Phalloidin (magenta), DAPI (blue) (top). Scanning-Electron Micrographs (SEM) of control and BMPR2^+/– ECs (bottom). Inlets show regions of interest. Respective quantifications of the number of filopodia per 100 µm of cell edge for immunofluorescence (upper) and SEM (lower) analysis are shown on the right. *p < 0.05; ***p < 0.001. Scale bar: 10 µm. f Immunofluorescence staining of HUVECs transfected with FITC-labeled control siRNA (siControl) or a 1:1 mix of FITC-labeled control siRNA and BMPR2-targeting siRNA (siBMPR2). Phalloidin (magenta), DAPI (blue), FITC (green). Inlets show regions of interest. Quantification of the number of filopodia per 100 µm of cell edge is shown below. ***p < 0.001. Scale bar: 20 µm. g Immunofluorescence staining of BMPR2^+/- ECs overexpressing GFP control (upper) or BMPR2-GFP for filopodia rescue (lower). Phalloidin (magenta), DAPI (blue), GFP (green). Insets show regions of interest. Quantification of the number of filopodia per 100 µm of cell edge is shown on the right. ***p < 0.001. Scale bar: 20 µm. h Single particle trajectories of individual BMPR2 molecules. Trajectories were recorded by Total Internal Reflection Fluorescence (TIRF) microscopy. For this, BMPR2-HA was overexpressed in Cos7 cells and labeled with individual Quantum-Dots (Qdot). Trajectories were recorded for 25 s and analyzed via tracking software. Trajectories were overlaid onto F-Actin signal (upon co-transfection of LifeAct-GFP) and color coded relative to their magnitude (immobile BMPR2: Blue; mobile BMPR2: Red). Arrowheads indicate different BMPR2 mobilities across different cellular sub-compartments (left). BMP2 induced confinement of BMPR2 (right): BMPR2 trajectories in Cos7 cells before (upper) and after stimulation with 3 nM BMP2 (lower). Reduction in diffusivity of individual BMPR2 molecules in µm²/sec is shown after stimulation with BMP2.