- Email: [email protected]
Cadherins and catenins in development
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Cadherins and catenins in development Otmar Huber, Christiane Bierkamp and Rolf Kemler* Cadherinsand cateninsrepresentkey moleculesduring development.Recentfindingsdemonstratethe involvement of cadherinsand cateninsin signalingpathways.In a workinghypothesis,signalingvia ~-cateninregulatesthe epithelial-mesenchymaltransitionin vertebratedevelopment. Addresses Max-Planck Institute for Immunobiology, Department of Molecular Embryology, StLibeweg 51, D-79108 Freiburg, Germany "e-mail: [email protected] Correspondence: Rolf Kemler
proteins, that is, desmocollins and desmogleins [16]. Quite detailed analysis, including site-directed mutations, has identified the binding sites in 13-catenin and plakoglobin that regulate protein-protein interactions [17-20]. The finding that [3-catenin and plakoglobin are homologous to the Drosophila Armadillo protein led to the identification of a whole Armadillo protein family, which includes p120, APC, and importin. Even more importantly, this homology suggested that [~-catenin and/or plakoglobin might also exhibit a signaling function [21°,22"], as will be discussed below.
Current Opinion in Cell Biology 1996, 8:685-691 © Current Biology Lid ISSN 0955-0674
Abbreviations APC adenomatous polyposis coli GSK313 glycogen synthase kinase 313 LEF-1 ZO-1
lymphoid enhancer factor-1 zonula occludens-1 protein
Figure 1
EGFreceptor Cadherin
ZO'I~
Desmoglein/desmocollin
~_~teni~ Plakoglobin
Introduction Cadherins represent a distinct family of single-transmembranedomain glycoproteins which serve as specific cell adhesion molecules acting in a Ca2÷-dependent manner, largely by homophilic protein-protein interaction [1]. For many new members of this protein family, identified by structural homology, an involvement in cell adhesion has not yet been shown, and in fact some may exhibit other biological functions [2°-6"]. T h e classical cadherins, including E-, N-, and P-cadherin, are the best studied representatives of the family; recently, the three-dimensional structures of the extracellular domains of E- and N-cadherin were published [7"'-9"']. Classical cadherins are defined by a highly conserved cytoplasmic domain and the capability to form complexes with the catenins--that is, (x-catenin, lS-catenin, plakoglobin (T-catenin), and p120 [I0]. Several lines of evidence indicate that cadherins and catenins are key molecules in adhesion-dependent morphogenetic processes during development. Before reviewing recent progress here, we will summarize briefly the molecular architecture of the cadherin-catenin complex and survey newly discovered interaction partners of catenins (Fig. 1). Catenins link cadherins to the actin filament network [10]; now it appears that they also interact with other transmembrane or cytoplasmic proteins as more general linkers or adapters. For example, 13-catenin associates with the epidermal growth factor (EGF) receptor [11], the tight junction protein ZO-1 (zonula occludens-1 protein) [12], and the adenomatous polyposis coli (APC) tumor suppressor protein [13,14]. Remarkably, the APC-I]-catenin interaction is regulated by glycogen synthase kinase 313 (GSK313), a component of the wingless/Wnt signaling pathway [15"°]. Plakoglobin also interacts with the desmosomal
APC~-'SK313 ~'/~.-catenin ~
APC © 1996CurrentOpinionin CellBiology
Catenins play multiple roles as linker or adapter proteins. This scheme shows the major interaction partners for I~-catenin and plakoglobin described so far [11,12,15°°,16-90].
In this review, we want to survey the three major arguments that cadherins and catenins represent key regulatory molecules during development: first, the expression patterns for cadherins correlate with distinct morphogenetic events; second, overexpression of variants of cadherins and catenins (using a dominant-negative approach) leads to perturbations in morphogenesis; and third, loss-of-function experiments demonstrate the importance of the cadherins and catenins in developmental processes.
Expressionpatternsduringdevelopment T h e first clue that cadherins might be important regulatory molecules during development came from the fact that their individual expression patterns are, very often, correlated with distinct morphogenetic events such as cell migration, cell sorting, organogenesis, and tissue remodeling [1]. E-cadherin is expressed very early on embryonic cells [231, and is also expressed later during development, mainly on epithelial cells. In mouse embryos, the first cell type that is negative for E-cadherin is the forming mesoderm at the gastrulation stage [24]. Mesoderm derivatives remain negative for E-cadherin, but the protein becomes re-expressed in cells undergoing
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a mesenchymal-epithelial transition. E-cadherin is also expressed in the mouse embryonic brain [25] and a subset of sensory neurons, in addition to in a specific type of adherence junction in Schwann cells [26]. Cadherins are detected in mesenchymal cells and their derivatives [27°-29°], a somewhat unexpected finding as mesenchyme is regarded as a loose tissue and cadherins have been thought to regulate cell adhesion in solid tissues. In fact, cad-11 is expressed rather early in mouse embryos in the mesenchymes surrounding organ-anlages, in the cephalic mesoderm, and during somite formation [27°-29°]. Although cad-ll is currently considered to be the major mesenchymal cadherin, M-cadherin and N-cadherin are expressed in other mesenchymal components such as muscle or condensing chondrocytes [30,31]. Most of the recently identified cadherins are expressed in the developing vertebrate brain [32]. T h e primordial neural tube continuously expresses N-cadherin, whereas R-, B-, E-, and T-cadherins are expressed only at later stages of brain development; this expression correlates with distinct regionalization of the central nervous system. Catenins, in contrast, are expressed rather ubiquitously, although some distinct differences can be recognized [24]. During mouse preimplantation development, both c~-catenin and [3-catenin are maternally provided as protein and mRNA, and the embryonic genes become activated at the late two-cell stage [23]. Plakoglobin exhibits a distinct expression profile, lacking the maternal component and appearing first at late morula stage. Importantly, [3-catenin transiently accumulates in nuclei on the dorsal side of Xenopus and zebrafish blastulae ([33°°]; see below).
Dominant-negative
approach
In general, overexpression of two types of dominant-negative constructs has been used to study the importance of either the extracellular or the cytoplasmic domain of cadherins. An overexpressed cadherin with most of the extracellular domain deleted but the cytoplasmic domain intact (Aextra) still binds to catenins and thereby reduces their availability for the endogenous cadherins. A cadherin with the extracellular domain intact and the cytoplasmic domain deleted (Acyto) appears to interact in a homophilic manner with the endogenous cadherin but is unable to form a functional cadherin-catenin complex. Injecting Aextra N-cadherin mRNA into early Xenopus embryos results in a dramatic inhibition of cell adhesion and a perturbation of the integrity of the ectodermal cell layer at midgastrulation [34]. Similarly, injecting Aextra mRNAs for N-cadherin, XB-cadherin, or both E-cadherin and EP-cadherin into specific blastomeres induces their local overproduction and results in disorganization of the tissues descending from the injected cells [35,36]. XB-cadherin is more effective than N-cadherin, and EP-cadherin is more effective than E-cadherin, in inducing these perturbations. An attractive interpretation of these results is that these
varying extents of interference reflect different affinities of the cadherins for catenins or other cytoskeletal structures. Overexpression of the second type of mutant constructs has unraveled the more specific functions of each cadherin [37,38]. Injection of Acyto N-cadherin mRNA leads to perturbation of neural tube formation; analogously, E-cadherin seems to be specifically required for maintaining the integrity of the ectoderm during epiboly at gastrulation [39]. Interestingly, overexpression of full-length C-cadherin, a close homolog of E-cadherin, fails to compensate for the loss of E-cadherin function, and ectopic expression of full-length C-cadherin or N-cadherin in wild-type cells can even cause ectodermal lesions [39,40]. Differential expression of cadherins thus evidently reflects specific requirements for distinct adhesive properties during different morphogenetic events. However, in these experiments injected mRNA overrides all transcriptional regulatory mechanisms that could normally regulate E-cadherin expression, sometimes causing novel dominant defects. Cadherins may also be involved in cell differentiation events: overexpression of Aextra N-cadherin in the presumptive muscle anlage of early Xenopus embryos results in suppression of muscle differentiation [41]. As muscle precursors develop from a community of cells, a role of cadherins in the community effect is suggested here. Transgenic mice overexpressing Aextra genes for N-cadherin and E-cadherin that are driven by various tissue type specific promoters can be used to study cadherin function in specific cell lineages of particular organs, such as the small intestine and pancreatic 13 cells [42°,43°]. A dramatic effect is observed when expression of Aextra N-cadherin is directed to mouse intestinal epithelial cells: cell-cell and cell-matrix contacts are disrupted; the differentiated polarized phenotype is lost; and precocious entry into a cell death program occurs, indicating a linkage between cell adhesion, signaling and apoptosis [43°,44°]. Ectopic expression of E-cadherin in a retinal pigment epithelial cell line which endogenously expresses other cadherin subtypes induces epithelial cell polarity and even causes de novo assembly of desmosomes [45°°]. This remarkable result points to an additional role for E-cadherin in addition to its known function in cell adhesion. Equally significant in this regard are recent observations that specific cadherins can stimulate the formation of specific tissues [46°°]. By gene targeting, embryonic stem (ES) cells lacking E-cadherin and exhibiting a cell adhesion defect were generated; this phenotype could be rescued by expressing either E-cadherin or N-cadherin cDNA driven by a constitutive promoter. Remarkably, such mutant ES cells rescued with E-cadherin were only capable of forming epithelia, whereas the same cells that were rescued with N-cadherin formed cartilage and neuroepithelium. These results give strong support for
Cadherinsand cateninsin developmentHuber, Bierkamp and Kemler 687
the idea of a role of cadherins in histogenesis, again suggesting that, in addition to playing a role in cell adhesion, cadherins participate more directly in a signaling pathway. T h e involvement of catenins in developmental processes has also been studied. Overexpression of wild-type 13catenin on the ventral side of Xenopus or zebrafish embryos induces the formation of a complete secondary body axis [47",48"]. Expression of deletion mRNA constructs of 13-catenin shows that the Armadillo (Arm)-repeat region is both necessary and sufficient to induce axis duplication, indicating participation in the generation of intracellular signals involved in the specification of dorsal mesoderm. Similar experiments carried out with plakoglobin again showed that the complete coding region of plakoglobin, or just the central Arm repeat region, is also sufficient to induce axis duplication [49"].
Loss-of-function analysis An alternative way to gain insights into the developmental function of cadherins and catenins is to inhibit their expression. Two different approaches can be used, namely gene-targeting experiments leading to nonfunctional (knocked-out) genes or antisense experiments to deplete the mRNA and reduce specific protein expression.
Mouse embryos lacking 13-catenin die in utero at gastrulation with a specific defect in the embryonic ectodermal cell layer, despite the rather ubiquitous expression of 13-catenin in wild-type cells at this developmental stage [57"]. Mutant cells detach from the embryonic ectodermal cell layer and are dispersed in the proamniotic cavity, and no mesoderm is~formed, as monitored by the absence of T-brachyury gene expression. Plakoglobin cannot substitute for the absent 13-catenin during mouse development even though it also b i n d s to E-cadherin and oc-catenin. In Xenopus, depletion of 13-catenin affects dorsal mesoderm induction [58]; this was explained by proposing a role for 13-catenin in signaling pathways. An antisense approach was also used to reduce the level of EP-cadherin mRNA and protein in the Xenopus embryo [59]. This depletion leads to a dose-dependent reduction of adhesion between blastomeres. With high concentrations of the most effective antisense oligonucleotides, embryos fail to undergo gastrulation and arrest at the blastula stage, showing the need for maternal EP-cadherin in this process.
13-catenin signaling in vertebrate development
In Drosophila the shotgun gene locus encodes the homolog of E-cadherin (DE-cadherin) [52,53",54"]. Null mutant embryos expressing no zygotic DE-cadherin exhibit no severe defects in general epithelial organization, because maternally derived DE-cadherin is sufficient, but formation of epithelia involving extensive morphogenetic movements (e.g. forming Malphigian tubules and tracheal ducts) is impaired. Complete absence of maternal and zygotic shotgun expression impairs formation of all epithelia.
T h e homology of 13-catenin and plakoglobin to the Drosophila segment polarity protein Armadillo has recently attracted much attention [21°,22"]. It was intriguing that these components of cell adherence junctions share a common structure with this component of the wingless signal transduction pathway; this suggested that these molecules have similar biochemical functions. This has now been confirmed. In Drosophila, Armadillo is found in adherence junctions together with (~-catenin and DE-cadherin [52] and probably plays another independent role in wingless signaling [21",60"]. As mentioned, accumulating evidence also implicates 13-catenin in a signaling pathway. Overexpression of 13-catenin in Xenopus embryos results in dorsal mesoderm induction, as does expression of Xwnt8, a vertebrate homolog of the wingless protein [47"]. Inhibition of 13-catenin expression suppresses mesoderm formation in both Xenopus and mouse [57",58]. Moreover, 13-catenin in cultured mouse cells and Armadillo in Drosophila accumulate in the cytoplasm upon wingless/Wnt signaling [60",61"] and both proteins are found in the nucleus in distinct developmental stages [33"°,62"].
Two additional members of the cadherin superfamily, the fat and dachsous gene products, were identified in Drosophila [55,56]. T h e y have large tandem arrays of cadherin domains: Fat contains 34 such domains and Dachsous contains 27. Both genes are involved in imaginal disc development and morphogenesis. Genetic interactions between dachsous and fat mutants suggested a model whereby a coupled equilibrium between homophilic and heterophilic interactions of the Dachsous and Fat proteins controls morphogenesis and cell proliferation [56].
Although the genetic analysis in Drosophila has defined the major components of the wingless signaling pathway, two important components have so far been missing, namely the putative receptor of wingless and the transcription factor which putatively interacts with Armadillo and controls the transcription of the target genes. Recent significant findings are that Drosophila Frizzled may act as a receptor for wingless [63"'] and that the nuclear factor lymphoid enhancer factor-1 (LEF-1) binds to 13-catenin (O Huber et al., unpublished data; see Note
Mouse embryos lacking expression of E-cadherin are affected very early in development [50,51"]. E-cadherin negative embryos die around the time of implantation, being unable to form an intact trophectoderm. Compaction at the morula stage (a process known to be mediated by E-cadherin) appears normal due to the presence of some maternal protein. Such reverse genetics clearly demonstrates the essential role of E-cadherin in development.
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added in proof). LEF-1, originally described in pre-B lymphocytes as an HMG-type transcription factor [64], is also highly expressed in mouse embryos at gastrulation stage [65]. LEF-1 fulfills all requirements for being one of the missing components in the wingless/Wnt signaling pathway: it binds to I~-catenin and translocates lS-catenin to the nucleus, and, most importantly, is able to induce dorsal mesoderm when expressed in Xenopus embryos. The interaction of 13-catenin with LEF-1 may restrict the binding of LEF-1 to fewer, selected DNA sites. Interestingly, the LEF-l-I~-catenin complex can bind to the promoter region of the E-cadherin gene, suggesting that the LEF-l-I~-catenin complex regulates E-cadherin transcription; this regulation may well turn out to be a part of the wingless/Wnt signaling pathway, which controls the epithelial-mesenchymal transition during vertebrate development. All these recent findings are assembled in the model depicted in Figure 2. During early mouse embryonic development the most important morphogenetic events are the formation of true epithelia--namely, the trophectoderm and the embryonic ectoderm cell layer. E-cadherin function is crucial for the formation of epithelia and requires the allocation of 13-catenin to
the adherence junctions. At gastrulation stage, some embryonic ectodermal cells generate the mesoderm. This epithelial-mesenchymal transition proceeds with loss of E-cadherin expression and increased expression of LEF-1 [24,65]. Upon Wnt signaling (or via other signals leading into the Wnt signaling pathway), 13-catenin accumulates in the cytoplasm; post-translational modifications of 13-catenin in the cytoplasm make 13-catenin accessible for interaction with LEF-1. In the nucleus, the lS-catenin-LEF-1 complex reduces E-cadherin transcription and possibly also induces mesenchymal gene expression. Thus, 13-catenin and LEF-1 could regulate the very early molecular events involved in generating premesenchymal cells. This working model, although very attractive, is far from complete. More information is needed on the wingless/Wnt receptor, on the cytoplasmic lS-catenin interaction partners such as GSK315 and/or APC [15ee,62"], and on the regulation of LEF-1 expression. Additional, as yet unconceived components probably remain to be discovered, but ~-catenin and LEF-1 probably represent key molecules in the wingless/Wnt signaling pathway. This signaling cascade may be evolutionarily conserved, as the pop-1 gene, which specifies mesodermal precursor cells in the nematode Caenorhabditis elegans, encodes an HMG-box protein homologous to LEF-1 [66].
Figure 2
_~ Wnt
© 1996 Current Opinion in Cell
Biology
Scheme for the epithelial-mesenchymal transition in vertebrate development. At gastrulation stage, the embryonic ectodermal cell layer (depicted as epithelial cells on the left) generates mesenchymal cells (on the right). The maintenance of an epithelial cell layer requires both a functional intercellular E-cadherin-catenin adhesion complex anchored to the actin-filament network (A), and the allocation of 13-catenin (13) to this adhesion complex (full versus dotted arrow in the left-hand cell). For simplicity, only E-cadherin (E) and I~-catenin are depicted as components of the adhesion complex. Upon Writ signaling, ~-catenin accumulates in the cytoplasm (reversed orientation of the full and dotted arrows in the middle cell) and interacts with LEF-1. The complex of LEF-1 and ~-catenin translocates to the nucleus (N), binds to the E-cadherin gene promoter (shown in the right-hand cell) and downregulates E-cadherin gene transcription, which initiates the formation of premesenchymal cells. The complex of LEF-1 and ~-catenin may also positively regulate the transcription of mesenchymal genes.
Cadherins and ¢atenins in development Hub°r, Bierkamp and Kemler 689
Conclusions Accumulating evidence underlines the importance of cadherins and catenins in development. There are at least two ways by which cadherins might control morphogenetic processes. First, the cadherin gene family appears to be much larger than originally expected. Thus, expression of cadherin subtypes with distinct adhesion specificities could represent an important regulatory mechanism. T h e X-ray crystallographic analysis that reveals differences in the three-dimensional structures of E- and N-cadherin represents an important breakthrough in this respect. Second, the fact that different cadherins can stimulate the formation of specific tissues points to a biological function of cadherins beyond cell adhesion and suggests that cadherins are more directly involved in a molecular dialog between the cell membrane and the nucleus. Catenins probably come into play here. I~-catenin in particular appears to be a key element in a novel signal transduction pathway which may well control the epithelial-mesenchymal transition during gastrulation in vertebrate development. In summary, cadherins and catenins, which have long been considered to be structural components of adherence junctions, are turning out to control morphogenesis more directly than originally expected, and this opens up an exciting area of research into cell adhesion molecules.
5. •
Lee SW: H-cadherin, a novel cadhedn with growth inhibitory functions and diminished expression in human breast cancer. Nat Med 1996, 2:776-782. See annotation [6"]. 6. •
FranklinJL, Sargent TD: Ventral neural cadherin, s novel cadherin expressed in a subset of neural tissues in the zebrsfish embryo. Dev Dyn 1995, 206:121-130. This paper, together with [2"-5"], describes newly discovered members of the cadherin gene family. 7. ••
Shapiro L, Fannon AM, Kwong PD, Thompson A, Lehmann MS, Gr0bel G, Legrand J-F, AIs-Nielsen J, Colman DR, Hendrickson WA: Structural basis of cell-cell adhesion by cadherins. Nature 1995, 374:327-337. See annotation [9"']. 8. ••
Overduin M, Harvey TS, Bagby S, Tong KI, Yau P, Takeichi M, Ikura M: Solution structure of the epithelial cadherin domain responsible for selective cell adhesion. Science 1995, 267:386-389. See annotation [9"']. 9. ••
Nagar B, Overduin M, Ikura M, Rini JM: Structural basis of calcium-induced E-cadherin rigidification and dimerization. Nature 1996, 380:360-364. Three very important papers [7"'-9"] elucidating the structural basis for the molecular interactions of N-cadherin and E-cadherin. 10.
Abede H, Schwartz H, Kemler R: Cadherin-catenin complex: protein interactions and their implications for cedherin function. J Cell Biochem 1996, 61:514-523.
11.
Hoschuetzky H, Aberle H, Kemler R: ~-cetenin mediates the interaction of the cadherin-catenin complex with epidermal growth factor receptor. J Cell Biol 1994, 127:1375-1380.
t 2.
RajasekaranAK, Hojo M, Huima T, Rodriguez-Boulon E: Catenins and zonula occludens-1 form a complex during early stages in the assembly of tight junctions. J Cell Biol 1996, 132:451-463.
13.
Rubinfeld B, Souza B, Albert I, MLiller O, Chamberlain SH, Masiarz FR, Munemitsu S, Polakis P: Association of the APC gene product with 13-catenin. Science 1993, 262:1731-t 734.
14.
Su LK, Vogelstein B, Kinzler KW: Association of the APC tumor suppressor protein with catenins. Science 1993, 262:1734-1737.
Note added in proof T h e material referred to in the text as 'O Huber et al., unpublished data' has now been accepted for publication [67].
Acknowledgements We thank Rosemary Schneider and Gesine Kleinw~ichter for typing the manuscript and Randy Cassada for helpful discussions and editorial assistance. This work was supported by the Max-Planck Society and the Minna-James-Heineman Foundation.
References and recommended reading
15. ••
Rubinfeld B, Albert I, Porfiri E, Fiol C, Munemitsu S, Polakis P: Binding of GSK3~ to the APC-~-cetenin complex and regulation of complex assembly. Science 1996, 272:1023-1026. This paper makes an important contribution to our knowledge of the molecular interactions in vertebrate cells that could regulate the cytoplasmic pool of ~-catenin. 16.
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Papers of particular interest, published within the annual period of review, have been highlighted as: • •• 1.
of special interest of outstanding interest Takeichi M: Morphogenetic roles of classic cadherins. Curt Opin Cell Biol 1995, 7:619-627.
2. •
Thomson RB, Igarashi P, Biemesderfer D, Kim R, Abu-Alfa A, SoleimaniM, Aronson PS: Isolation and cDNA cloning of Kspcadherin, a novel kidney-specific member of the cadherin multigene family. J Biol Chem 1995, 270:17594-17601. See annotation [6°]. 3. •
Tashiro K, Tooi O, Nakamura H, Koga C, Ito Y, Hikasa H, Shiokawa K: Cloning and expression studies of cDNA for s novel Xenopus cadherin (XmN-cadherin), expressed maternally end later neural-specifically in embryogenesis. Mech Dev 1995, 54:161-171. See annotation [6"] 4. •
Sugimoto K, Honda S, Yamamoto T, Ueki T, Monden M, Kaji A, Matsumoto K, Nakamura T: Molecular cloning and characterization of a newly identified member of the cadherin familly, PB-cadherin. J Biol Chem 1996, 271:11548-11556. See annotation [6"].
21. Peifer M: Cell adhesion and signal transduction: the Armadillo ~;ee connection. Trends Cell Biol 1995, 5:224-229. annotation [22"]. 22. Gumbiner BM: Signal trsnsduction by ~-catenin. Curt Opin Cell hese Bio/1995, 7:634-640. papers [21",22"] are thoughtful reviews on cell adhesion and signal transduction by Armadillo/~-catenin.
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Ohsugi M, Hwang S, Butz S, Knowles BB, Solter D, Kemler R: Expression and cell membrane localization of catenins during mouse preimplantetion development. Day Dyn 1996, 206:391-402.
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Butz S, Lame L: Expression of catenins during mouse embryonic development and in adult tissues. Cell Adhes Commun 1995, 3:337-352.
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MatsunamiH, Takeichi M: Fetal brain subdivisions defined by Rand E-cadherin expressions: evidence for the role of cadherin activity in region-specific, cell-cell adhesion. Dev Biol 1995, 172:466-478.
26.
FannonAM, Sherman DL, Ilyina-Gragerova G, Brophy PJ, Friedrich VL, Colman DR: Novel E-cadherin-mediated adhesion in peripheral nerve: Schwann cell architecture is stabilized by autotypic adherens junctions. J Cell Biol 1995, 129:189-202.
27. •
HoffmannI, Bailing R: Cloning and expression analysis of a novel mesodermally expressed cadherin. Dev Biol 1995, 169:337-346. See annotation [29"]. 28. •
Kimura'I', Matsunami H, Inoue T, Shimamura K, Uchida N, Ueno T, Miyazaki T, Takeichi M: Cadherin-11 expressed in association with mesenchymal morphogenesis in the head, somite, and limb bud of early mouse embryos. Dev Biol 1995, 169:347-358. See annotation [29"]. 29. •
SimonneauL, Kitagawa M, Suzuki S, Thiery JP: Cad-11 expression marks the mesenchymal phenotype: towards new functions for cadherins? Cell Adhes Commun 1995, 3:115-130. Together with [27",28"], this paper characterizes cad-11 as a mesodermal cadherin. 30.
Zeschnigk M, Kozian D, Kuch C, Schmoll M, Starzinski-Powitz A: Involvement of M-cadherin in terminal differentiation of skeletal muscle cells. J Cell Sci 1995, 108:2973-2981.
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Tavella S, Raffo P, Tacchetti C, Cancedda R, Castagnola P: N-CAM and N-cadherin expression during in vitro chondrogenesis. Exp Cell Res 1994, 215:354-362.
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Redias C: Cadherin expression in the developing vertebrate CNS: from neuromeres to brain nuclei and neural circuits. Exp Cell Res 1995, 220:243-256.
43. •
Hermiston M, Gordon Jl: Inflammatory bowel disease and adenomas in mice expressing a dominant negative N-cadherin. Science 1995, 270:1203-1206. In this paper and [42",44"], the authors describe transgenic mice models which were established to study the function of cadherins by heterotypic expression. 44.
Hermiston ML, Gordon JL: In vivo analysis of cadherin function in the mouse intestinal epithelium: essential roles in adhesion, maintenance of differentiation, and regulation of programmed cell death. J Cell Bio/1995, 129:489-506. See annotation [43"]. •
45. •,
MarrsJA, Andersson-Rsone C, Jeong MC, Cohen-Gould L, Zurzolo C, Nabi IR, Rodriguez-Boulan E, Nelson WJ: Plasticity in epithelial cell phenotype: modulation by expression of different cadherin cell adhesion molecules. J Cell Biol 1995, 129:507-519, See annotation [46"']. 46. •e
Larue L, Antos C, Butz S, Huber O, Delmas V, Dominis M, Kemler R: A role for cadherins in tissue formation. Development 1996, in press. These papers [45"',46"] give strong support for a role of cadherins in histogenesis and signal transduction. 47. •
FunayamaN, Fagotto F, McCrea P, Gumbiner BM: Embryonic axis induction by the armadillo repeat domain of ~-catenin: evidence for intracellular signaling. J Cell Bio/1995, 128:959-968.
See annotation [49"]. 48. •
Kelly GM, ErezyUmazDF, Moon RT: Induction of a secondary embryonic axis in zebrafish occurs following the overexpression of ~-catenin. Mech Dev 1995, 53:261-273. See annotation [49"]. 49. •
Karnovsky A, Klymkowsky MW: Anterior axis duplication in Xenopus induced by the over-expression of the cadherinbinding protein plakoglobin. Proc Nat/Acad Sci USA 1995, 92:4522-4526. These papers [47"-49"] show that overexpression of ~-catenin or plakoglobin results in dorsal mesoderm induction. 50.
Lame L, Ohsugi M, Hirchenhain J, Kemler R: E-cadherin null mutant embryos fail to form a trophectoderm epithelium. Proc Natl Acad Sci USA 1994, 91:8263-8267.
Schneider S, Steinbeisser H, Warga RM, Hausen P: Bets-catenin translocation into nuclei demarcates the dorsalizing centers in frog and fish embryos. Mech Day 1996, 57:191-198. Describes the first demonstration that, during normal frog and fish development, ~-catenin becomes transiently accumulated in the nuclei of cells.
Riethmacher D, Brinkmann V, Birchmeier C: A targeted mutation in the mouse E-cadherin gene results in defective preimplantetion developmenL Proc Natl Acad Sci USA 1995, 92:855-859. The authors of [50,51"] demonstrate, by gene-targeting experiments, the importance of E-cadherin in trophectoderm formation.
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Kintner C: Regulation of embryonic cell adhesion by the cadherin cytoplasmic domain. Cell 1992, 69:225-236.
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DufourS, Saint Jeannet JP, Broders F, Wedlich D, Thiery JP: Differential perturbations in the morphogenesis of anterior structures induced by overexpression of truncated XB- and Ncadherins in Xenopus embryos. J Cell Biol 1994, 127:521-535.
33. ••
36.
Broders F, Thiery J-P: Contribution of cadherin to directional cell migration and histogenesis in Xenopus embryos. Cell Adhes Commun 1995, 3:419-440.
37.
K,'ihl M, Finnemann S, Binder O, Wedlich D: Dominant negative expression of a cytoplasmically deleted mutant of XB/Ucadherin disturbs mesoderm migration during gastrulation in Xenopus laevis. Mech Dev 1996, 54:71-82.
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Lee C-H, Gumbiner M: Disruption of gastrulation movements in Xenopus by a dominant-negative mutant for C-cadherin. Dev Bio11995, 171:363-373.
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Levine E, Lee CH, Kintner C, Gumbiner BM: Selective disruption of E-cadherin function in early Xenopus embryos by a dominant negative mutant. Development 1994, 120:901-909.
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41.
Holt CE, Lemalre P, Gurdon JB: Cadherin-modlated cell interactions are necessary for the activation of MyoD in Xenopus mesoderm. Proc Nat/Acad Sci USA 1994, 91:10844-10848.
42. •~ee
Dahl U, Sj~din A, Semb H: Cadherins regulate aggregation of pancreatic ~-cells in vivo. Development 1996, 122:2895-2902. annotation [43"].
51. •
Oda H, Uemura T, Harada Y, Iwal Y, Takeichi M: A Drosophila homolog of cadherin associated with armadillo and essential for embryonic cell-cell adhesion. Dev Biol 1994, 165:716-726.
53.
UemuraT, Oda H, Kraut R, Hayashi S, Kataoka Y, Takeichi M: Zygotic Drosophila E-cadherin expression is required for processes of dynamic epithelial cell rearrangement in the Drosophila embryo. Genes Day 1996, 10:659-671. See annotation [54"]. •
54.
Tepass U, Gruszynski-DeFeo E, Haag TA, Omatyar L, T6r6k T, Hartanstein V: Shotgun encodes Drosophila E-cadherin and is preferentially required during cell rearrangement in the neuroectoderm and other morphogenetically active epithelia. Genes Dev 1996, 10:672-685. This paper, together with [52,53"], describes the characterization of Drosophila E-cadherin. •
55.
Mahoney PA, Weber U, Onofrechuk P, Biessmann H, Bryant PJ, Goodman CS: The fat tumor suppressor gene in Drosophila encodes a novel member of the cadherin gene superfamily. Cell 1991, 67:853-868.
5~.
Clark HF, Brentrup D, Schneitz K, Bieber A, Goodman C, Noll M: Dachsous encodes a member of the cadhedn superfamily that controls imaginal disc morphogenesis in Drosophila. Genes Dev 1995, 9:1530-1542.
57. •
Haegel H, Larue L, Ohsugi M, Fedorov L, Herranknecht K, Kemler R: Lack of ~-catenin affects mouse development at gastrulation. Development 1995, 121:3529-3537. Lack of ~-catenin affects mesoderm formation, as demonstrated by gene targeting in mice [57"] and by antisense experiments in Xenopus [58]. 58.
Heasman J, Crawford A, Goldstone K, Garner-Hamrick P, Gumbiner B, McCrea P, Kintner C, Noro CY, Wylie C: Overexpression of cadherins and underexpression of ~-catenin
Cadherins and catenins in development Huber, Bierkamp and Kemler 691
59.
inhibit dorsal mesoderm induction in eady Xenopus embryos. Cell 1994, 79:791-803. Heasman J, Ginsberg D, Geiger B, Goldstone K, Pratt T, YoshidaNoro C, Wylie C: A functional test for maternally inherited cadherin in Xenopus shows its importance in cell adhesion at the blastula stage. Development 1994, 120:49-57.
60. •
Orsulic S, Peifer M: An in vivo structure-function study of Armadillo, the 13-catenin homolog, reveals both separate and overlapping regions of the protein required for call adhesion and for wingless signaling. J Cell Bio11996, in press. See annotation [62"]. 61. •
Papkoff J, Rubinfald B, Schryver B, Polakis P: Wnt-1 regulates free pools of catenins and stabilizes APC-catenin complexes. Mo/ Cell Biol 1996, 16:2128-2134. See annotation [62"]. 62. Yost C, Torres M, Miller JR, Huang E, Kimelman D, Moon RT: The • axis-inducing activity, stability, and subcellular distribution of ~-cetenin is regulated in Xenopus embryos by glycogen synthase kinase 3. Genes Dev 1996, 10:1443-1454. These papers [60"-62"] describe the characterization of the molecular interactions of the components of the winglessANnt signaling pathway. See also [15"'].
63. oe
Bhanot P, Brink M, Samos CH, Hsieh J-C, Wang Y, Macke JP, Andrew D, Nathans J, Nusse R: A new member of the fr/zz/ed family from Drosophila functions as a Wingless receptor. Nature 1996, 382:225-230. Describes the first identification of Drosophila Frizzled as a wingless receptor. 64.
Travis A, Amsterdam A, Belanger C, Grosschedl R: LEF-1, a gene encoding a lymphoid-specific protein with an HMG domain, regulates T-cell receptor (~ enhancer function. Genes Dev 1991, 5:880-894.
65.
Oosterwegel M, Van de Wetering M, Timmermann J, Kruisbeek A, Destree O, Meijlink F, Clevers H: Differential expression of the HMG box factors TCF-1 and LEF-1 during murine embryogenesis. Development 1993, 118:439-448.
66.
Lin R, Thompson S, Priess JR: pop-1 encodes an HMG box protein required for the specification of mesoderm precursor in early C. elegans embryos. Cell 1995, 83:599-609.
67.
Huber O, Kom R, McLaughtin J, Ohsugi M, Herrmann BG, Kemler R: Nuclear localization of 13-catenin by interaction with transcription factor LEF-1. Mech Dev 1996, in press.
Cadherins and catenins in development Otmar Huber, Christiane Bierkamp and Rolf Kemler* Cadherinsand cateninsrepresentkey moleculesduring development.Recentfindingsdemonstratethe involvement of cadherinsand cateninsin signalingpathways.In a workinghypothesis,signalingvia ~-cateninregulatesthe epithelial-mesenchymaltransitionin vertebratedevelopment. Addresses Max-Planck Institute for Immunobiology, Department of Molecular Embryology, StLibeweg 51, D-79108 Freiburg, Germany "e-mail: [email protected] Correspondence: Rolf Kemler
proteins, that is, desmocollins and desmogleins [16]. Quite detailed analysis, including site-directed mutations, has identified the binding sites in 13-catenin and plakoglobin that regulate protein-protein interactions [17-20]. The finding that [3-catenin and plakoglobin are homologous to the Drosophila Armadillo protein led to the identification of a whole Armadillo protein family, which includes p120, APC, and importin. Even more importantly, this homology suggested that [~-catenin and/or plakoglobin might also exhibit a signaling function [21°,22"], as will be discussed below.
Current Opinion in Cell Biology 1996, 8:685-691 © Current Biology Lid ISSN 0955-0674
Abbreviations APC adenomatous polyposis coli GSK313 glycogen synthase kinase 313 LEF-1 ZO-1
lymphoid enhancer factor-1 zonula occludens-1 protein
Figure 1
EGFreceptor Cadherin
ZO'I~
Desmoglein/desmocollin
~_~teni~ Plakoglobin
Introduction Cadherins represent a distinct family of single-transmembranedomain glycoproteins which serve as specific cell adhesion molecules acting in a Ca2÷-dependent manner, largely by homophilic protein-protein interaction [1]. For many new members of this protein family, identified by structural homology, an involvement in cell adhesion has not yet been shown, and in fact some may exhibit other biological functions [2°-6"]. T h e classical cadherins, including E-, N-, and P-cadherin, are the best studied representatives of the family; recently, the three-dimensional structures of the extracellular domains of E- and N-cadherin were published [7"'-9"']. Classical cadherins are defined by a highly conserved cytoplasmic domain and the capability to form complexes with the catenins--that is, (x-catenin, lS-catenin, plakoglobin (T-catenin), and p120 [I0]. Several lines of evidence indicate that cadherins and catenins are key molecules in adhesion-dependent morphogenetic processes during development. Before reviewing recent progress here, we will summarize briefly the molecular architecture of the cadherin-catenin complex and survey newly discovered interaction partners of catenins (Fig. 1). Catenins link cadherins to the actin filament network [10]; now it appears that they also interact with other transmembrane or cytoplasmic proteins as more general linkers or adapters. For example, 13-catenin associates with the epidermal growth factor (EGF) receptor [11], the tight junction protein ZO-1 (zonula occludens-1 protein) [12], and the adenomatous polyposis coli (APC) tumor suppressor protein [13,14]. Remarkably, the APC-I]-catenin interaction is regulated by glycogen synthase kinase 313 (GSK313), a component of the wingless/Wnt signaling pathway [15"°]. Plakoglobin also interacts with the desmosomal
APC~-'SK313 ~'/~.-catenin ~
APC © 1996CurrentOpinionin CellBiology
Catenins play multiple roles as linker or adapter proteins. This scheme shows the major interaction partners for I~-catenin and plakoglobin described so far [11,12,15°°,16-90].
In this review, we want to survey the three major arguments that cadherins and catenins represent key regulatory molecules during development: first, the expression patterns for cadherins correlate with distinct morphogenetic events; second, overexpression of variants of cadherins and catenins (using a dominant-negative approach) leads to perturbations in morphogenesis; and third, loss-of-function experiments demonstrate the importance of the cadherins and catenins in developmental processes.
Expressionpatternsduringdevelopment T h e first clue that cadherins might be important regulatory molecules during development came from the fact that their individual expression patterns are, very often, correlated with distinct morphogenetic events such as cell migration, cell sorting, organogenesis, and tissue remodeling [1]. E-cadherin is expressed very early on embryonic cells [231, and is also expressed later during development, mainly on epithelial cells. In mouse embryos, the first cell type that is negative for E-cadherin is the forming mesoderm at the gastrulation stage [24]. Mesoderm derivatives remain negative for E-cadherin, but the protein becomes re-expressed in cells undergoing
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a mesenchymal-epithelial transition. E-cadherin is also expressed in the mouse embryonic brain [25] and a subset of sensory neurons, in addition to in a specific type of adherence junction in Schwann cells [26]. Cadherins are detected in mesenchymal cells and their derivatives [27°-29°], a somewhat unexpected finding as mesenchyme is regarded as a loose tissue and cadherins have been thought to regulate cell adhesion in solid tissues. In fact, cad-11 is expressed rather early in mouse embryos in the mesenchymes surrounding organ-anlages, in the cephalic mesoderm, and during somite formation [27°-29°]. Although cad-ll is currently considered to be the major mesenchymal cadherin, M-cadherin and N-cadherin are expressed in other mesenchymal components such as muscle or condensing chondrocytes [30,31]. Most of the recently identified cadherins are expressed in the developing vertebrate brain [32]. T h e primordial neural tube continuously expresses N-cadherin, whereas R-, B-, E-, and T-cadherins are expressed only at later stages of brain development; this expression correlates with distinct regionalization of the central nervous system. Catenins, in contrast, are expressed rather ubiquitously, although some distinct differences can be recognized [24]. During mouse preimplantation development, both c~-catenin and [3-catenin are maternally provided as protein and mRNA, and the embryonic genes become activated at the late two-cell stage [23]. Plakoglobin exhibits a distinct expression profile, lacking the maternal component and appearing first at late morula stage. Importantly, [3-catenin transiently accumulates in nuclei on the dorsal side of Xenopus and zebrafish blastulae ([33°°]; see below).
Dominant-negative
approach
In general, overexpression of two types of dominant-negative constructs has been used to study the importance of either the extracellular or the cytoplasmic domain of cadherins. An overexpressed cadherin with most of the extracellular domain deleted but the cytoplasmic domain intact (Aextra) still binds to catenins and thereby reduces their availability for the endogenous cadherins. A cadherin with the extracellular domain intact and the cytoplasmic domain deleted (Acyto) appears to interact in a homophilic manner with the endogenous cadherin but is unable to form a functional cadherin-catenin complex. Injecting Aextra N-cadherin mRNA into early Xenopus embryos results in a dramatic inhibition of cell adhesion and a perturbation of the integrity of the ectodermal cell layer at midgastrulation [34]. Similarly, injecting Aextra mRNAs for N-cadherin, XB-cadherin, or both E-cadherin and EP-cadherin into specific blastomeres induces their local overproduction and results in disorganization of the tissues descending from the injected cells [35,36]. XB-cadherin is more effective than N-cadherin, and EP-cadherin is more effective than E-cadherin, in inducing these perturbations. An attractive interpretation of these results is that these
varying extents of interference reflect different affinities of the cadherins for catenins or other cytoskeletal structures. Overexpression of the second type of mutant constructs has unraveled the more specific functions of each cadherin [37,38]. Injection of Acyto N-cadherin mRNA leads to perturbation of neural tube formation; analogously, E-cadherin seems to be specifically required for maintaining the integrity of the ectoderm during epiboly at gastrulation [39]. Interestingly, overexpression of full-length C-cadherin, a close homolog of E-cadherin, fails to compensate for the loss of E-cadherin function, and ectopic expression of full-length C-cadherin or N-cadherin in wild-type cells can even cause ectodermal lesions [39,40]. Differential expression of cadherins thus evidently reflects specific requirements for distinct adhesive properties during different morphogenetic events. However, in these experiments injected mRNA overrides all transcriptional regulatory mechanisms that could normally regulate E-cadherin expression, sometimes causing novel dominant defects. Cadherins may also be involved in cell differentiation events: overexpression of Aextra N-cadherin in the presumptive muscle anlage of early Xenopus embryos results in suppression of muscle differentiation [41]. As muscle precursors develop from a community of cells, a role of cadherins in the community effect is suggested here. Transgenic mice overexpressing Aextra genes for N-cadherin and E-cadherin that are driven by various tissue type specific promoters can be used to study cadherin function in specific cell lineages of particular organs, such as the small intestine and pancreatic 13 cells [42°,43°]. A dramatic effect is observed when expression of Aextra N-cadherin is directed to mouse intestinal epithelial cells: cell-cell and cell-matrix contacts are disrupted; the differentiated polarized phenotype is lost; and precocious entry into a cell death program occurs, indicating a linkage between cell adhesion, signaling and apoptosis [43°,44°]. Ectopic expression of E-cadherin in a retinal pigment epithelial cell line which endogenously expresses other cadherin subtypes induces epithelial cell polarity and even causes de novo assembly of desmosomes [45°°]. This remarkable result points to an additional role for E-cadherin in addition to its known function in cell adhesion. Equally significant in this regard are recent observations that specific cadherins can stimulate the formation of specific tissues [46°°]. By gene targeting, embryonic stem (ES) cells lacking E-cadherin and exhibiting a cell adhesion defect were generated; this phenotype could be rescued by expressing either E-cadherin or N-cadherin cDNA driven by a constitutive promoter. Remarkably, such mutant ES cells rescued with E-cadherin were only capable of forming epithelia, whereas the same cells that were rescued with N-cadherin formed cartilage and neuroepithelium. These results give strong support for
Cadherinsand cateninsin developmentHuber, Bierkamp and Kemler 687
the idea of a role of cadherins in histogenesis, again suggesting that, in addition to playing a role in cell adhesion, cadherins participate more directly in a signaling pathway. T h e involvement of catenins in developmental processes has also been studied. Overexpression of wild-type 13catenin on the ventral side of Xenopus or zebrafish embryos induces the formation of a complete secondary body axis [47",48"]. Expression of deletion mRNA constructs of 13-catenin shows that the Armadillo (Arm)-repeat region is both necessary and sufficient to induce axis duplication, indicating participation in the generation of intracellular signals involved in the specification of dorsal mesoderm. Similar experiments carried out with plakoglobin again showed that the complete coding region of plakoglobin, or just the central Arm repeat region, is also sufficient to induce axis duplication [49"].
Loss-of-function analysis An alternative way to gain insights into the developmental function of cadherins and catenins is to inhibit their expression. Two different approaches can be used, namely gene-targeting experiments leading to nonfunctional (knocked-out) genes or antisense experiments to deplete the mRNA and reduce specific protein expression.
Mouse embryos lacking 13-catenin die in utero at gastrulation with a specific defect in the embryonic ectodermal cell layer, despite the rather ubiquitous expression of 13-catenin in wild-type cells at this developmental stage [57"]. Mutant cells detach from the embryonic ectodermal cell layer and are dispersed in the proamniotic cavity, and no mesoderm is~formed, as monitored by the absence of T-brachyury gene expression. Plakoglobin cannot substitute for the absent 13-catenin during mouse development even though it also b i n d s to E-cadherin and oc-catenin. In Xenopus, depletion of 13-catenin affects dorsal mesoderm induction [58]; this was explained by proposing a role for 13-catenin in signaling pathways. An antisense approach was also used to reduce the level of EP-cadherin mRNA and protein in the Xenopus embryo [59]. This depletion leads to a dose-dependent reduction of adhesion between blastomeres. With high concentrations of the most effective antisense oligonucleotides, embryos fail to undergo gastrulation and arrest at the blastula stage, showing the need for maternal EP-cadherin in this process.
13-catenin signaling in vertebrate development
In Drosophila the shotgun gene locus encodes the homolog of E-cadherin (DE-cadherin) [52,53",54"]. Null mutant embryos expressing no zygotic DE-cadherin exhibit no severe defects in general epithelial organization, because maternally derived DE-cadherin is sufficient, but formation of epithelia involving extensive morphogenetic movements (e.g. forming Malphigian tubules and tracheal ducts) is impaired. Complete absence of maternal and zygotic shotgun expression impairs formation of all epithelia.
T h e homology of 13-catenin and plakoglobin to the Drosophila segment polarity protein Armadillo has recently attracted much attention [21°,22"]. It was intriguing that these components of cell adherence junctions share a common structure with this component of the wingless signal transduction pathway; this suggested that these molecules have similar biochemical functions. This has now been confirmed. In Drosophila, Armadillo is found in adherence junctions together with (~-catenin and DE-cadherin [52] and probably plays another independent role in wingless signaling [21",60"]. As mentioned, accumulating evidence also implicates 13-catenin in a signaling pathway. Overexpression of 13-catenin in Xenopus embryos results in dorsal mesoderm induction, as does expression of Xwnt8, a vertebrate homolog of the wingless protein [47"]. Inhibition of 13-catenin expression suppresses mesoderm formation in both Xenopus and mouse [57",58]. Moreover, 13-catenin in cultured mouse cells and Armadillo in Drosophila accumulate in the cytoplasm upon wingless/Wnt signaling [60",61"] and both proteins are found in the nucleus in distinct developmental stages [33"°,62"].
Two additional members of the cadherin superfamily, the fat and dachsous gene products, were identified in Drosophila [55,56]. T h e y have large tandem arrays of cadherin domains: Fat contains 34 such domains and Dachsous contains 27. Both genes are involved in imaginal disc development and morphogenesis. Genetic interactions between dachsous and fat mutants suggested a model whereby a coupled equilibrium between homophilic and heterophilic interactions of the Dachsous and Fat proteins controls morphogenesis and cell proliferation [56].
Although the genetic analysis in Drosophila has defined the major components of the wingless signaling pathway, two important components have so far been missing, namely the putative receptor of wingless and the transcription factor which putatively interacts with Armadillo and controls the transcription of the target genes. Recent significant findings are that Drosophila Frizzled may act as a receptor for wingless [63"'] and that the nuclear factor lymphoid enhancer factor-1 (LEF-1) binds to 13-catenin (O Huber et al., unpublished data; see Note
Mouse embryos lacking expression of E-cadherin are affected very early in development [50,51"]. E-cadherin negative embryos die around the time of implantation, being unable to form an intact trophectoderm. Compaction at the morula stage (a process known to be mediated by E-cadherin) appears normal due to the presence of some maternal protein. Such reverse genetics clearly demonstrates the essential role of E-cadherin in development.
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Cell-to-cell contact and extracellular matrix
added in proof). LEF-1, originally described in pre-B lymphocytes as an HMG-type transcription factor [64], is also highly expressed in mouse embryos at gastrulation stage [65]. LEF-1 fulfills all requirements for being one of the missing components in the wingless/Wnt signaling pathway: it binds to I~-catenin and translocates lS-catenin to the nucleus, and, most importantly, is able to induce dorsal mesoderm when expressed in Xenopus embryos. The interaction of 13-catenin with LEF-1 may restrict the binding of LEF-1 to fewer, selected DNA sites. Interestingly, the LEF-l-I~-catenin complex can bind to the promoter region of the E-cadherin gene, suggesting that the LEF-l-I~-catenin complex regulates E-cadherin transcription; this regulation may well turn out to be a part of the wingless/Wnt signaling pathway, which controls the epithelial-mesenchymal transition during vertebrate development. All these recent findings are assembled in the model depicted in Figure 2. During early mouse embryonic development the most important morphogenetic events are the formation of true epithelia--namely, the trophectoderm and the embryonic ectoderm cell layer. E-cadherin function is crucial for the formation of epithelia and requires the allocation of 13-catenin to
the adherence junctions. At gastrulation stage, some embryonic ectodermal cells generate the mesoderm. This epithelial-mesenchymal transition proceeds with loss of E-cadherin expression and increased expression of LEF-1 [24,65]. Upon Wnt signaling (or via other signals leading into the Wnt signaling pathway), 13-catenin accumulates in the cytoplasm; post-translational modifications of 13-catenin in the cytoplasm make 13-catenin accessible for interaction with LEF-1. In the nucleus, the lS-catenin-LEF-1 complex reduces E-cadherin transcription and possibly also induces mesenchymal gene expression. Thus, 13-catenin and LEF-1 could regulate the very early molecular events involved in generating premesenchymal cells. This working model, although very attractive, is far from complete. More information is needed on the wingless/Wnt receptor, on the cytoplasmic lS-catenin interaction partners such as GSK315 and/or APC [15ee,62"], and on the regulation of LEF-1 expression. Additional, as yet unconceived components probably remain to be discovered, but ~-catenin and LEF-1 probably represent key molecules in the wingless/Wnt signaling pathway. This signaling cascade may be evolutionarily conserved, as the pop-1 gene, which specifies mesodermal precursor cells in the nematode Caenorhabditis elegans, encodes an HMG-box protein homologous to LEF-1 [66].
Figure 2
_~ Wnt
© 1996 Current Opinion in Cell
Biology
Scheme for the epithelial-mesenchymal transition in vertebrate development. At gastrulation stage, the embryonic ectodermal cell layer (depicted as epithelial cells on the left) generates mesenchymal cells (on the right). The maintenance of an epithelial cell layer requires both a functional intercellular E-cadherin-catenin adhesion complex anchored to the actin-filament network (A), and the allocation of 13-catenin (13) to this adhesion complex (full versus dotted arrow in the left-hand cell). For simplicity, only E-cadherin (E) and I~-catenin are depicted as components of the adhesion complex. Upon Writ signaling, ~-catenin accumulates in the cytoplasm (reversed orientation of the full and dotted arrows in the middle cell) and interacts with LEF-1. The complex of LEF-1 and ~-catenin translocates to the nucleus (N), binds to the E-cadherin gene promoter (shown in the right-hand cell) and downregulates E-cadherin gene transcription, which initiates the formation of premesenchymal cells. The complex of LEF-1 and ~-catenin may also positively regulate the transcription of mesenchymal genes.
Cadherins and ¢atenins in development Hub°r, Bierkamp and Kemler 689
Conclusions Accumulating evidence underlines the importance of cadherins and catenins in development. There are at least two ways by which cadherins might control morphogenetic processes. First, the cadherin gene family appears to be much larger than originally expected. Thus, expression of cadherin subtypes with distinct adhesion specificities could represent an important regulatory mechanism. T h e X-ray crystallographic analysis that reveals differences in the three-dimensional structures of E- and N-cadherin represents an important breakthrough in this respect. Second, the fact that different cadherins can stimulate the formation of specific tissues points to a biological function of cadherins beyond cell adhesion and suggests that cadherins are more directly involved in a molecular dialog between the cell membrane and the nucleus. Catenins probably come into play here. I~-catenin in particular appears to be a key element in a novel signal transduction pathway which may well control the epithelial-mesenchymal transition during gastrulation in vertebrate development. In summary, cadherins and catenins, which have long been considered to be structural components of adherence junctions, are turning out to control morphogenesis more directly than originally expected, and this opens up an exciting area of research into cell adhesion molecules.
5. •
Lee SW: H-cadherin, a novel cadhedn with growth inhibitory functions and diminished expression in human breast cancer. Nat Med 1996, 2:776-782. See annotation [6"]. 6. •
FranklinJL, Sargent TD: Ventral neural cadherin, s novel cadherin expressed in a subset of neural tissues in the zebrsfish embryo. Dev Dyn 1995, 206:121-130. This paper, together with [2"-5"], describes newly discovered members of the cadherin gene family. 7. ••
Shapiro L, Fannon AM, Kwong PD, Thompson A, Lehmann MS, Gr0bel G, Legrand J-F, AIs-Nielsen J, Colman DR, Hendrickson WA: Structural basis of cell-cell adhesion by cadherins. Nature 1995, 374:327-337. See annotation [9"']. 8. ••
Overduin M, Harvey TS, Bagby S, Tong KI, Yau P, Takeichi M, Ikura M: Solution structure of the epithelial cadherin domain responsible for selective cell adhesion. Science 1995, 267:386-389. See annotation [9"']. 9. ••
Nagar B, Overduin M, Ikura M, Rini JM: Structural basis of calcium-induced E-cadherin rigidification and dimerization. Nature 1996, 380:360-364. Three very important papers [7"'-9"] elucidating the structural basis for the molecular interactions of N-cadherin and E-cadherin. 10.
Abede H, Schwartz H, Kemler R: Cadherin-catenin complex: protein interactions and their implications for cedherin function. J Cell Biochem 1996, 61:514-523.
11.
Hoschuetzky H, Aberle H, Kemler R: ~-cetenin mediates the interaction of the cadherin-catenin complex with epidermal growth factor receptor. J Cell Biol 1994, 127:1375-1380.
t 2.
RajasekaranAK, Hojo M, Huima T, Rodriguez-Boulon E: Catenins and zonula occludens-1 form a complex during early stages in the assembly of tight junctions. J Cell Biol 1996, 132:451-463.
13.
Rubinfeld B, Souza B, Albert I, MLiller O, Chamberlain SH, Masiarz FR, Munemitsu S, Polakis P: Association of the APC gene product with 13-catenin. Science 1993, 262:1731-t 734.
14.
Su LK, Vogelstein B, Kinzler KW: Association of the APC tumor suppressor protein with catenins. Science 1993, 262:1734-1737.
Note added in proof T h e material referred to in the text as 'O Huber et al., unpublished data' has now been accepted for publication [67].
Acknowledgements We thank Rosemary Schneider and Gesine Kleinw~ichter for typing the manuscript and Randy Cassada for helpful discussions and editorial assistance. This work was supported by the Max-Planck Society and the Minna-James-Heineman Foundation.
References and recommended reading
15. ••
Rubinfeld B, Albert I, Porfiri E, Fiol C, Munemitsu S, Polakis P: Binding of GSK3~ to the APC-~-cetenin complex and regulation of complex assembly. Science 1996, 272:1023-1026. This paper makes an important contribution to our knowledge of the molecular interactions in vertebrate cells that could regulate the cytoplasmic pool of ~-catenin. 16.
Witcher LL, Collins R, Puttagunta S, Mechanic SE, Munson M, Gumbiner B, Cowin P: Desmosomal cadherin binding domains in plakoglobin. J Biol Chem 1996, 271:10904-10909.
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Aberle H, Schwartz H, Hoschuetzky H, Kemler R: Single amino acid substitutions in proteins of the armadillo gene family abolish their binding to (x-catenin. J Biol Chem 1996, 271:1520-1526.
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Sacco PA, McGranahan TM, Wheelock MJ, Johnson KR: Identification of plakoglobin domains required for association with N-cadherin and (x-catenin. J Biol Chem 1995, 270:20201-20206.
19.
Ozawa M, Terada H, Pedraza C: The fourth armadillo repeat of plakoglobin (~-catenin) is required for its high affinity binding to the cytoplasmic domains of E-cadherin and desmosomal cedherin Dsg2, and the tumor suppressor APC protein. J Biochem t 995, 118:1077-1082.
20.
Wahl JK, Sacco PA, McGranahan-Sadler TM, Sauppe LM, Wheelock MJ, Johnson KR: Plakoglobin domains that define its association with the desmosomsl cedherins and the classical cadherins: identification of unique and shared domains. J Cell Sci 1996, 109:1143-1154.
Papers of particular interest, published within the annual period of review, have been highlighted as: • •• 1.
of special interest of outstanding interest Takeichi M: Morphogenetic roles of classic cadherins. Curt Opin Cell Biol 1995, 7:619-627.
2. •
Thomson RB, Igarashi P, Biemesderfer D, Kim R, Abu-Alfa A, SoleimaniM, Aronson PS: Isolation and cDNA cloning of Kspcadherin, a novel kidney-specific member of the cadherin multigene family. J Biol Chem 1995, 270:17594-17601. See annotation [6°]. 3. •
Tashiro K, Tooi O, Nakamura H, Koga C, Ito Y, Hikasa H, Shiokawa K: Cloning and expression studies of cDNA for s novel Xenopus cadherin (XmN-cadherin), expressed maternally end later neural-specifically in embryogenesis. Mech Dev 1995, 54:161-171. See annotation [6"] 4. •
Sugimoto K, Honda S, Yamamoto T, Ueki T, Monden M, Kaji A, Matsumoto K, Nakamura T: Molecular cloning and characterization of a newly identified member of the cadherin familly, PB-cadherin. J Biol Chem 1996, 271:11548-11556. See annotation [6"].
21. Peifer M: Cell adhesion and signal transduction: the Armadillo ~;ee connection. Trends Cell Biol 1995, 5:224-229. annotation [22"]. 22. Gumbiner BM: Signal trsnsduction by ~-catenin. Curt Opin Cell hese Bio/1995, 7:634-640. papers [21",22"] are thoughtful reviews on cell adhesion and signal transduction by Armadillo/~-catenin.
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23.
Ohsugi M, Hwang S, Butz S, Knowles BB, Solter D, Kemler R: Expression and cell membrane localization of catenins during mouse preimplantetion development. Day Dyn 1996, 206:391-402.
24.
Butz S, Lame L: Expression of catenins during mouse embryonic development and in adult tissues. Cell Adhes Commun 1995, 3:337-352.
25.
MatsunamiH, Takeichi M: Fetal brain subdivisions defined by Rand E-cadherin expressions: evidence for the role of cadherin activity in region-specific, cell-cell adhesion. Dev Biol 1995, 172:466-478.
26.
FannonAM, Sherman DL, Ilyina-Gragerova G, Brophy PJ, Friedrich VL, Colman DR: Novel E-cadherin-mediated adhesion in peripheral nerve: Schwann cell architecture is stabilized by autotypic adherens junctions. J Cell Biol 1995, 129:189-202.
27. •
HoffmannI, Bailing R: Cloning and expression analysis of a novel mesodermally expressed cadherin. Dev Biol 1995, 169:337-346. See annotation [29"]. 28. •
Kimura'I', Matsunami H, Inoue T, Shimamura K, Uchida N, Ueno T, Miyazaki T, Takeichi M: Cadherin-11 expressed in association with mesenchymal morphogenesis in the head, somite, and limb bud of early mouse embryos. Dev Biol 1995, 169:347-358. See annotation [29"]. 29. •
SimonneauL, Kitagawa M, Suzuki S, Thiery JP: Cad-11 expression marks the mesenchymal phenotype: towards new functions for cadherins? Cell Adhes Commun 1995, 3:115-130. Together with [27",28"], this paper characterizes cad-11 as a mesodermal cadherin. 30.
Zeschnigk M, Kozian D, Kuch C, Schmoll M, Starzinski-Powitz A: Involvement of M-cadherin in terminal differentiation of skeletal muscle cells. J Cell Sci 1995, 108:2973-2981.
31.
Tavella S, Raffo P, Tacchetti C, Cancedda R, Castagnola P: N-CAM and N-cadherin expression during in vitro chondrogenesis. Exp Cell Res 1994, 215:354-362.
32.
Redias C: Cadherin expression in the developing vertebrate CNS: from neuromeres to brain nuclei and neural circuits. Exp Cell Res 1995, 220:243-256.
43. •
Hermiston M, Gordon Jl: Inflammatory bowel disease and adenomas in mice expressing a dominant negative N-cadherin. Science 1995, 270:1203-1206. In this paper and [42",44"], the authors describe transgenic mice models which were established to study the function of cadherins by heterotypic expression. 44.
Hermiston ML, Gordon JL: In vivo analysis of cadherin function in the mouse intestinal epithelium: essential roles in adhesion, maintenance of differentiation, and regulation of programmed cell death. J Cell Bio/1995, 129:489-506. See annotation [43"]. •
45. •,
MarrsJA, Andersson-Rsone C, Jeong MC, Cohen-Gould L, Zurzolo C, Nabi IR, Rodriguez-Boulan E, Nelson WJ: Plasticity in epithelial cell phenotype: modulation by expression of different cadherin cell adhesion molecules. J Cell Biol 1995, 129:507-519, See annotation [46"']. 46. •e
Larue L, Antos C, Butz S, Huber O, Delmas V, Dominis M, Kemler R: A role for cadherins in tissue formation. Development 1996, in press. These papers [45"',46"] give strong support for a role of cadherins in histogenesis and signal transduction. 47. •
FunayamaN, Fagotto F, McCrea P, Gumbiner BM: Embryonic axis induction by the armadillo repeat domain of ~-catenin: evidence for intracellular signaling. J Cell Bio/1995, 128:959-968.
See annotation [49"]. 48. •
Kelly GM, ErezyUmazDF, Moon RT: Induction of a secondary embryonic axis in zebrafish occurs following the overexpression of ~-catenin. Mech Dev 1995, 53:261-273. See annotation [49"]. 49. •
Karnovsky A, Klymkowsky MW: Anterior axis duplication in Xenopus induced by the over-expression of the cadherinbinding protein plakoglobin. Proc Nat/Acad Sci USA 1995, 92:4522-4526. These papers [47"-49"] show that overexpression of ~-catenin or plakoglobin results in dorsal mesoderm induction. 50.
Lame L, Ohsugi M, Hirchenhain J, Kemler R: E-cadherin null mutant embryos fail to form a trophectoderm epithelium. Proc Natl Acad Sci USA 1994, 91:8263-8267.
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34.
Kintner C: Regulation of embryonic cell adhesion by the cadherin cytoplasmic domain. Cell 1992, 69:225-236.
52.
35.
DufourS, Saint Jeannet JP, Broders F, Wedlich D, Thiery JP: Differential perturbations in the morphogenesis of anterior structures induced by overexpression of truncated XB- and Ncadherins in Xenopus embryos. J Cell Biol 1994, 127:521-535.
33. ••
36.
Broders F, Thiery J-P: Contribution of cadherin to directional cell migration and histogenesis in Xenopus embryos. Cell Adhes Commun 1995, 3:419-440.
37.
K,'ihl M, Finnemann S, Binder O, Wedlich D: Dominant negative expression of a cytoplasmically deleted mutant of XB/Ucadherin disturbs mesoderm migration during gastrulation in Xenopus laevis. Mech Dev 1996, 54:71-82.
38.
Lee C-H, Gumbiner M: Disruption of gastrulation movements in Xenopus by a dominant-negative mutant for C-cadherin. Dev Bio11995, 171:363-373.
39.
Levine E, Lee CH, Kintner C, Gumbiner BM: Selective disruption of E-cadherin function in early Xenopus embryos by a dominant negative mutant. Development 1994, 120:901-909.
40.
Detrick J, Dickey D, Kintner C: The effects of N-cadherin misexpression on morphogenesis in Xenopus embryos. Neuron 1990, 4:493-506.
41.
Holt CE, Lemalre P, Gurdon JB: Cadherin-modlated cell interactions are necessary for the activation of MyoD in Xenopus mesoderm. Proc Nat/Acad Sci USA 1994, 91:10844-10848.
42. •~ee
Dahl U, Sj~din A, Semb H: Cadherins regulate aggregation of pancreatic ~-cells in vivo. Development 1996, 122:2895-2902. annotation [43"].
51. •
Oda H, Uemura T, Harada Y, Iwal Y, Takeichi M: A Drosophila homolog of cadherin associated with armadillo and essential for embryonic cell-cell adhesion. Dev Biol 1994, 165:716-726.
53.
UemuraT, Oda H, Kraut R, Hayashi S, Kataoka Y, Takeichi M: Zygotic Drosophila E-cadherin expression is required for processes of dynamic epithelial cell rearrangement in the Drosophila embryo. Genes Day 1996, 10:659-671. See annotation [54"]. •
54.
Tepass U, Gruszynski-DeFeo E, Haag TA, Omatyar L, T6r6k T, Hartanstein V: Shotgun encodes Drosophila E-cadherin and is preferentially required during cell rearrangement in the neuroectoderm and other morphogenetically active epithelia. Genes Dev 1996, 10:672-685. This paper, together with [52,53"], describes the characterization of Drosophila E-cadherin. •
55.
Mahoney PA, Weber U, Onofrechuk P, Biessmann H, Bryant PJ, Goodman CS: The fat tumor suppressor gene in Drosophila encodes a novel member of the cadherin gene superfamily. Cell 1991, 67:853-868.
5~.
Clark HF, Brentrup D, Schneitz K, Bieber A, Goodman C, Noll M: Dachsous encodes a member of the cadhedn superfamily that controls imaginal disc morphogenesis in Drosophila. Genes Dev 1995, 9:1530-1542.
57. •
Haegel H, Larue L, Ohsugi M, Fedorov L, Herranknecht K, Kemler R: Lack of ~-catenin affects mouse development at gastrulation. Development 1995, 121:3529-3537. Lack of ~-catenin affects mesoderm formation, as demonstrated by gene targeting in mice [57"] and by antisense experiments in Xenopus [58]. 58.
Heasman J, Crawford A, Goldstone K, Garner-Hamrick P, Gumbiner B, McCrea P, Kintner C, Noro CY, Wylie C: Overexpression of cadherins and underexpression of ~-catenin
Cadherins and catenins in development Huber, Bierkamp and Kemler 691
59.
inhibit dorsal mesoderm induction in eady Xenopus embryos. Cell 1994, 79:791-803. Heasman J, Ginsberg D, Geiger B, Goldstone K, Pratt T, YoshidaNoro C, Wylie C: A functional test for maternally inherited cadherin in Xenopus shows its importance in cell adhesion at the blastula stage. Development 1994, 120:49-57.
60. •
Orsulic S, Peifer M: An in vivo structure-function study of Armadillo, the 13-catenin homolog, reveals both separate and overlapping regions of the protein required for call adhesion and for wingless signaling. J Cell Bio11996, in press. See annotation [62"]. 61. •
Papkoff J, Rubinfald B, Schryver B, Polakis P: Wnt-1 regulates free pools of catenins and stabilizes APC-catenin complexes. Mo/ Cell Biol 1996, 16:2128-2134. See annotation [62"]. 62. Yost C, Torres M, Miller JR, Huang E, Kimelman D, Moon RT: The • axis-inducing activity, stability, and subcellular distribution of ~-cetenin is regulated in Xenopus embryos by glycogen synthase kinase 3. Genes Dev 1996, 10:1443-1454. These papers [60"-62"] describe the characterization of the molecular interactions of the components of the winglessANnt signaling pathway. See also [15"'].
63. oe
Bhanot P, Brink M, Samos CH, Hsieh J-C, Wang Y, Macke JP, Andrew D, Nathans J, Nusse R: A new member of the fr/zz/ed family from Drosophila functions as a Wingless receptor. Nature 1996, 382:225-230. Describes the first identification of Drosophila Frizzled as a wingless receptor. 64.
Travis A, Amsterdam A, Belanger C, Grosschedl R: LEF-1, a gene encoding a lymphoid-specific protein with an HMG domain, regulates T-cell receptor (~ enhancer function. Genes Dev 1991, 5:880-894.
65.
Oosterwegel M, Van de Wetering M, Timmermann J, Kruisbeek A, Destree O, Meijlink F, Clevers H: Differential expression of the HMG box factors TCF-1 and LEF-1 during murine embryogenesis. Development 1993, 118:439-448.
66.
Lin R, Thompson S, Priess JR: pop-1 encodes an HMG box protein required for the specification of mesoderm precursor in early C. elegans embryos. Cell 1995, 83:599-609.
67.
Huber O, Kom R, McLaughtin J, Ohsugi M, Herrmann BG, Kemler R: Nuclear localization of 13-catenin by interaction with transcription factor LEF-1. Mech Dev 1996, in press.