Fig. 5

SERCA function controls the rates of mesodermal migration and budding. (A) Wild-type 28?hpf Tg(kdrl:eGFP) zebrafish embryo demonstrates intersomitic vasculature seen by wide field (top, scale bar: 200??m) and higher magnification below (scale bar: 100??m). For A-H, images are inverted for improved clarity. (B-H) Paired confocal images show 3D reconstructions of the 14th-15th and 20th intersomitic vessels from embryos treated with (B) DMSO, (C) 1.25?µM CPA, (D) 5?µM CPA, (E) 10?µM CPA, (F) 20?µM CPA (G) 10?µM CPA washed out after 2?h, and (H) 20?µM CPA washed out after 2?h. Varying CPA dose results in dose-dependent reduction in vascular budding until at 20??M, budding is reversibly suspended. Budding resumes after CPA washout. Scale bar: 50?µm for all images. Red arrows indicate distal-most positioning of nuclei in B and E (see caption for I,J). (I,J) Plots show, for each treatment, the collective number of branches (red lines are medians) on vessels 13-16 which form in the middle of the treatment (I) and the proportion of branches with cell nuclei at tip positions (J): compare the positions of nuclei (arrowed) in B versus E. Error bars in J indicate the s.e.m. In a parallel dose-dependent manner, CPA reduces branch numbers (I) and distal migration of endothelial cells (J) with resumption of branching and migration upon CPA washout. (K-M) Vessel path length (K), linear vessel height (L), and the total number of intersomitic vessels per embryo (M) measured in 3D show similar CPA dose- and time-dependent reductions in branching. Data in K and L are shown for the 20th intersomitic vessel, which forms during the treatment (red lines are medians).