Fig. 4. SMAD2 phosphorylation and SMA in yolk sacs from wild-type and ALK5^–/– embryos. (A) PSMAD2 (PS2) was detected by immunohistochemistry in both the endothelial (EC) and mesothelial (mes) cell layer of wild-type (WT) yolk sacs. (B,C) In the Tie1-Cre-ALK5^fl/fl yolk sac, PSMAD2 was not detected in either of the cell layers (B), but phosphorylation of SMAD2 was observed in yolk sacs from ALK5^–/– embryos (C). (D) Smooth muscle actin expression in wild-type yolk sacs. (E,F) In the Tie1-Cre-ALK5^–/– yolk sac, SMA was not detected (E), but it was observed in ALK5^–/– yolk sacs (F). (G,H) Real-time reverse-transcriptase (RT)-PCR analysis of ALK4 RNA expression in wild-type, ALK5^–/– (G) and Tie1-Cre-ALK5^–/– (H) yolk sacs. Expression of ALK4 RNA was compared to β-actin (as loading control) in wild-type and mutant yolk sacs. The number of yolk sacs analyzed 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 a black horizontal line (^*P<0.05, ^**P<0.2). EC, endothelial cell layer; end, endoderm; mes, mesothelial cell layer. Scale bars, 0.5 mm.