Fig. 5

Notch signalling mediates early primary ISV specification. (A) Quantification of ECs upward speed (in microns/day) in ISVs of wild-type [n=12 aISV (67 cells), 13 vISV (103 cells)], NICD^OE(n=30 aISV, 29 NICD^OE cells) and MO-dll4 (n=9 vISV, 85 cells) embryos (32 to 54?hpf) at three different time points: (I) 2.5?h before secondary sprout connection; (II) during three-way connection; and (III) 2.5?h after resolution of the three-way connection. (B,C) Notch activity reporter Tg[tp1-MmHbb:kaede]^um15;Tg[kdr-l:ras-Cherry]^s916 imaged at 52?hpf (B) and at 6 dpf (C) in the same embryo, after conversion of the Kaede photoconvertible fluorescent protein at 29?hpf (Notch activity reporter shown in green, all ECs labelled in red). Red arrowheads indicate ISVs expressing high levels of the Kaede^green protein, both at 52?hpf and at 6?dpf. (D) Quantification of the ratio of arteries and veins, determined at 6 dpf, correlated to the Notch activity status at 52?hpf, after conversion of the Kaede photoconvertible fluorescent protein at 29?hpf [tp1 positive, negative or high (as indicated by red arrowheads in B)] (n=20 embryos; *P<0.0001, two-way ANOVA). (E) Quantification of the percentage of tp1-positive (tp1+) and tp1-negative (tp1?) ISVs in untreated (n=20 embryos) versus 2×tricaine-treated (tric 2×) embryos (n=12 embryos) at 52?hpf, after conversion of the Kaede photoconvertible fluorescent protein at 29?hpf. Flow inhibition between 29 and 52?hpf does not affect tp1 promoter activity during this period. (F) Quantification of the ratio of arteries and veins, determined at 6 dpf, correlated to the Notch activity status at 52?hpf (tp1 positive or tp1 negative), in untreated (n=20 embryos) versus 2×tricaine-treated embryos (n=12 embryos). Flow inhibition does not affect the balance of arteries and veins formed from tp1-positive ISVs, but tp1-negative ISVs form significantly more arteries after tricaine treatment (*P=0.027, two-way ANOVA). (G) Ipsilateral neighbourhood analysis of Notch activity status of vessels with two neighbours in 52?hpf embryos after conversion of the photoconvertible Kaede protein at 29?hpf. Graph shows the frequency of finding a tp1-positive or -negative ISV, given the Notch activity status of the neighbouring ISVs [tp1 positive (+) or tp1 negative (?)] (n=19 embryos, 199 ISVs) (*P<0.0001, one-way ANOVA). (H) Quantification of the ratio of arterial ISVs with activated Notch signalling first detectable (as indicated by tp1 signal) before connection of the secondary sprout (i.e. during phase I) or after connection of the secondary sprout (i.e. during phase II) (n=22 ISVs). Data are mean±s.e.m. NS, not significant. Scale bars: 100??m.