Figure 5

(A–C) In situ hybridization at 24 and 30 hpf showing reduced expression of lyve1 (A) and nr2f2 (B) following roscovitine treatment. ephb4a expression remains unchanged (C). (D and E) Confocal images of 28-hpf Tg(fli1:EGFP;prox1a:kalt4:UAS:uncTagRFP) embryos showing reduced numbers of prox1+ cells (light-blue arrowheads) in the PCV upon roscovitine treatment. Co-localization channel is shown in yellow; quantified in (F). Data show mean ± SEM (unpaired t test). (G–L) Selected images from a time-lapse series depicting the dynamics of PCV sprouting in the presence of roscovitine. Two ECs (colored in red and blue) leave the PCV as part of a single sprout (G) that generates two vessel types. The leading EC (red) connects to an arterial ISV to generate a vISV (I and J); the following cell (blue) divides (J) and gives rise to 2 daughter cells (K and L, yellow arrowheads), with one joining a vISV and the second one incorporating into the nascent PAC. (M) Schematic model depicting cellular events inducing cell-cycle-arrest-induced sprouting. Dorsal cells of the PCV sense high levels of Vegfc (purple) secreted from the hypocord and the DA. Activation of Vegfr3/Flt4-ERK signaling in these cells leads to cell-cycle-arrest-induced sprouting.