The epithelial phenotypes we observed in the Villin-Cre;Ihhflox/flox
mice are overall consistent with other mouse models that disrupt Hh signaling during gut morphogenesis, such as mice overexpressing the Hh inhibitor Hhip, or mice with a conditional deletion of Ptch1.12, 13
However, none of the previous studies addressed the critical question of whether paracrine Hh signaling affects ISC self-renewal or whether the epithelial phenotypes are due to the disruption of trans-amplifying/progenitor cells near the crypt base. Our study showed that in Villin-Cre;Ihhflox/flox
mice there is a clear expansion of the ISC compartment, providing solid evidence that Ihh regulates ISCs during gut development. Furthermore, our findings indicate that Ihh is the key Hh ligand mediating the observed epithelial phenotypes in the small intestine and colon after birth. Additionally, our studies with conditional epithelial Smo
knockout mice demonstrated that Ihh regulates ISC fates strictly in a paracrine fashion. This statement is corroborated by the fact that there is a profound and consistent disruption of the mesenchymal compartment, especially at the crypt base surrounding ISCs in Villin-Cre;Ihhflox/flox
mice. Altogether these data suggest that ablation of Ihh
leads to the significant deterioration of the microenvironment surrounding ISCs, which in turn leads to the expansion of ISCs and altered epithelial cell differentiation programs. In addition, previous studies have suggested that the mesenchymal cells surrounding ISCs are the major source of Wnt and function primarily to maintain ISC proliferation. Here, we propose a different role of the ISC microenvironment, which is to restrain crypt size and prevent abnormal stimulation. Thus, a delicate balance between the proliferative and restrictive activity by mesenchymal cells surrouding ISCs likely exists to refine the shape, size, and function of the gut epithelium to form proper crypt-villus structures.
Pericryptal myofibroblasts are generally considered the key mesenchymal cells that regulate ISCs. On the other hand, cells within the muscularis mucosae layer, despite its vicinity to the ISCs, are not known to be involved in regulating ISCs. In our current study, we find the development of muscularis mucosae cells is strictly dependent on epithelial Ihh signaling, as deletion of Ihh leads to total ablation of the muscularis mucosae in both the colon and small intestine. Our studies suggest that loss of the muscularis mucosae layer may contribute to ISC expansion and deregulation of intestinal epithelial cell differentiation. These findings provide a basis for further investigation of the role of the muscularis mucosae in ISC regulation. For example, in vitro co-culture of muscularis mucosae cells with Lgr5+ ISCs will provide additional evidence of how the muscularis mucosae modulates ISC self-renewal and expansion. In addition, using genomic approaches, one could identify secretory factors produced by the muscularis mucosae. The functions of these secretory factors in regulating ISCs should then be further explored using either knockout mouse models or through in vitro analysis of Lgr5+ ISCs.
What are the molecular mechanisms behind the expansion of the ISC compartment upon Ihh loss during gut morphogenesis? Our genomic analyses indicate that Ihh likely regulates ISC self-renewal and cell fate determination via multiple mechanisms. The reduction of BMP signaling upon Ihh deletion could constitute one possible mechanism that leads to the described phenotype, as BMP signaling normally acts to inhibit ISC self-renewal and repress crypt formation in the gut.7, 26
Nevertheless, the reduced BMP signaling does not account for all the phenotypes observed in Villin-Cre;Ihhflox/flox
mice. For example, in villin-noggin
mice in which BMP signaling is completely abrogated, no morphological alternations are detected till 4-weeks of age.26
Other factors that likely contribute to the severe phenotypes seen in mutant mice are the complete loss of muscularis mucosae cells and the disruption of the ECM that surrounds ISCs. One can imagine muscularis mucosae cells likely provide solid structural support for ISCs at the crypt base. It is also likely that muscularis mucosae cells secrete additional factors that maintain proper ISC number and crypt structure such as the Wnt antagonist Sfrp2
, a down-regulated gene in Villin-Cre;Ihhflox/flox
mice identified by our microarray analysis. Additionally, our microarray analysis showed a profound loss of ECM gene expression in Villin-Cre;Ihhflox/flox
mice at the RNA level. The ECM components were further impaired by the up-regulation of MMPs, the major enzymes that degrade ECM proteins. Altogether, these mesenchymal compartment changes provide a pro-growth microenvironment for ISCs, promoting ISC expansion and subsequent expansion of the transit-amplifying compartment.
These findings imply a very interesting possibility that Ihh deletion, resulting in the loss of structural ECM integrity, the loss of the muscularis mucosae, and expansion of ISCs may predispose to neoplastic transformation. This is in contrast to many other organs where abnormal activation of the Hh pathway promotes tumor development.27, 28
Indeed, the role of the Hh pathway in colorectal cancer remains controversial with numerous conflicting reports.18, 24, 29-31
Clearly, the precise role of Hh signaling in colorectal tumorigenesis needs to be further clarified.
In a recent study, Zacharias et al
reported that chronic Hh inhibition in adult mice resulted in villus atrophy and profound inflammatory responses in the intestine, resembling human celiac disease.22
These data suggest that Hh signaling acts as an important anti-inflammatory factor in the gut. In our study, we also noted the presence of acute inflammation in the intestine of Villin-Cre;Ihhflox/flox
mice, but mostly in a patchy manner and centered around regions lined by vacuolated cells at the crypt-villus junction in the small intestine and crypt-surface in the colon. We speculate that in the absence of generalized inflammation, the acute inflammatory changes in Villin-Cre;Ihhflox/flox
mice most likely results from a weakened mucosal barrier caused by defective enterocyte differentiation. It is possible that the inflammatory changes may also be aggravated by proinflammatory responses released by stromal cells in the absence of Ihh signaling. Our microarray analysis also demonstrated an increased acute inflammatory response in Villin-Cre;Ihhflox/flox
mice. For example, surface markers expressed by neutrophils and/or macrophages such as CD11β/ITGAM, CD14 and CD177, and proinflammatory cytokines or chemokines, such as IL1β, CCL6, CCL9, CCL25 and CXCL5 were all up-regulated in Villin-Cre;Ihhflox/flox
mice (Supplemental Table 1
). Many of these genes overlap with the genes identified by Zacharias et al
. However, we detect no resemblance of the inflammatory process in Villin-Cre;Ihhflox/flox
mice to human chronic inflammatory bowel diseases. Specifically, lymphocytic infiltration which is characteristically seen in celiac disease or Crohn's disease, and significant cryptitis or crypt abscess formation which is typically seen in ulcerative colitis are all absent in Villin-Cre;Ihhflox/flox
mice. Altogether, the analysis suggests that Hh signaling may play distinct roles in regulating inflammatory responses during gut development versus homeostasis.