Multiple Roles of Canonical Wnt Signaling in Cell Cycle Progression ...

[Cell Cycle 3:6, 701-703; June 2004]; ©2004 Landes Bioscience

Multiple Roles of Canonical Wnt Signaling in Cell Cycle Progression and Cell Lineage Specification in Neural Development Extra Views

Received 04/07/04; Accepted 04/08/04

Previously published online as a Cell Cycle E-publication: http://www.landesbioscience.com/journals/cc/abstract.php?id=912

KEY WORDS wnt, β-catenin, neural stem cell, neural crest, sensory neuron, cell cycle, fate decision

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dishevelled peripheral nervous system central nervous system sonic hedgehog

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Wnt proteins form a family of secreted lipid-modified molecules that control numerous developmental processes such as tissue induction, morphogenesis, cell proliferation, cell fate decisions, and differentiation.1 Canonical Wnt signaling involves the binding of Wnts to Frizzled receptors and subsequent activation of Dishevelled (Dsh), which prevents ubiquitin-mediated degradation of cytoplasmic β-catenin and allows its translocation into the nucleus. Activation of target genes is achieved by binding of β-catenin to TCF/LEF transcription factors and is modulated by cross-talk of Wnt/β-catenin signaling with various other signal transduction pathways including signaling by Notch, TGFβ factors, FGFs, sonic hedgehog (shh), and Cadherin.2-4 Several studies have revealed a role of Wnt signaling in stem cell expansion. In embryonic stem cells, activation of Wnt regulates self-renewal5 and inhibits neural differentiation.6 Likewise, Wnt signal activation maintains hematopoietic stem cells in long-term culture and leads to sustained reconstitution of hematopoietic lineages in vivo.7-9 Further stem cell types controlled by Wnt are found in the skin10 and in the intestinal crypts.11-13 In the central nervous system, wnt3a is required for neural progenitor proliferation and hippocampal development,14 while absence of both wnt1 and wnt3a affects the expansion of dorsal neural cells including the neural crest.15 Accordingly, Wnt signal activation by ectopic expression of Wnt or of constitutively activated β-catenin reduces neuronal differentiation and increases progenitor numbers, which leads to a massive enlargement of neural tissue in certain regions of the central nervous system (CNS).16-18 However, it is less clear whether the observed phenotypes are due to effects on multipotent, self-renewing neural stem cells or on transient amplifying progenitors. Israsena and colleagues19 reported an increase in secondary neurosphere formation (indicating self-renewal activity of sphereforming cells) from β-catenin-overexpressing cells that had been isolated from the ganglionic eminence. In contrast, Wnt family members promoted maturation and proliferation of neural progenitors from the cortex, apparently without increasing the proportion of progenitors that generated secondary or tertiary spheres.20 These differences might be due to region-specific or context-dependent responses to Wnt signaling. To further address the role of the canonical Wnt pathway in neural stem cells, we have recently generated mice either carrying a loss-of-function or gain-of-function mutation of β-catenin specifically in neural crest stem cells.21,22 During development, neural crest cells emerge from the dorsal part of the neural tube and emigrate to various locations within the embryo to generate most structures of the peripheral nervous system (PNS) as well as some nonneural tissues such as pigmented melanocytes, smooth muscle cells in the outflow tract of the heart, and craniofacial bones, cartilage, and connective tissues.23 Upon loss of β-catenin, melanocytes and sensory ganglia fail to develop in vivo.21 Moreover, cell culture analysis revealed that neural crest stem cells lacking β-catenin are unable to acquire a sensory neural fate, while migration and, in particular, proliferation are unaffected. Complementary to these results, sustained β-catenin activity in neural crest stem cells results in the formation of excess sensory neuronal cells in vivo that form at the expense of virtually all other neural crest derivatives.22 Again, the increased formation of sensory neurons is not due to increased proliferation of neural crest cells but rather to a bias of mutant cells to give rise to sensory neurons.

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ABBREVIATIONS

Signaling by the canonical Wnt pathway has multiple functions in stem cells. It can either control stem cell expansion or—as we have recently demonstrated with neural crest stem cells—influence cell lineage decisions by promoting specific fates at the expense of others. Thus, the role of canonical Wnt in stem cells is dependent on cell-intrinsic properties that determine how a cell responds to Wnt. The molecular basis for the functional diversity of Wnt in different stem cell types remains to be elucidated.

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Correspondence to: Lukas Sommer; Institute of Cell Biology; Department of Biology; Swiss Federal Institute of Technology; ETH-Hönggerberg; Zurich CH-8093 Switzerland; Tel.: +41.1.633.34.92; Fax: +41.1.633.10.69; Email: sommer @cell.biol.ethz.ch

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Figure 1. In neural crest stem cells, Wnt/β-catenin activation induces fate decisions without affecting proliferation. In other stem cells (including embryonic and hematopoietic stem cells as well as neural progenitors of the CNS) canonical Wnt signaling maintains a stem cell state by promoting selfrenewal. The molecular mechanisms underlying this differential responsiveness to Wnt remain to be elucidated. Shown are sensory neurons aggregating in ganglion-like structures induced by Wnt1-treatment of neural crest stem cells in culture (Kleber M, Sommer L).

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A role of Wnt signaling in fate acquisition has also been supported by other studies. Wnt signaling promotes myogenic specification and controls the differentiation of specific lineages in the skin and the intestine.11,24,25 In contradiction to other studies,17,19,20 Muroyama and coworkers showed a requirement of wnt1/wnt3a in the specification of interneurons in the dorsal spinal cord and reported increased neuronal differentiation in Wnt3a-treated neurosphere cultures, without observing a mitogenic effect of Wnt signaling.26,27 The effect of a growth factor on the generation of specific cell types to the detriment of others could be explained by selective mechanisms that control the survival or proliferation of a subpopulation of progenitor cells; alternatively, the growth factor might instruct a multipotent stem cell to choose a particular fate at the expense of other possible fates. The only way to unambiguously distinguish between these possibilities is to analyze the developmental potential of single cells out of a defined cell population, and to determine whether the majority of the cells behave in the same manner in response to the growth factor in question. Therefore, to address the role of Wnt/ β-catenin in neural crest stem cells, we prospectively identified cells with neural crest stem cell features and challenged these cells with Wnt1 as well as other growth factors previously shown to act instructively on neural crest stem cells.22,28 These clonal analyses demonstrated the existence of a population of early neural crest stem cells able to generate sensory neurons in response to Wnt signaling, and autonomic neurons and other fates in response to other appropriate signals. The sizes of Wnt-treated clones was not increased, indicating that Wnt does not selectively support the proliferation of a putative sensory progenitor cell. Thus, unlike in other stem and progenitor cells, Wnt/β-catenin instructively promotes cell fate decision rather than stem cell expansion in neural crest stem cells.22 This does not exclude further activities of Wnt signaling in neural crest development. Wnts induce neural crest in the dorsal neuroepithelium, are involved in melanocyte formation, and appear to regulate proliferation of cardiac neural crest-derived cells.29-31 These different responses to Wnt signaling are likely due to changes in intrinsic properties that the cells acquire during development.32,33 Such intrinsic determinants might also contribute to the differential interpretation of Wnt signaling by different types of stem cells. In addition to the use of noncanonical Wnt pathways,34 alterations in TCF/LEF transcription factors interacting with β-catenin might be involved.35 While differences in cell-intrinsic cues conceivably influence the biological activity of Wnt in distinct stem and progenitor cell types, one and the same stem cell type might also be able to differentially respond to Wnt depending on the extracellular microenvironment. In neural crest development, factors counteracting Wnt/β-catenininduced sensory neurogenesis likely exist, because not all neural crest stem cells adopt a sensory fate in normal development although virtually all of these cells appear to be responsive to Wnt activity.22 With respect to the reported roles of Wnt in CNS stem and progenitor cells, the modulation of Wnt activity by FGFs has to be considered in more detail. FGF2 increases the levels of active β-catenin in neural progenitors from the ganglionic eminence.19 β-catenin overexpression maintains these cells in a proliferative state when cultured as neurospheres in the presence of FGF2, while it increases the proportion of neurons in adhesive cultures in the absence of FGF2. In cortical cells, however, Wnt signaling promotes maturation and proliferation, but only the maturation effect is FGF-dependent.20 Moreover, FGF together with Wnt activity is required to induce late features of the dorsal telencephalon.36 These data indicate that there will be no simple solution to the question of how—on the molecular

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level—canonical Wnt signaling elicits different cellular responses. One approach to address the issue consists of trying to systematically identify the downstream components of canonical Wnt signaling in different contexts. Several Wnt target genes have been described so far (see http://www.stanford.edu/~rnusse/pathways/targets.html). These include genes such as cyclin D1 and c-myc that control cell cycle progression;37,38 neurogenins and NRSF/REST that are involved in neurogenesis;19,39 ephrins and cell adhesion molecules that control cell-cell interactions;40-42 and FGF4, linking FGF with Wnt signaling.43 It is the challenge for the future to elucidate the composition of the signaling networks that allow Wnt/β-catenin to regulate these and potentially other target genes in a stage- and location-dependent manner.

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Shtutman M, Zhurinsky J, Simcha I, Albanese C, D’Amico M, Pestell R, et al. The cyclin D1 gene is a target of the β-catenin/LEF-1 pathway. Proc Natl Acad Sci USA 1999; 96:5522-7. 38. Tetsu O, McCormick F. β-catenin regulates expression of cyclin D1 in colon carcinoma cells. Nature 1999; 398:422-6. 39. Nishihara S, Tsuda L, Ogura T. The canonical Wnt pathway directly regulates NRSF/REST expression in chick spinal cord. Biochem Biophys Res Commun 2003; 311:55-63. 40. Jamora C, DasGupta R, Kocieniewski P, Fuchs E. Links between signal transduction, transcription and adhesion in epithelial bud development. Nature 2003; 422:317-22. 41. Conacci-Sorrell ME, Ben-Yedidia T, Shtutman M, Feinstein E, Einat P, Ben-Ze’ev A. NrCAM is a target gene of the β-catenin/LEF-1 pathway in melanoma and colon cancer and its expression enhances motility and confers tumorigenesis. Genes Dev 2002; 16:2058-72. 42. 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