Fig. 5. Effect of TGFß1 in smooth muscle actin expression in Eng mutant yolk sacs. Caldesmon and -smooth muscle actin was detected by immunohistochemistry in sectioned yolk sacs, stained as whole mounts. (A) Caldesmon was expressed in mesothelial cells of wild-type yolk sacs but was not detected in Eng mutant yolk sacs (B). (C) In wild-type embryos, -sma was observed in cells surrounding the vessels while in (D) Eng knockout yolk sac its expression was absent. (E) -sma expression was partially restored after 3 hours of TGFß treatment (red arrowheads). In Eng mutant yolk sacs -sma was absent (F) while after treatment with high concentrations of TGFß1 (5 ng/ml) for 8 hours its expression was clearly restored (G, red arrowheads). (H) Real-time RT-PCR analysis of -sma RNA expression in wild-type and Eng mutant yolk sacs. Expression of -sma mRNA was compared with ß-actin (as loading control) in wild-type and Eng mutant yolk sac. E9.5 yolk sac endoderm was used as a negative control. The number of yolk sacs analysed is shown (n). Contamination with genomic DNA was not evident in samples without RT (not shown). The Wilcoxon Test was used for statistical analysis of significance; medians are shown as black horizontal bar (^*P<0.1; ^**P<0.05). Scale bar: 0.5 mm. Abbreviations: EC, endothelial cell layer; end, endoderm; mes, mesothelial cell layer.