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PNAS 109 (39): 15817-15822

Copyright © 2012 by the National Academy of Sciences.

Hedgehog-responsive mesenchymal clusters direct patterning and emergence of intestinal villi

Katherine D. Waltona,1, Åsa Kolteruda,b,1, Michael J. Czerwinskia, Michael J. Bella,c, Ajay Prakasha, Juhi Kushwahaa, Ann S. Grossea, Santiago Schnellc, and Deborah L. Gumucioa,2

aDepartment of Cell and Developmental Biology, University of Michigan Medical School, Ann Arbor, MI 48109; bDepartment of Biosciences and Nutrition, Karolinska Instituet, Novum, SE-141 83 Huddinge, Sweden; and cDepartment of Molecular and Integrative Physiology, Department of Computational Medicine and Bioinformatics, and Brehm Center for Diabetes Research, University of Michigan Medical School, Ann Arbor, MI 48105

Figure 01
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Fig. 1.. Mesenchymal clusters are responsive to Hh and PDGF/PDGFRα signaling and are intimately associated with the epithelium. (A–H) X-Gal (blue) and eosin (pink) staining of Ptc1LacZ/+ (A–D) and Gli1LacZ/+ (E–H) intestines. Red arrows indicate mesenchymal clusters. (I) TEM demonstrating association of the cluster with the epithelial basement membrane. (J) Magnification of the boxed region in I. (K and L) Cluster cells express PDGFRα (anti-EGFP, green), and Gli1 or Ptc1 (anti-β-gal, red) in PDGFRαEGFP/+;Gli1LacZ/+ (K) or PDGFRαEGFP/+;Ptc1LacZ/+ (L) intestines. (Scale bars, 50 μm, unless indicated otherwise.)


Figure 02
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Fig. 2.. Cluster formation in PDGFRαEGFP/+ intestines occurs in an anterior-to-posterior and dorsal-to-ventral wave. (A) Average distance of the cluster wavefront compared with average total small intestine length at E14.5, E15.5, and E16.5. (B) Boxed area in D, showing the wavefront of cluster formation in the proximal ileum (arrow) at E15.5. (C) Cluster formation extends throughout the small intestine by E16.5 (magnified from Fig. S1). (D) Three-dimensional reconstruction of five 1-μm z-slices from a tiling array of two-photon images of an E15.5 intestine. The figure is a composite to form the full figure. (E) At the wave front, clusters predominate on the dorsal side (marked by omentum, arrows). (F) Average cluster size (mean ± SD) of the largest 500 clusters per intestinal region at E15.5 (P < 0.0001 for all comparisons). (G) Average length (mean ± SD) of the longest villi (top 5%) in each region at E15.5 to E18.5. Average longest villus lengths are significantly different among regions at each stage and among regions between stages (P < 0.001 for all comparisons); n = 7,020.


Figure 03
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Fig. 3.. Multiple rounds of villus emergence occur during fetal development. (A) Section of an E16.5 intestine (H&E), showing alternating villus lengths. (B) Boxed area in A. (C) Longitudinal section of an E15.5 intestine (H&E) with alternating villus lengths. (D) Average number of villi (mean ± SD) per section in duodenum, jejunum, and ileum at E15.5 to E18.5. (Scale bars, 50 μm.)


Figure 04
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Fig. 4.. Hh signaling controls cluster formation and villus size. (A–J) E13.5 PDGFRαEGFP/+ intestines, cultured for 2 d with vehicle (A), 10 μM tomatidine (B, E, and H), 5 μM cyclopamine (C, F, and I), or 1 μM SAG (D, G, and J). (A–D) Five-micrometer longitudinal confocal sections of whole intestines. (E–G) Sections of intestines with anti–E-cadherin (green) outlining epithelium and DAPI (blue). (H–J) Sections with anti-GFP showing PDGFRα+ cluster cells (green) and DAPI (blue). (K–N) E14.5 intestines, cultured for 2 d with the same treatments as in A–D, respectively. (O–R) Vibratome sections of jejunum (O and P) or ileum (Q and R) of E14 PtcLacZ/+ intestines cultured with tomatidine (O and Q) or cyclopamine (P and R) for 2 d. (S) SAG treated intestines have significantly larger clusters (green) than control intestines (red, blue). (*P < 0.0001.) Average cluster sizes were no treatment: 1,601 μm2 ± 155.6 (SEM), n = 110; tomatidine: 1,416 μm2 ± 143.9 (SEM), n = 139; SAG: 2,220 μm2 ± 136.5 (SEM), n = 143.


Figure 05
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Fig. 5.. Mesenchymal clusters of Hh-responsive cells form by aggregation. (A–F) Sections of 2-h BrdU-labeled E15.0 (A–C) or E15.5 (D–F) duodenum stained with anti-BrdU (green), anti–E-cadherin (red), and DAPI (blue). White circles outline clusters. (C and F) Magnifications of boxed areas in B and E, respectively. Note the postmitotic cluster (asterisk in B). (G) BrdU labeling indices (mean ± SEM) at different stages in clusters (gray) vs. unclustered mesenchyme (white). (H and I) Anti-Arl13b marks primary cilia in mesenchymal clusters (green) and DAPI marks nuclei (blue). (Scale bars, 50 μm.)


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