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Fig. 1. Hh signaling in the neonatal small intestine is paracrine. Hh signals are
attenuated in villin-Hhip mice. (A) In situ hybridization for
Ihh in E18.5 small intestine shows predominant expression in
intervillus epithelium. (B) Immunohistochemical staining (brown) for Shh and
Ihh with anti-Hh antibody reveals Hh protein in lamina propria at the villus
base. (C) X-gal stain of small intestine from a P0
Ptch1+/nLacZ mouse demonstrates that subepithelial
mesenchymal cells and scattered SMCs in the ME closest to the epithelium
(arrow) respond to Hh signals. (D,E) Isolated epithelium (D) and mesenchyme
(E) from E18.5 small intestine. (F) RT-PCR analysis of known epithelial
(villin) and mesenchymal (Madcam1, Actg2) markers demonstrates the
purity of each fraction. Shh and Ihh mRNAs are exclusively
epithelial, while Ptch1, Ptch2, Gli1, Gli2, Gli3, Hhip, and
Bmp4 are mesenchymal. Each RT-PCR was done on three independently
isolated fractions and total small intestine (S.I.). HPRT levels
appear uniform. (G) Immunostain for Hhip (brown) in P0 wild-type small
intestine (WT, top) shows expression in submucosa and muscularis externa of
newborn jejunum. P0 villin-Hhip mice (TG, bottom) exhibit ectopic
expression of Hhip in the epithelium. (H, top) Q-RT-PCR for the
villin-Hhip transcript reveals a 45-fold range of expression of the
transgene in P0-P5 jejunum from eight founders. villin-Hhip mRNA
levels are expressed relative to founder #8, set equal to 1.0. (H, bottom)
Q-RT-PCR shows that Hh-inhibition results in significant downregulation of Hh
target genes, Ptch1 and Gli1, relative to wild-type
littermates (set at 100%). Q-RT-PCR data are normalized to threshold cycle
values for HPRT. Error bars equal s.e.m. for triplicate analysis.
Founders are either on C57BL/6 background (indicated as B6), or hybrid
background (B6 xSJL). Scale bars: 50 µm.
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