Figure 7. TB4 restores appropriate vasculogenic gene and protein expression in the Hand1-null yolk sac.

Real-time qRT–PCR on E8.5 yolk sacs demonstrated the mis-expression of vascular genes in Hand1 homozygous mutant yolk sacs (−/−) compared with heterozygous yolk sacs (+/−) and their restoration after treatment with synthetic TB4. Elevated levels of Vegf, Flt1 and Pecam expression in Hand1-null yolk sacs were returned to control levels after TB4 treatment (a). Differential expression of Pecam was confirmed at the protein level by western blotting with α-PECAM on lysates from E8.5 Hand1-heterozygous and Hand1-null yolk sacs treated with either vehicle (−TB4) or TB4 (+TB4): TB4 treatment restored elevated PECAM levels in Hand1 mutants to control levels as determined in heterozygous littermates (b). Genes implicated in vasculogenic endothelial cell proliferation, migration and differentiation (Tgfβ1, TgfβrII, Smad5, Endoglin and Smad2) were also rescued to the control levels observed in Hand1 heterozygous yolk sacs (c). Elevated expression of Notch1, 2 and 4 in mutants was also reduced to control levels after administration of TB4, to restore appropriate VEGF signalling and vascular patterning (d). Error bars are s.e.m., where n = mean of three separate qRT–PCR experiments on three pooled yolk sacs carried out in triplicate. **P<0.01; ***P<0.001 (Student's t-test).