This study has established that Math1 is sufficient to induce the program of secretory cell development in the mouse intestine. Transgenic expression of Math1 resulted in almost complete transformation of the intestine into a secretory epithelium. The loss of enterocytes and expansion of the secretory cell lineage suggested that Math1 regulates the cell fate choice of a bipotential progenitor. The importance of Math1 for intestinal lineage determination had previously been suggested from the loss of secretory cell types in Math1-deficient mice (Shroyer et al., 2007
; Yang et al., 2001
). Since Math1 is expressed in mature secretory cells as well as in cells in the progenitor zone (Pinto et al., 2003
; Yang et al., 2001
), the question remained whether its primary role was maintenance of the differentiated secretory cell phenotype or induction of differentiation. Our study showed that Math1 expression can override the normal developmental program to induce secretory cell differentiation, suggesting that a primary role of this transcription factor is determination of cell fate choice. Interestingly, expression of Math1 stimulated apparently normal terminal differentiation processes since we observed expression of differentiation markers specific for each secretory cell type, including Muc2 (goblet cells), hormones (enteroendocrine cells), and cryptdins (Paneth cells). Accordingly, the expression of transcription factors downstream of Math1 that are known to be important for specific secretory cell types were increased robustly in Vil-Math1 transgenic mice, including Neurog3 () (Jenny et al., 2002
; Lopez-Diaz et al., 2007
) and Gfi1 (data not shown) (Shroyer et al., 2005
Notch signaling has been shown to play a primary role in the regulation of cell lineage determination in the intestinal epithelium. Our study and others point to Notch regulation of Math1 transcription as the critical determinant of cell fate. Treatment with γ-secretase inhibitors to block Notch signaling has been shown to induce intestinal phenotypes similar to those evident in the Vil-Math1 transgenic mice (Milano et al., 2004
; Searfoss et al., 2003
; van Es et al., 2005
). Similarly, genetic models with disrupted Notch signaling exhibited a generalized secretory cell expansion together with loss of enterocytes (Crosnier et al., 2005
; Jensen et al., 2000
; Riccio et al., 2008
; van Es et al., 2005
). Math1 expression was increased in these models of Notch disruption, consistent with our finding that Math1 expression controls absorptive versus secretory cell fate. Accordingly, genetic models with increased Notch signaling exhibited decreased Math1 and loss of intestinal secretory cell types (Fre et al., 2005
; Stanger et al., 2005
). To determine if increased Math1 mRNA had an effect on the Notch signaling pathway in the Vil-Math1 transgenics, we analyzed expression of the Notch target gene Hes1. No change in Hes1 mRNA levels were observed in Vil-Math1 transgenic founders (Supplementary Fig. 5
), suggesting that the epithelial changes that we observed in our studies were due to increased levels of Math1 and not due to inhibition of Notch signaling. Together these studies and our data point to Math1 as the key effector regulating the choice of secretory versus absorptive cell fate choice in the intestine.
Transgenic Math1 expression affected intestinal proliferation in complex ways. We observed an overall reduction in epithelial cell proliferation, which suggested that Math1 stimulated differentiation and withdrawal from the cell cycle. Loss of Notch signaling in the intestine, and thus increased Math1, had previously been shown to convert proliferating crypt progenitors into post-mitotic cells with derepression of the cyclin-dependent kinase inhibitors p27Kip1
(Riccio et al., 2008
). Increased cell-cycle inhibitor expression was proposed to result from loss of Hes1 repressor action on the promoters of these genes (Riccio et al., 2008
). However, our observations suggest that increased Math1 may also contribute to the Notch affect on proliferation. Although Vil-Math1 transgenics exhibited decreased epithelial cell proliferation, intestinal Hes1 mRNA expression was not changed, suggesting that the reduced proliferation and increased differentiation of progenitors was likely due to changes in Math1 and not Hes1. Finally, Math1 was observed to affect the expression of intestinal stem cell markers, with decreased expression of Lgr5 and increased expression of Prom1, consistent with Math1 promoting the differentiation of Lgr5-positive crypt base columnar stem cells to Prom1-positive committed progenitor cells.
In addition to the profound changes to the epithelium, there were marked changes to the mesenchyme in Vil-Math1 transgenic mice, with a general cellular expansion and increased proliferation observed in both submucosa and villus cores. Since Math1 expression in our transgenic model was limited to epithelial cells, the mesenchymal changes were indirect, suggesting that the epithelial cell changes altered signaling to control the differentiation of the mesenchyme. The epithelial cell changes were multifaceted, thus signaling changes could result from changes to the intervillus progenitor zone, the loss of enterocytes, and/or expansion of secretory cell types. It is likely that multiple signaling pathways were affected by these cellular changes, including Wnt and Hedgehog.
In conclusion we have demonstrated that Math1 is a key transcriptional determinant of cell fate choice in the intestine. Proper Math1 levels are required for the normal patterning of epithelial cell proliferation as well as the distribution of secretory versus absorptive cells. The associated expansion of secretory cell types and loss of enterocytes observed when Math1 levels were increased suggests that this transcription factor directs the cell fate choice of a bipotential progenitor. Furthermore, the epithelial cell changes induced by increasing Math1 had consequences for the mesenchyme, presumably due to alterations in epithelial-mesenchymal crosstalk. Although radial patterning was essentially preserved, the mesenchyme of Vil-Math1 transgenic mice was grossly expanded, with increased proliferation and differentiation of several mesenchymal cell types. Thus, Math1 expression is critical for coordinated differentiation and morphogenesis of both epithelium and mesenchyme.