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First published online 8 December 2004
doi: 10.1242/dev.01576

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Epithelial hedgehog signals pattern the intestinal crypt-villus axis

Department of Cell and Developmental Biology, University of Michigan Medical School, Ann Arbor, MI 48109-0616, USA

View larger version (114K): [in a new window]   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.

View larger version (93K): [in a new window]   Fig. 2. High-level Hh inhibition interferes with epithelial remodeling and villus formation. (A-D) H-E staining. (A) Wild-type animals exhibit organized and well-developed villi. (B-D) Founders #4, 5, 6 (villin-HhipHi) display regions of flattened epithelium. (E-H) Ki67 staining for proliferative cells (brown nuclei). (E) In wild-type mice, Ki67 positive cells are restricted to the intervillus base. The flattened epithelium of founders #4 (F), #5 (G) and #6 (H) is highly proliferative. Stratified epithelium is also evident in these founders (F, arrows, insets). Scale bars: 50 µm for A-H; 20 µm for F.

View larger version (102K): [in a new window]   Fig. 4. Hh signals are required for proper enterocyte differentiation. (A) The enterocyte marker, iFABP, is expressed only in differentiated villus tip epithelium in P0 wild-type small intestine (brown). (B) In P0 transgenic small intestine, iFABP is missing from broad regions of villus epithelium (red dotted line). (C) Ki67 immunostaining of a section adjacent to the one shown in B reveals that regions lacking iFABP contain proliferating cells (blue dotted line). (D) The brush border kinase, cGKII (red), is expressed at the apical surface of differentiated epithelial cells, as seen in wild-type P0 mice. (E) cGKII expression is severely reduced in villin-Hhip P0 mice. (F) Transmission electron microscopy of villus tip epithelial cells from wild-type P0 small intestine reveals a well-formed brush border. (G) Villin-Hhip mice have a shortened, poorly formed brush border (arrows) and large lipid droplets are seen at the apical end of epithelial cells (asterisk). (H-J) Immunofluorescence staining for actin (green) stains the epithelial brush border and smooth muscle of the small intestine; the neuronal marker peripherin (red) stains enteric neurons. (H) Wild-type P0. (I) In high-expressing founders, neuronal cells are well represented, but muscle layers are obviously reduced (arrows). Brush border actin staining is negligible. (J) Low-expressing founders exhibit normal peripherin staining and normal musculature. A-I are from neonates and J is from P5 mouse. Scale bars: 50 µm.

View larger version (92K): [in a new window]   Fig. 3. Low-level Hh inhibition causes abnormal villus patterning and increased epithelial proliferation (A,B) H-E staining shows that villin-Hhip mice demonstrate broad extensively branched villi. Ectopic precrypt structures are seen on villus tips (A,B, inset). (C,D) Ki67 staining. The epithelium is highly proliferative (brown nuclei). Ectopic crypt-like structures contain proliferating cells (D, inset). (E,F) Wholemount view of villus structures. Wild-type P3 duodenum contains characteristic finger-like villi (E), whereas villin-Hhip P3 founders exhibit extensively branched villus structures (F). Scale bars: 50 µm (20 µm for insets in B and D).

View larger version (111K): [in a new window]   Fig. 5. Tcf4/ß-catenin activity is enhanced in villin-Hhip mice. (A,C,E) Wild-type mice. (B,D,F) villin-Hhip mice. Tcf4/ß-catenin target genes Cdx1 (A), Cd44 (C), and Ephb2 (E) are normally present in the wild-type P0 intervillus epithelium. Cd44 is upregulated in polyps, as seen in this spontaneous lesion in wild-type duodenum (C, inset). In P0 villin-Hhip mice, Cdx1 (B), Cd44 (D), and Ephb2 (F), expression domains are expanded onto villus tip epithelium. Cd44 and Ephb2 expression is also evident in ectopic crypt-like structures in this P3 mouse (D,F, inset, arrows). (G) Q-RT-PCR of P0, P3, P5 jejunum RNA for c-myc mRNA. All founders had significant increases in c-myc expression levels, relative to WT (set equal to 1.0). Error bars represent s.e.m. for triplicate analysis. Overlap of ß-catenin (green) and propidium iodide (magenta) is seen as white. Note that in P0 wild-type mice, ß-catenin is present exclusively at cell-cell junctions in villus tip epithelium (H, arrowheads), while nuclear ß-catenin (white) is restricted to proliferating cells in intervillus regions (I, arrows). In P0 villin-Hhip transgenic mice, increased levels of cytoplasmic and nuclear ß-catenin are observed in villus tip epithelium (J,K, arrows). Scale bars: 50 µm for A-F,I,K; 100 µm for C (inset); 10 µm for H,J.

View larger version (82K): [in a new window]   Fig. 6. ISEMFs express Ptch1 and are responsive to Shh stimulation. (A,B) ISEMF cell lines from rat jejunum (MIC101), ileum (MIC216), colon (MIC316), and human colon (CCD-18Co) respond to 24- or 72-hour treatment with Shh-conditioned medium (CM) from Cos7 cells transfected with pcDNA3.1-Shh (Shh CM) or pcDNA3.1 (control CM). Q-RT-PCR analysis reveals upregulation of Gli target genes Ptch1 (A) and Gli1 (B). Error bars represent s.e.m. of triplicate analysis. (C) X-gal stain marks nuclear LacZ expression from Ptch1 locus in cells beneath intervillus epithelium in P0 Ptch1+/nLacZ small intestine. (D-F) Confocal microscopy of immunofluorescence for -SMA (red) and desmin (green) identifies ISEMFs beneath epithelium. False-color overlay of X-gal stain (blue) reveals that ISEMF cells surrounding precrypts (arrows) express Ptch1. X-gal-positive nuclei are also found in scattered surface SMCs of the muscularis extrema (C,D, arrowhead) and in rare desmin-positive cells in the lamina propria (F, arrowhead). Scale bar: 20 µm.

View larger version (147K): [in a new window]   Fig. 7. Ectopic ISEMFs and expanded SMCs in villus tips of villin-Hhip mice. Immunofluorescence for -SMA (A-I, red), desmin (A,B,D,E,G,H, green), or Ki67 (C,F,I, green). (A-C) Wild-type mice. Myofibroblasts (-SMA positive, desmin negative) are primarily associated with epithelium in intervillus regions (A) and few are found in villus cores (B, red arrow). SMCs in villus tips express desmin and -SMA (A,B, yellow arrow); SMC precursors and pericytes express desmin only (B, green arrow); Epithelial proliferation (C, green) is restricted to intervillus epithelium in WT p3 intestine. (D-I) villin-Hhip mice. ISEMFs are found subjacent to epithelium throughout the villus axis (D,G, red; E,H, red, arrows). The villus tip core (outlined in B,E,H) also contains an increased number of desmin-positive SMCs (D,E,G,H, green). Ectopic epithelial proliferation is often adjacent to ectopic ISEMFs (F, arrows) in P3 (F) or P5 (I) mice. Nuclei are counterstained with DAPI (blue). Scale bars: 50 µm; 20 µm for confocal images (B,E,H).