The intestinal epithelium is constantly and rapidly renewing throughout the lifespan of vertebrates, thereby representing a major target for tumorigenesis. This epithelium can be divided into two functionally distinct compartments. The crypt of Lieberkühn constitutes the proliferative compartment and contains stem/progenitor cells, as well as, in the small intestine, terminally differentiated Paneth cells. Multipotent stem cells, located near the bottom of crypts, generate new cells, which migrate upwards while differentiating into enterocytes, goblet, and enteroendocrine cells. Proliferation stops at the crypt–villus junction, and terminally differentiated cells are located on the neighboring villus, which constitute the differentiated compartment. In the small intestine, a fourth cell type, the Paneth cell, migrates downward and settles at the bottom of the crypts as postmitotic, differentiated cells. The balance among proliferation, differentiation, migration, and cell death must be tightly regulated to maintain homeostasis of this epithelium.
We reported the expression of Sox9, an HMG-box transcription factor, specifically, in the rapidly proliferating stem/progenitor cells found at the bottom third of Lieberkühn crypts throughout the length of the intestine and in the Paneth cells of the small intestine, as well as in human tumors of the intestinal epithelium (Blache et al., 2004
). Sox9 was first identified as a key regulator of cartilage and male gonad development. Heterozygous Sox9
mutations are responsible for the campomelic dysplasia syndrome, a skeletal dysmorphology syndrome characterized by skeletal malformation of endochondral bones and by male-to-female sex reversal in the majority of genotypically XY individuals (Foster et al., 1994
; Wagner et al., 1994
). Sox9 has also been implicated in the development of cranial neural crest derivatives (Spokony et al., 2002
), in the neural stem cell switch from neurogenesis to gliogenesis (Stolt et al., 2003
) and in heart (Akiyama et al., 2004a
), hair (Vidal et al., 2005
), and pancreas (Seymour et al., 2007
) development. In each of these tissues, Sox9 expression is restricted to specific cell types, suggesting a complex transcriptional regulation. In addition, the currently identified Sox9 target genes, for instance, in the cartilage and in the gonad, display tissue-specific expression (Ng et al., 1997
; de Santa Barbara et al., 1998
), indicating that Sox9 may regulate distinct sets of genes in the different tissues in which it is expressed.
In the intestinal epithelium, the function of Sox9 remains unresolved, although in vitro studies suggested a role in the control of cell differentiation (Blache et al., 2004
). In vitro and in vivo data indicate that Sox9
is a transcriptional target of Wnt signaling. For instance, Sox9 expression is abrogated in Tcf4-null embryos, and it is strongly expressed in colorectal carcinoma cell lines containing activating mutations in components of the Wnt pathway (Blache et al., 2004
The Wnt pathway plays a central role among the extracellular signals required to maintain the homeostasis of the intestinal epithelium. In particular, deletion of the gene encoding Tcf4, another HMG-box transcription factor (Korinek et al., 1998
), or overexpression of the inhibitor Dickkopf (Pinto et al., 2003
; Kuhnert et al., 2004
), resulted in a loss of the proliferative compartment and in impaired differentiation of secretory cell lineages. Conversely, mutation of the gene encoding Apc, a negative regulator of the pathway, resulted in crypt expansion, abrogation of cell migration, and amplification of the Paneth cell population (Sansom et al., 2004
; Andreu et al., 2005
). In addition, deletion of the Wnt receptor Frizzled-5 revealed an essential role of the Wnt–Frizzled-5 pathway in the maturation of Paneth cells (van Es et al., 2005
). The sorting process of epithelial cells along the crypt–villus axis also depends on the Wnt pathway, via a modulation of Ephrin–Eph receptor interactions (Batlle et al., 2002
). The Wnt signaling pathway can thus induce diverse cellular responses in the intestinal epithelium. In addition to these physiological functions, the Wnt pathway is centrally implicated in cancer, as mutations in components of this pathway have been identified in the majority of human colorectal carcinoma (Morin et al., 1997
). Such mutations mimic activation of the pathway by Wnt ligands (i.e., stabilization of β-catenin) and result in constitutive transcriptional activity of the β-catenin–Tcf4 complex and in aberrant expression of its target genes (Korinek et al., 1997
). Despite the central importance of this pathway in the physiopathology of the intestinal epithelium, little is known about the molecular mechanisms involved in restricting this wide spectrum of potential functions to elicit a specific and adequate response from Wnt-stimulated cells.
The fact that Sox9
is transcriptionally regulated by the β-catenin–Tcf4 complex (Blache et al., 2004
), together with the particular expression of Sox9 in the compartment of the intestinal epithelium that contains Wnt-stimulated cells, suggests distinct functions in proliferating stem/progenitor cells and in the postmitotic Paneth cells (Fig. S1 A, available at http://www.jcb.org/cgi/content/full/jcb.200704152/DC1
). To address the different aspects of Sox9 function during the turnover of the intestinal epithelium, including its possible role in specifying the cell response to Wnt signals, we specifically inactivated the corresponding gene in the intestinal epithelium.