Depletion of the Colonic Epithelial Precursor Cell Compartment Upon Conditional Activation of the Hedgehog Pathway

doi:10.1053/j.gastro.2009.02.068

Gastroenterology

Volume 136, Issue 7, June 2009, Pages 2195-2203.e7

https://doi.org/10.1053/j.gastro.2009.02.068 Get rights and content

Background & Aims

The intestinal epithelium is a homeostatic system in which differentiated cells are in dynamic equilibrium with rapidly cycling precursor cells. Wnt signaling regulates intestinal epithelial precursor cell fate and proliferation. Homeostatic systems exist by virtue of negative feedback loops, and we have previously identified the Hedgehog (Hh) pathway as a potential negative feedback signal in the colonic epithelium. Indian hedgehog (Ihh) is produced by the differentiated enterocytes and negatively regulates Wnt signaling in intestinal precursor cells. We studied the role of members of the Hh signaling family in the intestine using a conditional genetic approach.

Methods

We inactivated the Hh receptor Patched1 (Ptch1) in adult mice, resulting in constitutive activation of the Hh signaling pathway. Effects on colonic mucosal homeostasis were examined. Colon tissues were examined by immunohistochemistry, in situ hybridization, transmission electron microscopy, and real-time polymerase chain reaction.

Results

Ihh but not Sonic hedgehog (Shh) was expressed in colonic epithelium. Expression of Ptch1 and Gli1 was restricted to the mesenchyme. Constitutive activation of Hh signaling resulted in accumulation of myofibroblasts and colonic crypt hypoplasia. A reduction in the number of epithelial precursor cells was observed with premature development into the enterocyte lineage and inhibition of Wnt signaling. Activation of Hh signaling resulted in induction of the expression of bone morphogenetic proteins (Bmp) and increased Bmp signaling in the epithelium.

Conclusions

Hh signaling acts in a negative feedback loop from differentiated cells via the mesenchyme to the colonic epithelial precursor cell compartment in the adult mouse.

Section snippets

Mice

We used previously generated Ptch1^flox/flox11 and Rosa26CreERT2¹² mouse lines. The Ptch1^flox/flox mice had a loxP site inserted into intron 7 and intron 9 of the Ptch1 gene. The Rosa26CreERT2 mutant mouse strain expressed a fusion gene encoding Cre recombinase and a modified ligand-binding domain for the estrogen receptor under control of the endogenous Rosa26 promoter. The 2 mouse lines were crossed to obtain Ptch1^flox/floxCreERT2^+/− mice. Eight-week-old mice were injected intraperitoneally

Conditional Deletion of Ptch1 Exons 8 and 9 Activates Hh Targets in the Mesenchyme

Because conflicting data have been reported on the colonic expression of Ihh and Shh, possibly due to problems with antibody specificity, we reexamined their expression using a combination of immunohistochemistry and in situ hybridization. We found that Ihh messenger RNA (mRNA) was expressed by the differentiating epithelial cells in the midcrypt region (Figure 1A,left). In contrast to our previous in situ hybridizations of the human colon,⁷ we could not detect Shh mRNA in the mouse colon (

Discussion

Hhs are expressed throughout the adult gastrointestinal tract. Experiments that addressed the role of Hh signaling in the intestine up to now have either addressed its function during development and in the early postnatal period16, 32, 33 or relied on the use of cyclopamine, a pharmacologic Hh signaling inhibitor.² Here we show that Ihh is the main Hh in the colon and signals from the epithelial cells to the mesenchyme. Conditional deletion of Ptch1 results in enhanced Hh signaling in the

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: Conflicts of interest The authors disclose no conflicts.

: Funding Supported by a VENI grant from the Netherlands Organization for Scientific Research and by a grant from the Dutch Digestive Foundation.

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Basic—Alimentary TractDepletion of the Colonic Epithelial Precursor Cell Compartment Upon Conditional Activation of the Hedgehog Pathway

Background & Aims

Methods

Results

Conclusions

Section snippets

Mice

Conditional Deletion of Ptch1 Exons 8 and 9 Activates Hh Targets in the Mesenchyme

Discussion

Lab Invest

Gastroenterology

Cell

Blood

Cell

Cell

Am J Pathol

Dev Biol

Gastroenterology

Gastroenterology

Gastroenterology

Gastroenterology

Gastroenterology

Hedgehog signaling in development and homeostasis of the gastrointestinal tract

Physiol Rev

Indian Hedgehog is an antagonist of Wnt signaling in colonic epithelial cell differentiation

Nat Genet

Expression of Sonic hedgehog pathway genes is altered in colonic neoplasia

J Pathol

Down-regulation of a morphogen (sonic hedgehog) gradient in the gastric epithelium of Helicobacter pylori-infected Mongolian gerbils

J Pathol

Sonic hedgehog expression correlates with fundic gland differentiation in the adult gastrointestinal tract

Gut

Patched2 modulates tumorigenesis in patched1 heterozygous mice

Cancer Res

Distinct roles of PTCH2 splice variants in Hedgehog signalling

Biochem J

Basic—Alimentary Tract
Depletion of the Colonic Epithelial Precursor Cell Compartment Upon Conditional Activation of the Hedgehog Pathway