EC-mural cell interactions promote deposition and maintenance of the vascular basement membrane. (A-C) Mural cells promote vascular basement membrane deposition in vitro. (A) Quantification of basement membrane components collagen IV and fibronectin in 3D collagen matrix assays with ECs alone (black) or EC-pericyte co-cultures (gray) using detergent-free immunostaining protocols to assess only the extracellular deposition of these proteins. Representative images of collagen IV staining in EC only (B) and EC-pericyte co-cultures (C); n>5 individual immunostained collagen plugs. (D-J) vSMCs promote vascular basement membrane deposition and stability in vivo. (D-I) Representative images of collagen IV (D,F,H) or fibronectin (E,G,I) immunostaining of the dorsal aorta in transverse paraffin sections of 5 dpf no heat shock double transgenic Tg(HSP70:gal4; UAS:pdgfrbDN-YFP) animals (D,E), 1-5 dpf heat-shocked control Tg(HSP70:gal4) animals (F,G) or 1-5 dpf heat-shocked pdgfrbDN-expressing double transgenic Tg(HSP70:gal4; UAS:pdgfrbDN-YFP) animals (H,I). (J) Quantification of 5 dpf dorsal aorta collagen IV or fibronectin immunostaining intensity using the relative basement membrane staining intensity of sections such as those shown in D-I. All data are shown as a percentage of the no heat shock control condition. n=10 fish total, combined data from three individual experiments. (K) Quantification of basement membrane protein levels by western blot analysis (n=3 blots, from three individual experiments) of each of the conditions shown in D-J. Data are represented as a percentage of the no heat shock control condition. (L) Representative western blot images are shown versus a tubulin loading control. (M) The ability of mural cells to regulate maintenance of full-length basement membrane components was analyzed by collecting protein lysates under non-reducing conditions. No heat shock control, heat shock control (not carrying the pdgfrbDN cassette) and pdgfrbDN, heat shock treatment lysates were collected for analysis of protein fragmentation levels of collagen IV and laminin proteins. Representative western blots are shown, with increased fragmentation (highlighted by arrows) of the proteins noted only in the pdgfrbDN heat shock condition, where mural cell coverage has been disrupted. Location of the full-length proteins is marked with an asterisk. Representative western blots from lysates of ten pooled zebrafish embryos and two individual experiments. Scale bars: 50 µm. Values are mean±s.e.m.