Fig. 3

Fig. 3

β-catenin dependent Wnt signaling positively regulates the number of ECs, while negatively regulating the number of erythrocytes. (A) Schematic representation of the experiment. (B) EC volume and cell numbers were analyzed by GFP expression in Tg(kdrl:GFP)^s843 embryos. (C) Erythrocyte volume and cell numbers were analyzed by GFP expression in Tg(gata1:GFP)^la781 embryos. Confocal images with the insets representing the overview, and the larger images displaying the specific volume used for quantification. Nuclei were visualized using TOPRO-3 (white). Inhibition of Wnt signaling by IWR treatment led to a reduction in EC volume (B, n = 10) and EC cell numbers (B, n = 19), and to an increase in erythrocyte volume (C, n = 6) and erythrocyte cell numbers (C, n = 22) compared to DMSO treated control embryos (volumes: B, n = 11; C, n = 8; cell numbers: B, n = 30; C, n = 25). Wnt activation (BIO) increased the EC volume (B, n = 8) and the EC number (B, n = 10), while decreasing the erythrocyte volume (C, n = 8) and the erythrocyte number (C, n = 36) compared to DMSO treated control embryos. Values represent mean±SD. *p<0.05, **p<0.01, ***p<0.001; Student's t-test. (D,E) Alternative analysis of the EC population by GFP expression of Tg(etv2:GFP)^ci1 (D) and of the erythrocyte cell population using whole mount in situ hybridization with an antisense tal1 probe (E). As observed by kdrl or gata1 expression (B,C), Wnt inhibitor treatment (IWR-1) resulted in reduced EC volume, but in an expanded erythrocyte cell population. Treatment with Wnt activator (BIO) increased the EC volume and decreased the erythrocytes population, respectively. Note, that for EC volume analysis of Tg(etv2:GFP)^ci1 the GFP staining in the muscle tissue was cropped and not included in the analysis. (F) Analysis of the EC volume using Tg(kdrl:GFP)^s843 and erythrocyte volume using Tg(gata1:GFP)^la781 in embryos treated with either Wnt inhibitor (IWR-1), proliferation inhibitor (AHU) or both (AHU+IWR-1). Inhibition of proliferation by AHU decreased the volume of kdrl:GFP and gata1:GFP compared to untreated control embryos, but had no effect on the ratio of both populations (indicated as gata1:GFP/kdrl:GFP). Hence, Wnt signaling specifically affects cell fate specification of ECs and erythrocytes and does not act via asymmetric cell division. (F, n ≥ 8), Values represent mean±SEM. *p<0.05, **p<0.01, ***p<0.001; Student's t-test.