AT THE EDGE OF DEVELOPMENTAL BIOLOGY: ADVANCES AND MYSTERIES ABOUT THE WNT GENES

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1 AT THE EDGE OF DEVELOPMENTAL BIOLOGY: ADVANCES AND MYSTERIES ABOUT THE WNT GENES Bénédicte Sanson, University of Cambridge, Department of Genetics, Downing Site, Cambridge CB2 3EH, UK, What is developmental biology? The past decade have witnessed amazing progress in our understanding of how animals arise from a single cell, the fertilised egg. Looking at embryos was in the past the occupation of embryologists, who carefully described the development of a wide range of organisms. It is now the occupation as well of geneticists and biochemists, since development of all organisms is under the control of genes, and genes code for proteins that have specific structural or enzymatic properties. The meeting of these three disciplines of biology have generated a very dynamic field of research, which is called developmental biology. This field has attracted considerable attention with the realisation that many genes and biochemical pathways essential to the development of embryos are precisely those disrupted in human cancers and in many genetic diseases. Moreover, workers in the field have come to the astonishing realisation that many genes involved in the development of organisms as different as flies and humans are essentially identical. This implies that studying less complex organisms that are easily accessible to experimentation can yield relevant information for the development of humans, as well as for the understanding of human diseases. This idea has stimulated the study of a few model organisms, scattered across the scale of evolution: these are the vertebrates mouse, chick, Zebra fish and Xenopus (a frog), and the invertebrates Drosophila (the fruit fly) and Caenorhabditis elegans (a worm). The Drosophila model has distinguished itself with the identification of many genes involved in early development, achievement that has been recognised in 1995 by the attribution of the Nobel Prize of Medicine to three pioneers of Drosophila developmental genetics: E. Lewis, C. Nusslein-Volhard and E. Wieschaus. Work in Drosophila as well as in other models, is based on disrupting the normal function of a given gene, using genetic and molecular techniques. The careful observation of the consequences of this disruption on the growing organism allows the scientist to deduce the normal function of the gene. To take a very simple example, if the inactivation of a given gene results in an embryo with no nervous system, one can conclude that this gene is required for the development of neural structures. This approach has revealed the fundamental role of a small number of signalling molecules in the making of animals. These molecules emanate from specific groups of cells to influence the fate and behaviour of other cells during development. Many of these signals have been identified. They belong to a few classes of secreted proteins: examples include FGFs (Fibroblast Growth Factors), TGF-βs (Transforming Growth Factors β), Hedgehog factors and Wnt factors. In addition to having funny names, these molecules share the ability to act as developmental regulators: they can trigger changes in gene expression inside the nucleus of the receiving cells, thus influencing the identity and fate of cells. They can also influence the behaviour of the cell in the developing organism, by altering the cell motility, polarity, shape etc. Great progress has been made in our knowledge of how these signalling molecules work, and what the components of their signalling pathways are. A signalling pathway is a chain of commands activated inside cells in Glossary: Gene expression: to be active or expressed, a gene has to be transcribed into a RNA in the cell nucleus (process of transcription). The RNA is then translated into a protein (process of translation) with a specific activity. Homeodomain genes: Genes coding for proteins containing a specialised part, the homeodomain, that binds to DNA. They are required for the transcription of specific group of genes and they play an important role during development. Promoter: region of DNA where the transcription of a gene is initiated. Proto-oncogene; gene which when disrupted, causes cancer. The disrupted gene is called oncogene. Ligand: any molecule that binds to a specific site on another molecule. Ligands can be extracellular proteins that bind to receptors. Receptor: protein at the surface of the cell that binds a specific extracellular ligand and initiates a response in a cell. Kinase: protein that has an activity which consists to add a phosphate to another protein (process of phosphorylation). These enzymes have often a role in relaying a signal in signalling pathways. Morphogenesis: ensemble of processes building the form of an embryo. antero-posterior axis, dorso-ventral axis: define the main axis of a growing organism. The head forms at the anterior end, the tail at the posterior end, etc. response to an exterior signal. In most pathways, the signalling molecules bind to receptors at the cell surface which in response send a signal across the cell membrane. This signal is then relayed in a cascade of biochemical

2 events inside the cell. This cascade ends up, for example, changing the expression of genes in the nucleus. The Wnt genes This review focuses on one family of signalling molecules: the Wnts. The first gene of this group was discovered in Drosophila in 1973 by Sharma [1], and named Wingless (Wg) after the phenotype of the mutant fly. The homologue of Wingless in vertebrates was found to be int-1, first identified as a proto-oncogene causing mammary tumours in mouse [Reviewed in 2]. The mouse gene was then renamed Wnt1, by contraction of Wingless and int-1. Many other Wnt genes have now been identified in humans, as well as in all the animals studied in developmental biology. One remarkable fact about these genes is that they have fundamental roles at many stages of development in all organisms. For example, early in development, Wnt1 is required for the formation of the brain in mammals. In the frog embryo, Wnt activity is necessary in the formation of the dorsal axis. In the fly embryo, Wingless is key in the establishment of segment polarity. Wnt role is not limited to early development, and many organs are dependent from them for their formation. Studies of the Wnt genes in the model organisms have revealed a common signalling pathway activated by these molecules. The first part of this review describes the current picture of the Wnt pathway, and asks if this canonical pathway can explain all the properties of the Wnt regulators. The second part of this review considers how these secreted factors spread through developing tissues to instruct cell changes. The range of the Wnt molecules has been disputed for a long time, when it was not clear if they could reach distant sites across large fields of cells. The current view is that the Wnt factors are indeed capable of travelling long distances in developing tissues, but this movement is unlikely to be just through passive diffusion. On the contrary, the transport of these secreted factors from one cell to the next is probably regulated, and finding how these factors travel is likely to develop into an exciting field of research. 1 THE WNT/WG SIGNALLING CASCADE Figure 1: The Wnt/Wg signalling pathway. (A) Components uncovered by genetic analysis in Drosophila. Armadillo protein accumulates in the cytoplasm of cells in response to Wingless signalling. Armadillo is also found associated to Cadherins in adherens junctions at the cell membrane. (B) Simplified view of the Wnt canonical pathway: Wnts bind to receptors of the Fz family; this activates Dsh which inhibits the Zw3/Axin/APC complex and stops Arm degradation. Arm becomes then available to bind nuclear factors of the TCF/Pan family and activates the transcription of target genes in the nucleus. 1.1 Evidence from Drosophila genetics: the Dsh/zw3/Arm trio Back in September 1994, when I started my postdoctoral project, only three genes were known to participate in the Wnt pathway: dishevelled (Dsh), zeste white 3 (zw3) and Armadillo (Arm). Genetic analysis in Drosophila had uncovered these genes and shown that all three are necessary in the cell receiving the Wingless signal (Figure 1A) [Reviewed in 3]. While Dsh and Arm act positively in the pathway, zw3 works as a negative regulator. The cloning of these genes showed that they all have homologues in higher organisms. Dsh encodes a new type of protein with domains highly conserved during evolution and is localised in the cytoplasm. Zw3 is a serine/threonine kinase homologous to the Glycogen Synthase Kinase 3 (GSK-3) in mammals, whereas Armadillo is the homologue of Beta-catenin, a major component of vertebrate adherens junctions. These three components were giving a promising start for the discovery of a Wnt signalling pathway conserved during evolution. But obviously some elements were missing. First, no candidate for a receptor had been uncovered by the genetic studies. Also, since apparently none of the components were detected in the nucleus, a link was missing to explain how the Wnt pathway was able to regulate gene transcription. Attention then turned to Armadillo, which was shown by genetic experiments in Drosophila to be the most downstream component of the cascade (Figure 1A). It was known also that, upon Wingless signalling, the Armadillo protein increases in stability and accumulates in the cytoplasm of the cell. It became clear that understanding the mechanism of the Wnt pathway meant understanding the function of Armadillo. 1.2 Role of Armadillo: cell adhesion or nuclear signalling? The discovery that Armadillo is the homologue of Betacatenin was rather puzzling. Beta-catenins are an essential cytoplasmic component of adherens junctions in vertebrates. Adherens junctions are adhesive plaques at the cell membrane which allow cells to stick to each other in organs or in epithelial sheets. Since hunting for a signalling pathway meant most likely looking for a biochemical cascade going from the cell surface to the

3 nucleus, the idea emerged that Armadillo might have signalling properties independent from its role in cellcell adhesion. Our work, in concert with the work of other groups, has provided evidence for this second function of Armadillo in signalling. We found that the major component of adherens junction in Drosophila, DE-Cadherin, is not a component of the Wg signalling pathway [4] (See figure 1A). Moreover, the adhesion complexes seemed to compete with the Wg signalling cascade for the availability of Armadillo. This suggested that Armadillo had indeed two distinct functions: one in cellular adhesion, and one in Wg signalling. This idea was finally demonstrated by the characterisation of Armadillo mutants which have lost their function in adhesion but still signal in the Wg pathway [5]. The mystery of the signalling function of Armadillo was solved shortly afterwards. In response to Wingless signalling, Armadillo interacts with a nuclear protein named Pangolin (Pan) or D-TCF to regulate the transcription of target genes [6-8]. This discovery echoed previous work in vertebrates pointing that Beta-catenins have signalling properties and that they associate with nuclear proteins of the Lef-1/TCF family [Reviewed in 3]. 1.3 Current picture of the Wnt signalling pathway The role of the Armadillo/Beta-catenin proteins in nuclear signalling is now well established. In the absence of the Wnt/Wingless signal, the nuclear factors of the Lef1/TCF/Pangolin family keep the Wnt target genes repressed [Reviewed in 9]. Upon activation of the pathway, Armadillo/Beta-catenin proteins increase in concentration in the cytoplasm, shuttle to the nucleus, and bind to Lef1/TCF/Pan factors to change the expression of target genes (Figure 1B). Concomitant to the discovery of the nuclear components of the Wnt pathway, an important finding was made at the other end of the signalling cascade. For the first time, a candidate for a Wnt/Wingless receptor was found [10]. This gene, Dfrizzled-2, belongs to a family of proteins named after one of its members, Drosophila frizzled (Fz), which was identified years before as a gene required for normal cell polarity in epithelia. It turned out that these proteins, which are receptors with seven transmembrane domains, are able to transduce the Wnt signal in cell culture. When expressed in cells, Fz receptors stimulate the accumulation of Armadillo in the cytoplasm, in a Wnt-dependent manner [10]. Genetic analysis in Drosophila has confirmed recently that Fz genes are required for Wingless signalling: the complete removal of both frizzled-2 and frizzled receptors in the embryo abolishes Wingless signalling [11]. The search for such receptors had been unsuccessful for years, probably because the functions of these receptors are redundant [12, 13]. Hence, the identification of the Frizzled family fills a major hole in the Wnt signalling cascade (Figure 1B). How the transduction of a signal through Fz receptors leads to Dsh activation and inactivation of the ZW3/GSK-3 kinase is, however, still unclear. The next step is by contrast quite well understood. The inactivation of the ZW3/GSK-3 kinase in fact inactivates a complex that targets the Armadillo protein for destruction in absence of Wg signalling [Reviewed in 14]. Thus, when cells are not exposed to the Wnt signal, this degradation complex keeps the concentration of Armadillo low in the cytoplasm. This complex contains three major components: ZW3/GSK-3, APC and Axin (Figure 1B). APC (Adenomatous Polyposis Coli gene) was originally identified as a human tumour suppresser gene: mutations in this gene cause a predisposition to colon cancer. Axin is a new protein identified first in vertebrates and very recently, in Drosophila [15]. The complex ZW3/GSK-3-APC-Axin phosphorylates Armadillo/Beta-catenin, and targets it for ubiquitination, which is followed by degradation by the proteasome [Reviewed in 14]. When the WG pathway is stimulated, the activity of the ZW3-APC-Axin complex is inhibited. The concentration of Armadillo raises in the cytoplasm, and Armadillo then shuttles to the nucleus to interact with the Lef1/TCF/Pangolin nuclear proteins. This is, however, not the whole story: current research is yielding additional players to the pathway [For an online update, see 16]. Their discovery provides information of how Wnt signalling is fine-tuned. For example, at the surface of the cell, interaction of Wnts with the Frizzled receptors is likely to be regulated by proteoglycans [17]. Proteoglycans are molecules that contain a protein core on which sugar chains are bound. These sugar chains are thought to bind Wnts, and this binding might facilitate the interaction of Wnts with Fz receptors. In support of this hypothesis, genetic analysis in Drosophila has shown that dally, a gene encoding the protein core of a major proteoglycan, is required for Wingless signalling [18, 19]. Inside the cell, additional components of the degradation box for Armadillo/Beta-catenin have been found. Interestingly, some of these factors are also required for the degradation of components of other key developmental signalling pathways such as the hedgehog pathway [Reviewed in 14]. Finally, in the nucleus, several new components have been shown to act in concert with Lef/TCF/Pangolin factors to repress Wnt target genes in absence of the Wnt signal [Reviewed in 20]. 1.4 Does the canonical pathway do it all? The above description shows how recent research unravels a picture more and more precise of a Wnt canonical pathway, conserved in all complex organisms from Caenorhabditis elegans to humans. Now, can this cascade explain all functions associated with Wnt activities? In Drosophila, one of the main functions of Wingless is to regulate the activity of key developmental genes, such as the homeodomain genes engrailed or ultrabithorax. These regulations have a direct impact on determining cell identities during Drosophila development. This is true in vertebrates as well. For example, in Xenopus, targets of Wnt activity include

4 siamois, an homeodomain gene which has a major role in dorsal axis formation. Molecular dissection of the promoter areas of the ultrabithorax or siamois genes has identified regulatory elements bound directly by TCF/Beta-catenin complexes [Reviewed in 3]. Thus, in those cases, it is clear that activation of the Wnt canonical pathway regulates directly these genes and therefore influences directly animal development. For other properties of the Wnt genes, however, the relationship between this pathway and the effects of Wnt signalling is not so clear. The Wnt factors mediate changes that affect the shape, polarity, growth or adhesion of cells [Reviewed in 2]. For example, earlier work has indicated that Wnt signalling modify the adhesive properties of cells in culture [21, 22]. In the canonical pathway, it is clear that Wnt signalling acts independently of the Cadherin adhesion complexes. Nevertheless, since Wnt controls the level of cytoplasmic Armadillo, it is still possible it affects Cadherin/Beta-Catenin complexes in addition to changing gene transcription through Lef-1/TCF/Pan proteins. But for now, how Wnts regulate cell adhesion remains unexplained. In the worm Caenorhabditis elegans, a Wnt signal is required at the four-cell stage for the asymmetric division of the cell named EMS. One aspect of this asymmetric division is a ninety-degree rotation of the mitotic spindle (the machinery that ensure equal repartition of the duplicated chromosomes in the daughter cells). This requires Wnt signalling, but strikingly, does not require any changes in gene transcription [23]. Evidence indicates that activation of upstream components of the Wnt pathway may have a direct effect on the cytoskeleton in this case. Those examples point out that some of the actions of Wnt signalling on the architecture of the cell might be direct, bypassing changes in gene expression. Elucidating how Wnt signals affect morphogenetic events remains a major challenge in the field. One possibility is that the canonical pathway branches at one or several of its downstream components to influence the behaviour of cells. In fact, all main members of this pathway are potential branch-point for effects outside the nucleus. For example, Dishevelled is a component of another pathway that controls cell polarity in Drosophila and vertebrates. Dsh activity in this pathway requires distinct parts of the protein from those involved in the Wingless cascade [24]. Recent results show that Dsh activates the Jun kinase pathway to promote changes in cell polarity [25]. Armadillo is a multi-potent protein found in different complexes outside the nucleus, including the Cadherin adhesion complexes. It is also the target of multiple regulations: in a genetic screen in Drosophila, we found seventeen known genes that control Armadillo levels [26]. Another possibility for Wnt signals to trigger changes in morphogenesis is through alternative pathways. In Xenopus, a member of the Wnt family, Wnt-5a, alters morphogenetic movements in the early embryo. Work from the Moon lab suggests that this Wnt factor acts through an alternative pathway, which may involve phosphatidyl-inositol signalling and stimulation of Protein Kinase C [27, 28] [see also 16]. 1.5 Conclusions Recent research has found a pathway for Wnt signalling. We have a receptor, a cascade of components inside the cytoplasm, and a downstream component shuttling to the nucleus to change gene expression. This sequence of events explains quite well how the Wnt factors govern changes in gene expression in developing organisms. However, how Wnts factors control cell behaviour and hence morphogenesis, is still far from understood. The Wnt canonical pathway may branch in the cytoplasm and alter directly the cytoskeleton of the cell, independently of changes in gene expression in the nucleus. Alternatively, Wnts may activate other pathways which specifically govern cell behaviour. To understand the complex role of Wnts in development, one might have to change the way we are looking at signalling cascades. Rather than looking at sequential events happening from the cell surface to the nucleus, we might have to envision networks of events inside cells. In this view, responding to a developmental signal would be a bit like firing a neuronal network, generating not a linear response, but an ensemble of integrated responses. 2 MOVEMENT OF WNTS IN DEVELOPING TISSUES 2.1 Long-range or short range? In 1994, the range of action of the Wnt factors was still debated [Reviewed in 29]. Some scientists favoured the view that Wnt proteins travel long distances through fields of cells. Consistent with this idea, Wnts showed apparent long-range effects in developing tissues. On the other hand, others argued that these long-range effects are indirect, and that Wnts act only at short distances, their action being relayed in large field of cells by other signalling molecules. This hypothesis was based partly on the fact that Wnt factors are secreted glycoproteins that bind strongly to the cell surface, making the idea of longrange diffusion difficult to envision. Also, short-range effects had been documented in the Drosophila embryo: here, Wingless acts only in adjacent cells to maintain the expression of the engrailed gene [30]. Now, it is widely accepted that Wnts can act directly at long distances. One of the key experiments which supports this view has been done in the Drosophila wing disc. Whereas the normal Wingless protein displays longrange effects, an engineered membrane-tethered form of Wingless was shown to act only on neighbouring cells to activate target genes [31]. Thus, long-range signalling by normal Wingless requires its physical movement from one cell to the next. Wnt factors share this long range of action with other secreted signalling molecules, such as Decapentaplegic (a Drosophila member of the TGF-β family) and Hedgehog [Reviewed in 32]. How Wnts and other signalling molecules reach distant sites is, however, still mysterious.

5 2.2 Range of Wingless in Drosophila Drosophila development provides a paradigm for studying the range of Wnts. Research focuses on the range of Wingless in two main tissues: the wing disc, which is an epithelial sac in the larval body that transforms into an adult wing after metamorphosis [Reviewed in 33]; and the segmented ectoderm of the embryo s trunk [Reviewed in 34] (See figure 2). In the wing disc, Wingless is expressed in a stripe of 3-4 cells, straddling the boundary between ventral and dorsal compartments (Figure 2A). High levels of Wg protein are found in those cells, whereas lower levels are detected in surrounding tissues, where the protein concentrates in small dots [32]. These dots are detected at a distance of about 10 cell diameters from the source of expression. Effects of Wingless signalling, however, are found at even longer distances. Three direct targets of Wingless have been discovered in the disc: achaetescute, distal-less and vestigial. These genes are activated at different distances from the Wingless source, achaetescute being expressed the closest to the Wingless stripe (Figure 2A). Their activation by Wingless is direct and requires Wg to travel across the field of cells [31, 35]. In the embryo, Wingless is expressed in stripes onecell wide in each presumptive segment of the trunk. These stripes abut the boundary between anterior and posterior compartments in each segment. We investigated the range of Wingless in the anterior direction from its source. One function of Wingless in segmental patterning is to specify the cells that secrete a smooth cuticle after epithelial differentiation. In these cells, the formation of small hair called denticles is antagonised by Wingless signalling, through repression of the gene shaven-baby [36]. We found that Wingless acts at a distance of 4 to 5 cells in the anterior direction to specify the smooth fate of epithelial cells [37, 38]. In the case of the wing disc, the range of Wingless is symmetrical about its expression domain: both achaetescute, distal-less and vestigial are activated equally in the ventral and dorsal direction from the Wingless source (Figure 2B). This is not the case in the embryonic segments: whereas Wingless acts over up to 4-5 cell diameters in the anterior direction, only adjacent cells are affected by Wingless in the posterior direction [38]. To show this asymmetry, we used two markers for Wingless action: specification of smooth cuticle and repression of the gene rhomboid. Posterior to the Wingless source, only the adjoining stripe of cells secretes a smooth cuticle. Likewise, rhomboid expression is unaffected posterior to the source, whereas it is completely repressed in the anterior direction (Figure 2B). Figure 2: Range of Wingless in Drosophila. (A) In the wing disc, Wingless is expressed in a small stripe of cells at the dorso-ventral (D/V) boundary (left). Wingless protein forms a symmetric gradient which activates the transcription of achaete scute (ac), Distalless (Dll) and vestigial (vg) (rigth). (B) On the ventral side of the embryo, Wingless is expressed in a row of cells abutting the antero/posterior (A/P) boundary in each segment (left). Anterior to its source, Wingless represses the transcription of rhomboid (rho) and shaven-baby (svb) (rigth). Posterior to its source, Wingless spreading is slowed down at the A/P boundary and stopped at the segment boundary (S). An asymmetric gradient of Wingless protein forms and this allows the expression of rho and svb posterior to the Wg source. This asymmetry follows in part from asymmetric distribution of the Wingless protein. First, Wingless movement appears restricted at the antero/posterior boundary. Second, further movement is blocked at the presumptive segment boundary, two cells diameter away. We found that the existence of this barrier requires another secreted molecule, hedgehog. In the absence of hedgehog, this barrier no longer forms and

6 conversely, over-activation of the hedgehog pathway decreases Wingless range [38]. These successive blocks of Wingless ligand movement generate an asymmetric gradient, which determines the antero-posterior polarity of each segment (Figure 2B). 2.3 Mechanism for Wnt transport: an unsolved mystery The existence of asymmetric gradients of Wingless protein in the embryo suggests that the movement of this extracellular protein across a field of cells is regulated. For now, we can only speculate about the mechanisms that control Wingless movement. One possibility is that Wingless reaches distant sites by active transport through cells, and that this transport is regulated [Reviewed in 34]. In mutants for the gene shibire, which codes for the GTPase Dynamin, Wingless s range decreases and becomes limited to cells immediately adjacent to the Wingless expressing cells [39]. Since Dynamin is required for the formation of endocytic vesicles inside cells, this suggests that Wingless is secreted normally in these mutants, but cannot travel from one cell to the next. This hypothesis implies that Wingless is transported through cells by the shuttling of endocytic vesicles, a process called transcytosis. This theory is attractive, and fits with the observation that Wingless proteins are detected in dots which have a vesicle-like structure, but it still remains to be proven. Other ways of controlling the range of extracellular signalling molecules is through their interaction with receptors at the surface of the cells. A number of studies in Drosophila indicate that receptor levels can affect ligand distribution in fields of cells [40-42]. Both receptors for hedgehog and decapentaplegic slow down their ligand movement. The case of Wingless is different: increase in concentration of its receptor DFrizzled2 stabilizes the Wingless protein, without any apparent effect on its movement [42]. Other types of receptors that could possibly affect the range of signalling molecules are low affinity receptors of the proteoglycan type (See section 1-3). Recent work has shown that proteoglycans stimulate hedgehog, decapentaplegic and Wingless signalling [17-19]. They are thought to bind these secreted proteins at multiple sites on their sugar chains. Thus, they could concentrate these ligands at the cell surface, and help their interaction with signalling receptors such as the Fz receptors. A corollary of this hypothesis is that they could slow down the movement of signalling molecules, by sequestrating them at the cell surface. 3 CONCLUSIONS In the last decade, there has been enormous progress in the understanding of how Wnts regulate gene expression. A precise picture for the signalling pathway used by these extracellular molecules is now available, and more details are constantly being added by current research. Now, the next challenge lies in understanding how these factors trigger changes in cyto-architecture. This question is not limited to the Wnt pathway: while morphogenetic changes are as important as changes in gene expression for the making of animals, they are still not well understood. Elucidating the molecular bases of morphogenesis is likely to constitute the next goal for many developmental biologists. Another mystery is how Wnts and other extracellular molecules travel to reach distant sites in developing tissues. While it is now clear that many of those signalling molecules can act directly at a distance, we need to understand if this movement requires passive or active transport, and how it is regulated. ACKNOWLEDGEMENTS I am grateful to Jean-Paul Vincent, who has been a very stimulating supervisor and collaborator for my postdoctoral projects. My post-doctoral stay at the MRC- LMB, Cambridge, UK was funded first by an EMBO fellowship (09/94-08/96), and then by a Marie Curie fellowship (09/96-08/98). I am now funded by a Wellcome Trust RCD Award. I thank Marc Bickle and Daniel St Johnston for comments on the manuscript. REFERENCES [1] R.P. Sharma, D. I. S., (1973). 50: p [2] R. Nusse, and H.E. Varmus, Cell, (1992). 69: p [3] K.M. Cadigan, and R. Nusse, Genes Dev., (1997). 11: p [4] B. Sanson, et al., Nature, (1996). 383: p [5] S. Orsulic, and M. Peifer, J. Cell Biol., (1996). 134: p [6] E. Brunner, et al., Nature, (1997). 385: p [7] J. Riese, et al., Cell, (1997). 88: p [8] M. van de Wetering, et al., Cell, (1997). 88: p [9] M. Bienz, Curr. Opin. Cell Biol., (1998). 10: p [10] P. Bhanot, et al., Nature, (1996). 382: p [11] P. Bhanot, et al., Development, (1999). 126: p [12] J.R. 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