Gastroenterology
Volume 133, Issue 2 , Pages 710-712, August 2007

Intestinal Development: The Many Faces of Wnt Signaling

  • Archana Kapoor
    • ,
  • H. Joyce Li
    • ,
  • Andrew B. Leiter

      Affiliations

    • Corresponding Author InformationAddress requests for reprints to: Andrew Leiter, MD, Division of Gastroenterology/Department of Medicine, University of Massachusetts Medical School, 364 Plantation Street, Worcester, Massachusetts 01605. fax: (508) 856-4770.

Division of Gastroenterology, Department of Medicine, University of Massachusetts Medical School, Worcester, Massachusetts

Article Outline

 

See “Phases of canonical Wnt signaling during the development of mouse intestinal epithelium” by Kim B-M, Mao J, Taketo MM, et al on page 529; and “SOX9 is required for the differentiation of Paneth cells in the intestinal epithelium” by Mori-Akiyama Y, van den Born M, van Es JH, et al on page 539.

The mammalian intestinal epithelium perpetually renews itself every 3–5 days from a large reservoir of pluripotent stem cells in the lower crypt.1 Intestinal stem cells divide and produce an actively proliferating population of transit amplifying cells that give rise to each of the 4 principal epithelial cell types in the small intestine. As immature cells migrate up the crypt–villus axis, they exit the cell cycle in the villus compartment and differentiate into either absorptive enterocytes, goblet cells, or enteroendocrine cells. By contrast, the Paneth cell lineage, which produces antimicrobial peptides, continues to proliferate and migrates downward to the crypt base. The crypt–villus unit is the basic developmental unit of the intestine where mature, terminally differentiated cells in the villi are topographically separated from immature proliferating cells in the crypts, making the small intestine an excellent model for studying development.

Small intestinal epithelial homeostasis, cell proliferation control, and cell lineage differentiation are regulated by a number of signaling pathways conserved throughout metazoan evolution. These pathways include the Wnt (wingless), Notch, hedgehog, and bone morphogenic protein (TGF-β/BMP) pathways. The potential importance of Wnt signaling in intestinal epithelial development was first recognized with the discovery that patients with familial adenomatous polyposis inherited one defective allele for the adenomatous polyposis (APC) gene. Mutations in the second APC allele resulted in abnormal Wnt signaling, leading to eventual development of colorectal cancer. Subsequent work has shown that sporadic colorectal cancers are frequently associated with somatic mutations that result in abnormal Wnt signaling.2

Studies in the fruit fly, Drosophila melanogaster, first revealed the importance of Wnt (Wingless) signaling in normal tissue development, regulating cell fate determination, cell proliferation, and cell polarity. A growing body of studies has confirmed the importance of Wnt signaling in normal mammalian development, especially with respect to the intestine. Two papers published in this issue of Gastroenterology explore the role of Wnt signaling in intestinal cell differentiation and proliferation. Both studies utilize transgenic mouse models that provide new insight into multiple effects of the Wnt pathway in the intestine.

Wnt proteins, encoded by approximately 20 different genes, comprise an evolutionary conserved family of soluble, cysteine-rich glycoproteins3 in mammals.4 Initiation of canonical Wnt signaling occurs when Wnt proteins bind to Frizzled proteins, a family of 7 transmembrane receptors, and a transmembrane coreceptor, LRP5/6. Signals generated in the absence of the coreceptor do not involve β-catenin and are referred to as the noncanonical pathway.5

Although the canonical Wnt pathway is relatively quiescent in mature intestinal epithelial cells, it plays a critical role in both normal development and tumorigenesis. When Wnt signaling is not activated, β-catenin is phosphorylated by glycogen synthase kinase 3 (GSK3β) and casein kinase 1. Phosphorylated β-catenin is then recruited to a complex containing APC, Axin, and other proteins (Figure 1) that are targeted for rapid ubiquitination and proteosomal degradation. Binding of Wnt to its receptor, Frizzled, and LRP coreceptor leads to a signaling cascade involving the activation of Disheveled, which recruits Axin to the plasma membrane to inhibit phosphorylation of β-catenin. Unphosphorylated β-catenin is redistributed throughout the cytoplasm, and translocated into the nucleus. Nuclear β-catenin associates with one of several members of T-cell factor (TCF)/lymphoid enhancer (LEF) binding protein family of transcription factors.6 This family of Wnt transcriptional effectors includes TCF1, TCF3, TCF4, and LEF-1, as well as isoforms arising from alternate promoter use and/or RNA splicing. Nuclear β-catenin serves as a coactivator for TCF/LEF proteins by displacing corepressor proteins and recruiting additional proteins including pygopus and Bcl9-2, resulting in increased transcription and expression of Wnt target genes, including a number of genes expressed in immature proliferating cells. Examples of Wnt target genes associated with cell proliferation include c-Myc, cyclin D, and Sox 9.

  • View full-size image.
  • Figure 1. 

    The canonical Wnt pathway and signaling through β-catenin. In the absence of Wnt ligand, β-catenin is phosphorylated by glycogen synthase kinase-3 (GSK3) and targeted for degradation through destruction complexes containing APC, Axin, and other proteins (left). As a result, Wnt target genes remain repressed. During active signaling, Wnt proteins bind to their receptors and inhibit phosphorylation of β-catenin by GSK3, thus preventing its degradation in APC complexes (right). As a result, stabilized β-catenin is redistributed from the cell membrane to the nucleus, where it interacts with TCF family transcription factors as a co-activator to activate target gene transcription. β-cat, β-catenin; APC, adenomatous polyposis coli; DVL, disheveled.

Active Wnt signaling in the adult small intestine is normally confined to proliferating immature cells and Paneth cells in the lower crypt. A number of genetic models suggest a critical role for the Wnt pathway in maintaining proliferation required to support the high turnover rate of the intestinal mucosa. Mice with an inactivated TCF4 gene, the major intestinal TCF/LEF transcription factor, failed to develop crypts and several epithelial lineages in the intestine. The precursor to crypts, the intervillus zone cells, was devoid of proliferating cells. These observations indicated that TCF4 and canonical Wnt signaling were essential for maintenance of both stem cells and cell proliferation in crypts.7

At about embryonic day 14 in the mouse, the intestine undergoes major changes from a pseudostratified epithelium to a tube lined with a monolayer of columnar cells, which undergo further morphogenesis to form villi. Crypts subsequently develop from cells in the intervillus zone. In this issue of Gastroenterology, Kim et al8 examined the developing mouse intestine to identify cells with active Wnt signaling. Their results demonstrate previously unappreciated differences in the location and components of Wnt signaling in the fetal intestine.

The first surprising observation by Kim et al was that proliferating intervillus cells in the fetal gut showed no evidence of canonical Wnt signaling. Instead, they found that postmitotic cells in developing villi were the major sites of Wnt activity. Unlike the adult intestine, the fetal intestine uses a different Wnt transcriptional effector, TCF3, rather than, TCF4, the major TCF/LEF family member expressed in the adult intestine. As a result, Wnt signals in the developing intestine appear to activate different Wnt target genes. Thus, Wnt signaling may serve a number of different functions in the intestine, depending on the developmental context and the specific pathway components involved. The importance of Wnt signaling for maintenance of cell proliferation during development requires additional study. The function of newly identified Wnt pathway activation in postmitotic cells remains to be elucidated.

The role of Wnt signaling in the specification and differentiation of Paneth cells has not been well established. The identification of nuclear β-catenin in Paneth cells suggests that Wnt signaling persists in this lineage. Activation of the Wnt pathway by conditional APC deletion in intestine resulted in increased numbers of Paneth cells, suggesting that this pathway is associated with Paneth cell development.9 It has been more difficult to obtain direct proof that the Wnt pathway is required for Paneth cell differentiation. Mice lacking TCF4 failed to develop Paneth cells,10 as did transgenic mice expressing the Wnt inhibitor Dkk1 throughout the intestine under control of the villin gene.11 However, in these studies, mice failed to develop crypts and showed developmental defects in other lineages with the loss of Wnt function, raising the possibility that the loss of Paneth cells could have resulted from stem cell depletion rather than a specific requirement for Wnt signals. To address the role of the Wnt pathway in Paneth cell differentiation, van Es et al10 generated mice lacking Frizzled5, which was believed to be the only Wnt receptor expressed in Paneth cells. Frizzled5-null mice developed Paneth cells that were mispositioned to the villi and upper crypt, leading to the conclusion that Wnt signals were required for Paneth cell maturation only, but not for their specification.10 However, another study subsequently showed expression of 2 other Frizzled family genes, Fz6 and Fz7 throughout the crypt base that may compensate for the loss of Fz5.12

The findings reported in this issue of Gastroenterology by Mori-Akiyama et al13 in mice lacking intestinal expression of the transcription factor Sox9 firmly establish that canonical Wnt signaling is needed for Paneth cell specification. Recently, our group, using conditional deletion of β-catenin combined with recombination-based lineage tracing, reached a similar conclusion.14

Sox9 belongs to the Sox family of transcription factors related to gender-determining factor gene SRY and TCF family of transcription factors.15 Sox family proteins contain highly conserved high mobility group domain that recognizes an A/T rich DNA binding motif: (A/T)(A/T)CAA(A/T)G. The roles of Sox proteins in the differentiation are well established for a number of tissues, including neural crest, heart, cartilage, and testes.16 Sox9 is expressed in the lower crypt region of the small intestine, overlapping areas with active Wnt signaling. It is also a direct transcriptional target of β-catenin–TCF4 complexes.17 Mori-Akiyama et al13 report in this issue of Gastroenterology that Sox9 is required for Paneth cell differentiation by conditionally knocking out the Sox9 gene in the intestine. In the absence of Sox9, mice fail to develop Paneth cells or to express early differentiation markers for this lineage. The other secretory lineages, goblet and enteroendocrine cells, were unaffected by the loss of Sox9, indicating that the loss of this Wnt target protein was specific for Paneth cells and did not disrupt stem cells.

A second important finding reported by Mori-Akiyama et al is that loss of Sox9 resulted in modest expansion of the proliferating crypt compartment in contrast to the failure to develop crypts seen with more generalized loss of Wnt function in TCF4-null mice. This important observation suggests that a different subset of Wnt target genes is important for stem cell maintenance and crypt progenitor cell proliferation. Recent work suggests c-Myc, a well-defined target of the Wnt pathway, is the major effector in controlling Wnt-induced cell proliferation.18, 19

Wnt signaling regulates many functions in different tissues. How some functions, but not others, are activated is not well understood. A very large number of genes are capable of participating in the Wnt pathway including >19 different ligands (Wnts), ≥8 different receptors (Frizzled), 2 coreceptors, a number of soluble inhibitors to their receptors and coreceptors, 4 different TCF transcription factors and their alternately spliced isoforms, and several β-catenin binding proteins (Pygopus, BCL9-2).20 Thus, there are a potentially limitless number of combinations of Wnt ligands, receptors, and effectors to determine different subsets of functions through selective activation of some Wnt target genes but not others.

The 2 articles appearing in this issue of Gastroenterology illustrate examples of how the outcomes of Wnt signaling may control a limited number of functions in the intestine that may depend in part on the tissue and developmental context. Mori-Akiyama et al show that one Wnt target gene, Sox9, controls specification of Paneth cells, with relatively little effect on other important Wnt functions, such as stem cell maintenance. Likewise, Kim et al show that the Wnt pathway is activated in distinct cell populations of the developing gut, utilizing a different set of effector transcription factors to activate expression of different Wnt target genes compared to the adult intestine. Both articles exemplify the kinds of new experimental approaches that will lead to a better understanding of roles of Wnt signaling in both normal gastrointestinal tract development and the development of common neoplasms like colorectal cancer.

Back to Article Outline

References 

  1. Stappenbeck TS, Wong MH, Saam JR, et al. Notes from some crypt watchers: regulation of renewal in the mouse intestinal epithelium. Curr Opin Cell Biol. 1998;10:702–709
  2. Bienz M, Clevers H. Linking colorectal cancer to Wnt signaling. Cell. 2000;103:311–320
  3. Wodarz A, Nusse R. Mechanisms of Wnt signaling in development. Annu Rev Cell Dev Biol. 1998;14:59–88
  4. Reya T, Clevers H. Wnt signalling in stem cells and cancer. Nature. 2005;434:843–850
  5. Veeman MT, Axelrod JD, Moon RT. A second canon (Functions and mechanisms of beta-catenin-independent Wnt signaling). Dev Cell. 2003;5:367–377
  6. Waterman ML. Lymphoid enhancer factor/T cell factor expression in colorectal cancer. Cancer Metastasis Rev. 2004;23:41–52
  7. Korinek V, Barker N, Moerer P, et al. Depletion of epithelial stem-cell compartments in the small intestine of mice lacking Tcf-4. Nat Genet. 1998;19:379–383
  8. Kim B-M, Mao J, Taketo MM, et al. Phases of canonical Wnt signaling during the development of mouse intestinal epithelium. Gastroenterology. 2007;133:529–538
  9. Andreu P, Colnot S, Godard C, et al. Crypt-restricted proliferation and commitment to the Paneth cell lineage following Apc loss in the mouse intestine. Development. 2005;132:1443–1451
  10. van Es JH, Jay P, Gregorieff A, van Gijn ME, et al. Wnt signalling induces maturation of Paneth cells in intestinal crypts. Nat Cell Biol. 2005;7:381–386
  11. Pinto D, Gregorieff A, Begthel H, et al. Canonical Wnt signals are essential for homeostasis of the intestinal epithelium. Genes Dev. 2003;17:1709–1713
  12. Gregorieff A, Pinto D, Begthel H, et al. Expression pattern of Wnt signaling components in the adult intestine. Gastroenterology. 2005;129:626–638
  13. Mori-Akiyama Y, van den Born M, van Es JH, et al. SOX9 is required for the differentiation of Paneth cells in the intestinal epithelium. Gastroenterology. 2007;133:539–546
  14. Wang Y, Giel-Moloney M, Rindi G, et al. Enteroendocrine precursors differentiate independently of Wnt and form serotonin expressing adenomas in response to active β-catenin. Proc Natl Acad Sci U S A. 2007;104:11328–11333
  15. Schafer AJ, Dominguez-Steglich MA, Guioli S, et al. The role of SOX9 in autosomal sex reversal and campomelic dysplasia. Philos Trans R Soc Lond B Biol Sci. 1995;350:271–277discussion 277–278
  16. Dong C, Wilhelm D, Koopman P. Sox genes and cancer. Cytogenet Genome Res. 2004;105:442–447
  17. Blache P, van de Wetering M, Duluc I, et al. SOX9 is an intestine crypt transcription factor, is regulated by the Wnt pathway, and represses the CDX2 and MUC2 genes. J Cell Biol. 2004;166:37–47
  18. Muncan V, Sansom OJ, Tertoolen L, et al. Rapid loss of intestinal crypts upon conditional deletion of the Wnt/Tcf-4 target gene c-Myc. Mol Cell Biol. 2006;26:8418–8426
  19. Sansom OJ, Meniel VS, Muncan V, et al. Myc deletion rescues Apc deficiency in the small intestine. Nature. 2007;446:676–689
  20. Kikuchi A, Yamamoto H, Kishida S. Multiplicity of the interactions of Wnt proteins and their receptors. Cell Signal. 2007;19:659–671

PII: S0016-5085(07)01170-5

doi:10.1053/j.gastro.2007.06.029

Refers to article:

  • Phases of Canonical Wnt Signaling During the Development of Mouse Intestinal Epithelium , 04 May 2007

    Byeong–Moo Kim, Junhao Mao, Makoto M. Taketo, Ramesh A. Shivdasani
    Gastroenterology August 2007 (Vol. 133, Issue 2, Pages 529-538)

  • SOX9 Is Required for the Differentiation of Paneth Cells in the Intestinal Epithelium , 22 May 2007

    Yuko Mori–Akiyama, Maaike van den Born, Johan H. van Es, Stanley R. Hamilton, Henry P. Adams, Jiexin Zhang, Hans Clevers, Benoit de Crombrugghe
    Gastroenterology August 2007 (Vol. 133, Issue 2, Pages 539-546)

Gastroenterology
Volume 133, Issue 2 , Pages 710-712, August 2007

Article Tools

* Image Usage & Resolution