Secondary sprouts in vash1 morphants exhibit more Prox1+ cells and higher proliferation rates. (A,B) Secondary sprouts in Tg[kdr-l:ras-Cherrys916,fli1a:nEGFPy7] embryos, with membrane- and nuclei-labelled endothelial cells (EC) in control (A) and vash1 KD embryos (B). White arrowheads indicate nuclei in secondary sprouts (outlined by dashed line). (C,D) Quantification of the number of endothelial nEGFP-labelled nuclei in secondary sprouts immediately before connection to the ISV (C), and cell division frequency in the secondary sprout before and after connecting to the ISV, from 30 to 70 hpf (D). Quantifications are from three biological replicates. **P<0.0021, ****P<0.0002 (Mann–Whitney test in C, t-test in D). (E,F) Prox1-positive (Prox1+ve) EC were identified by immunostaining in Tg[fli1a:nEGFPy7] embryos in control and vash1 morphants. In controls, migrating cells in secondary sprouts are Prox1+ve and their neighbouring EC in the PCV are Prox1-negative (E). In vash1 morphants, both EC in the secondary sprout and the neighbouring EC still in the PCV are Prox1+ve (F). Analysed EC are highlighted (dashed line), and neighbouring EC are connected with a line. (G) Quantification of incidence of nEGFP+ Prox1+ve neighbouring EC per seven somites per embryo in both control and vash1 MO-injected embryos. **P<0.0021 (Mann–Whitney test). Pictures are representative of three replicated experiments.
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