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Fig. S1. Allocation of secretory lineages in intestinal epithelium is unaffected by Hh-inhibition. (A,B) Alcian Blue stain for sulfated mucins in goblet cells (blue). (C,D) Immunostaining for the general enteroendocrine marker chromogranin A (dark brown). (E,F) Immunostaining for the specific enteroendocrine marker CCK (red). No obvious changes were observed in any of these secretory lineages among villin-Hhip transgenic mice (B,D,F), compared with wild-type littermates (A,C,E). Perinatal lethality of villin-Hhip mice precluded analysis of Paneth cells, which appear several days after birth. Scale bars: 50 µm.
Fig. S2. Bmp signals, which are partly dependent on Hh signals, are not crucial for establishing villus polarity. (A) Q-RT-PCR assay for Bmp4, Bmp2, Bmp7 mRNA levels in P0, P3 and P5 jejunum from villin-Hhip founders, represented relative to respective wild-type levels (set at 100%). Asterisks indicate founders on C57BL/6 background. (B) RT-PCR for Bmp4, Bmp2, Bmp7 in E18.5 small intestine (S.I.) and separated epithelial and mesenchymal components reveals mesenchymal-restricted expression of Bmp4, epithelial-restricted expression of Bmp7, and expression of Bmp2 in both tissues. HPRT levels appear uniform throughout. (C) Recombinant mouse Shh N-terminal polypeptide (Shh-N) induces expression of Bmp4, but not Bmp2, in cultured E18.5 small intestine mesenchyme at 0.1, 0.5 and 2.5 µg/ml Shh-N after 48-hour stimulation. Fold change is relative to 0 µg/ml stimulation, set equal to one. (D) Shh-N induces expression of Bmp4 in E18.5 small intestine mesenchyme after 12, 36 and 48 hours of stimulation, whereas treatment with vehicle has little effect. Fold change is relative to time zero (T0), set equal to one. (E,F) Immunohistochemistry staining reveals Noggin protein (brown) in p0 jejunum in a villin-Noggin transgenic founder (E), whereas wild-type littermates exhibit negligible staining (F). Ki67 immunohistochemistry indicates that the location and levels of intervillus proliferation are normal in intestine from villin-Noggin founders (not shown).
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