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Fig. 7. Smad2 phosphorylation and TGFß1 protein expression in
tie-1-Cre/TßRIIfl/fl and
tie-1-Cre/ALK5fl/fl double transgenic mutant yolk
sacs. PSmad2 was analysed by immunohistochemistry in sectioned whole mounts of
both untreated (A-C) and TGFß1-treated (D-F) yolk sacs. Phosphorylation
of Smad2 was observed in wild-type yolk sacs (A,D). In the yolk sacs of
tie-1-Cre/TßRIIfl/fl (B) and
tie-1-Cre/ALK5fl/fl (C) mice, PSmad2 was not
present in the mesothelial cell layer (green arrow) but phosphorylation of
Smad2 was restored after TGFß1 treatment (E,F, green arrows) for 1 hour.
To determine the expression of functional Cre in vivo, lacZ
expression was analysed in double transgenic yolk sacs. ß-Gal staining
was observed in the endothelial cells of
tie-1-Cre/TßRIIfl/fl (B and E, red
arrows) and tie-1-Cre/ALK5fl/fl (not shown).
TGFß1 protein expression was analysed by immunohistochemistry on
whole-mount staining of yolk sacs from (G) wild-type, (H)
tie-1-Cre/TßRIIfl/fl mutant embryos
and (I) tie-1-Cre/ALK5fl/fl mutant embryos. In
wild-type yolk sacs, normal expression of TGFß1 was observed in the
endothelial (black arrow) and mesothelial (green arrow) cell layers, while in
both tie-1-Cre/TßRIIfl/fl and
tie-1-Cre/ALK5fl/fl mutant yolk sacs, TGFß1
was no longer detected in the mesothelial cell layer (green arrows). Scale
bars: 0.5 mm. Abbreviations: EC, endothelial cell layer; end, endoderm; mes,
mesothelial cell layer.
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