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Fig. 1. Smad2 and Smad3 are independently regulated and co-expressed in the early embryo. (A-D) Western blot analysis of adult organ and cell lines. (A) Thymus extracts from homozygous Smad3^null animals (–/–) lack detectable Smad3 protein. (B) Equivalent Smad2 protein levels are found in wild-type, Smad3^+/– and Smad3^–/– spleen, thymus and liver extracts. (C) CCE ES cells, STO fibroblasts and spleen express Smad2 protein, whereas KT15 Smad2^Robm1 homozygous ES cell lines contain no Smad2. (D) Similar Smad3 levels are observed in thymus, CCE and KT15 Smad2-deficient ES cells, and STO fibroblasts. (E) Semi-quantitative RT-PCR analysis of Smad3, Smad2, Smad4 and hypoxanthine phosphoribosyltransferase (Hprt) expression in blastocysts (E3.5) and gastrulation stage embryos (E6.5 and 7.5). Smad2, Smad3 and Smad4 are co-expressed at all stages examined. (F) Quantitative analysis of Smad2 and Smad3 expression levels by ribonculease protection assay of CCE ES cell, embryo and adult thymus total RNA. Smad2 transcripts are approximately twofold more abundant than Smad3 transcripts in ES cells and E7.5 embryos. Smad3 levels equalize with Smad2 as development progresses, and by E10.5/11.5 the ratio of Smad3:Smad2 transcripts is nearly 1:1. (G-I) Smad3 whole-mount in situ hybridization. (G) Mouse embryos at mid- to late primitive streak stages show low levels of Smad3 expression throughout the embryo. The highest level of expression is seen in the extra-embryonic ectoderm of the posterior amniotic fold (paf) and its later derivative the chorion (ch). The visceral yolk sac endoderm (ve) is negative for Smad3. (H,I) Smad3 expression levels increase within the embryo proper by the early somite stage (E8.0-8.5), and are observed in the midline, node (n) and somites (s).

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