Fig. S3

PGE2 affects the expression of HSC and vascular markers by increasing β-catenin levels
Embryos were heat shocked at 5 somites and then exposed to DMSO, 10μM dmPGE2, or 10μM indomethacin as indicated from 10 somites until 36 hpf.
(A – E) Quantitative PCR was performed on cDNA obtained from whole embryo extracts. The primers are listed in Supplementary Table 1. An asterisk (*) indicates a statistically significant difference compared to the wild-type control, ANOVA, p<0.001, n≥5 sets of 50 embryos/condition; data shown as mean ± SD.
(A and B) wnt and PGE2 pathway modulation affected the expression of HSC markers runx1 and cmyb.
(C and D) wnt and PGE2 pathway modification altered the expression of the vascular marker flk1 and arterial marker ephrin B2.
(E) The wnt target cyclin D1 was also affected by the interaction of wnt and PGE2 signaling.
(F - J) wnt8 induction and dmPGE2 treatment synergistic regulated runx1/cmyb+ HSCs; exposure of wild-type embryos to dmPGE2 enhanced HSC number (G), as did induction of wnt8 (H). Combinatorial treatment (I) lead to increased runx1/cmyb staining in 25/41 embryos. (J) In 5/41 embryos, runx1/cmyb expression was reduced compared to wildtype after combined treatment with wnt8 and dmPGE2, indicating a potential negative effect of overactivation of the wnt pathway on HSC formation.
(K) Western blot analysis of total β-catenin in zebrafish embryo homogenates (12 embryos/lane) at 36 hpf. Induction of wnt8 as well as dmPGE2 treatment increased β- catenin, while indomethacin decreased levels in both wild-type and wnt8 embryos.
(L) qPCR for β-catenin revealed a lack of transcriptional upregulation following wnt8 induction or dmPGE2 treatment (ANOVA, p=0.937, n=6 sets of 50 embryos/condition).