Fig. 4

Fig. 4

Exogenous E2 Signaling Disrupts VEGF/NOTCH Cascades in the AGM

(A) E2 exposure had no impact on deltaC expression by WISH at 18 hpf but strongly reduced arterial expression at 24 and 36 hpf (n > 30/treatment); an arrowhead indicates artery.

(B) Expression of notch3 was similarly regulated by E2 at 18, 24, and 36 hpf (n > 30/treatment); an arrowhead indicates artery.

(C) qPCR confirmed E2-mediated reductions in deltaC and notch3 expression at 36 hpf (mean of triplicate experiments ± SEM; one-tailed t test deltaC, notch3^∗p < 0.05).

(D) Decreased Notch:GFP was observed in the artery (white bracket indicates artery walls) after E2 treatment from 12–36 or 12–23 hpf; no change occurred with exposure from 26–36 hpf (n ≥ 52).

(E) NICD induction increased expression of runx1/cmyb and rescued HSPCs in E2-treated embryos (n ≥ 31/treatment).

(F) Expression of VEGFAa was strongly decreased in zebrafish following E2 treatment at both 18 and 24 hpf (n ≥ 25/treatment); an arrow points to somite staining.

(G) qPCR confirmed that expression of VEGFAa was decreased at 24 hpf (mean of triplicate experiments ± SEM; one-tailed t test ^∗∗∗p < 0.001).

(H) Induction of VEGFAa increased runx1 expression and rescued the effect of E2 exposure; treatment with DAPT decreased expression of runx1 and blocked the VEGF-mediated rescue of HSPCs (n ≥ 20/treatment); an arrowhead indicates artery.