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Fig. 5 TGF-β signaling acts downstream of Gdf11 promoting pronephros and cloaca formation. (A) Volcano plot highlighting differentially expressed genes in WT and gdf11−/− pronephros. (B) Gene Ontology analysis of biological processes using down-regulated differentially expressed genes. (C) Western blot showing key proteins of the TGF-β/Smad signaling pathway in WT and gdf11−/− mutant pronephros/cloaca lysates. (D) Comparative expression of key TGF-β/Smad pathway proteins following Gdf11 overexpression or SRI-011381 treatment in HEK 293T cells. (E) Subcellular localization of Smad2 in Flag-Gdf11-infected cells with or without SRI-011381 treatment. Scale bar: 50 μm. (F) Partial rescue of pronephros developmental defects in gdf11 mutants by SRI-011381 treatment. (G) Partial rescue of cloaca developmental defects in gdf11 mutants by SRI-011381 treatment. (H) Partial rescue of cilia in pronephric duct in gdf11 mutants by SRI-011381 treatment. Scale bar: 50 μm.