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Fig. 7 Additive function of actin polymerization and hydrostatic pressure in driving endothelial cell (EC) migration and sprouting angiogenesis. (A–D) Representative maximum intensity projection confocal z-stacks of 28 hpf wildtype (A, B) and aqp1a.1rk28/rk28;aqp8a.1rk29/rk29 (C, D) embryos treated with 0.1% DMSO (A, C) or 0.8 µg/ml Lat. B (B, D) from 20 hpf to 28 hpf (for each condition: wildtype, n = 10 embryos; aqp1a.1rk28/rk28;aqp8a.1rk29/rk29, n = 12 embryos, two independent experiments). Scale bar, 20 µm. (E) Intersegmental vessel (ISV) length in 28 hpf wildtype and aqp1a.1rk28/rk28;aqp8a.1rk29/rk29 embryos treated with 0.1% DMSO or 0.8 µg/ml Lat. B (wildtype: n = 60 vessels from 10 control embryos, n = 66 vessels from 10 Lat. B-treated embryos; aqp1a.1rk28;aqp8a.1rk29: n = 126 vessels from 12 control embryos, n = 112 vessels from 11 Lat. B-treated embryos, two independent experiments). Statistical significance was determined by Brown–Forsythe ANOVA test with Sidak’s multiple-comparisons test; ns, p>0.05, **p<0.01, and ****p<0.0001. (F) Model of endothelial tip cell migration. Tip cells generate membrane protrusions by actin polymerization and increased hydrostatic pressure via Aquaporin-mediated water inflow. In the absence of actin polymerization and presence of Aquaporins, hydrostatic pressure deforms membranes to generate membrane protrusions. When Aquaporin function is lost, membrane protrusions is generated by actin polymerization. Fewer membrane protrusions are formed when only one mechanism is utilized, resulting in slower EC migration.