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Fig. 6. TGFß1 mRNA and protein expression in wild-type and Eng mutant yolk sacs. Expression of Tgfb1 mRNA was examined by whole-mount in situ hybridization in wild type (not shown) and (A) Eng mutant yolk sacs, and detected specifically in endothelial cells. (B) Higher magnification of A, showing Tgfb1-positive ECs (red arrow). TGFß1 protein was detected by immunohistochemistry following whole-mount staining and sectioning of yolk sacs from (C) wild-type and (D) Eng mutant embryos. In the wild-type yolk sac, TGFß1 was present in the endoderm as well as in both endothelial and mesothelial cell layers (C,D). (E,F) Higher magnification of the yolk sac layers. Positive endothelial cells are indicated by black arrows and positive mesothelial cells are indicated by green arrows. (E) In the Eng null yolk sac, TGFß1 was present in the endoderm but was almost absent in the endothelial and mesothelial cell layers. A TGFß1-positive endothelial cell is indicated by black arrow and a TGFß1-positive mesothelial cell is indicated by a green arrow. (F) Higher magnification of the yolk sac. TGFß1-negative endothelial cell is indicated by black arrow, TGFß1-negative mesothelial cell is indicated by green arrow and TGFß1-positive mesothelial cell is indicated by red arrow. (G) Real-time RT-PCR analysis of Tgfb1 mRNA expression in wild-type and Eng mutant yolk sacs. Expression of Tgfb1 mRNA compared with ß-actin (loading control) in wild-type and Eng mutant yolk sacs. The number of yolk sacs analysed is shown (n). Samples were corrected for loading difference using ß-actin primers. 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 stripe (*P>0.5). (H) The decreased expression of TGFß1 in Eng–/– yolk sacs was confirmed by Western blot analysis. *Unspecific band. Scale bars: 0.5 mm. Abbreviations: EC, endothelial cell layer; end, endoderm; mes, mesothelial cell layer.





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