REVIEWS. The cadherin superfamily in neuronal connections and interactions

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1 The cadherin superfamily in neuronal connections and interactions Masatoshi Takeichi Abstract Neural development and the organization of complex neuronal circuits involve a number of processes that require cell cell interaction. During these processes, axons choose specific partners for synapse formation and dendrites elaborate arborizations by interacting with other dendrites. The cadherin superfamily is a group of cell surface receptors that is comprised of more than 100 members. The molecular structures and diversity within this family suggest that these molecules regulate the contacts or signalling between neurons in a variety of ways. In this review I discuss the roles of three subfamilies classic cadherins, Flamingo/ CELSRs and protocadherins in the regulation of neuronal recognition and connectivity. Dendritic field The area covered by dendritic arborizations of a neuron. RIKEN Center for Developmental Biology, Minatojima-Minamimachi, Chuo-ku, Kobe , Japan. takeichi@cdb.riken.jp doi: /nrn2043 Published online 29 November 2006 Morphogenesis of neurons and the formation of connections with their intended targets are controlled by sequential, complex cell cell interactions. Developing neurons extend dendrites and axons. These dendrites generate complex arborizations, the pattern of which is regulated by interactions with other dendrites derived from adjacent neurons 1. Such arborizations contribute to the formation of dendritic fields, which are specific for each neuronal type. Under the guidance of attractive or repulsive factors 2, axonal growth cones migrate towards their target neurons and eventually make contact with them to form synapses 3, in a process that must require elaborate recognition mechanisms to ensure their correct pairing. Neuronal interactions and recognition are thought to be mediated, at least in part, by cell surface proteins, defined as cell cell adhesion molecules, the primary functions of which are to bring the apposed cell membranes into contact via their homophilic or heterophilic interactions. Many of these molecules are also known to have signalling activities. It is notable that even certain molecules that can be defined as signalling receptors or ligands, such as Notch Delta, neuroligin neurexin and Eph ephrin, can also promote cell cell adhesion 4 6. Therefore, cell surface molecules involved in adhesion and signalling might not be clearly distinguishable. Cadherins are a family of molecules with activities of this complexity. They constitute a superfamily that is comprised of more than 100 members in vertebrates, grouped into subfamilies that are designated as classic cadherins, desmosomal cadherins, protocadherins, Flamingo/CELSRs and FAT 7,8. The biological functions of the family members seem to have diverged: some of them, including classic cadherins and desmosomal cadherins, are well defined as adhesion molecules, but most of the other members do not necessarily show strong adhesive activities. With a few exceptions, cadherins are transmembrane proteins. Their extracellular domain contains repetitive subdomains called cadherin repeats, which contain sequences that are involved in calcium binding 9,10 (FIG. 1). The number of these repeats varies greatly among the members, from The cadherin repeats are involved in cis- or trans-interactions between the extracellular domains of the molecules, at least in the case of classic cadherins, and these interactions lead to homophilic binding between cadherin molecules. By contrast, the intracellular domain is not conserved among the subfamilies. Therefore, a general picture of the actions of cadherin superfamily molecules is emerging, in which they interact with each other or with other molecules at cell cell interfaces through their cadherin repeats, but generate different signals in the cytoplasm through distinct intracellular domains, thereby leading to diverse functions ranging from cell cell adhesion to signalling. Here, I give an overview of recent progress in the study of the roles of both classic and non-classic cadherins in neuronal interactions and recognition. With regards to the non-classic cadherins, detailed functional analyses have so far been achieved only for limited members or subfamilies, including Flamingo/CELSRs and some of the protocadherins. Accordingly, only these subfamilies have been chosen for discussion. It is well known that classic cadherins are important in synapse formation and stability; however, this specific issue has been reviewed recently 11, and so is not discussed in detail here. NATURE REVIEWS NEUROSCIENCE VOLUME 8 JANUARY

2 Vertebrate classic cadherins (e.g. N-cadherin, E-cadherin) Drosophila E-cadherin Drosophila N-cadherin Mammalian CELS Drosophila Flamingo Mammalian α-protocadherin Mammalian γ-protocadherin Cadherin repeat EGF-like domain Laminin globular-like domain Adherens junction Protein complexes that occur at cell cell junctions. They are composed of the cadherin catenin complexes, and characterized by accumulation of actin filaments at their cytoplasmic side. Neuroepithelial stage The earliest stage in the developing CNS. The neuroepithelium is a layer of cells with epithelium-like morphologies, which give rise to a diverse array of neural cells during development. Non-chordate-specific domain HormR domain GPS domain Plasma membrane Extracellular Intracellular Figure 1 Schematic drawings of cadherin superfamily members. Illustration shows representative molecules of the three subfamilies: classic cadherins, Flamingo/CELSRs and protocadherins. All vertebrate classic cadherins share a common primary structure. Drosophila melanogaster classic cadherins differ in their primary structures not only from the vertebrate versions but also between the subtypes E and N. Despite these differences, the structure and functions of their cytoplasmic domains seem to be conserved between species, as well as between subtypes. The vertebrate and D. melanogaster homologues of the Flamingo/CELSR subfamily are similar to each other in their domain organization. For α- and γ-protocadherins, only a single isoform is shown; other isoforms are identical to the one shown here in terms of their overall primary structure. D. melanogaster homologues for the α- and γ-protocadherins are not known. EGF, epidermal growth factor; GPS, G protein-coupled receptor proteolytic site; HormR, hormone receptor. Classic cadherins and invertebrate homologues Classic cadherins have been most clearly defined as adhesion molecules, because blocking cadherin activity with inhibitory antibodies or gene mutations facilitates the separation of cells or disrupts tissue architecture in various organs 12. In vertebrates, classic cadherins are characterized by the presence of five cadherin repeats in their extracellular domain and a conserved cytoplasmic domain to which two cytoplasmic proteins, p120 catenin and β-catenin, bind 7,13. The cadherin-coupled β-catenin further associates with α-catenin, which is known to be an actin-binding protein 14,15. The cadherin catenin complex forms the adherens junction at the apical portion of cell cell junctions, in which a number of cytoskeletal or signalling proteins are concentrated to organize a cell cell signalling centre 13. A given cadherin typically interacts with the same cadherin subtype, exhibiting homophilic interactions, although it can also bind other limited subtypes heterophilically 16,17. Approximately 20 members have been identified as part of the classic cadherin subfamily, which is further subdivided into types I and II. Most members of the subfamily are expressed in the nervous system, showing a distribution associated with neuronal connectivity. For example, each of the type II cadherins is expressed among restricted neuronal groups that are synaptically connected to each other, with the localization being concentrated in synaptic contacts 18,19. Invertebrates have similar classic cadherin-like molecules that, through their cytoplasmic domain, can bind catenins 7, However, these molecules differ from their vertebrate counterparts in terms of primary structure, in particular by having larger extracellular domains 23 (FIG. 1). Vertebrate E-cadherin and Drosophila melanogaster E-cadherin (DE-cadherin) seem to be functionally homologous, because both are expressed in epithelial cells and are essential for organization of the adherens junctions between these cells. Likewise, vertebrate N-cadherin and D. melanogaster N-cadherin (DN-cadherin) both function in the nervous system. Nevertheless, DE- and DN-cadherin have approximately 7 and 17 cadherin repeats, respectively, as compared with 5 repeats in their vertebrate counterparts. Furthermore, their extracellular domains contain unique sequences inserted at the proximal ends that are not present in the vertebrate classic cadherins; despite this, the DE- and DN-cadherin cytoplasmic domain is relatively similar to that of the vertebrate, as they can also bind catenins. Therefore, classic cadherins seem to have structurally diverged between the species, yet have preserved similar functions. Among the classic cadherins, N-cadherin and DN-cadherin are broadly expressed in the nervous system, and their roles in neural development have been extensively studied. N-cadherin in neuronal interactions Vertebrate N-cadherin is expressed from the beginning of neural development (the neuroepithelial stage) 24, and its expression persists in differentiated neurons in various species. This molecule is also expressed in non-neural tissues, including cardiac and skeletal muscle cells 25. Conventional knockout of the mouse N-cadherin gene causes early embryonic lethality, mainly because of heart defects 26,27. Zebrafish N-cadherin mutants, however, survive longer, exhibiting various malformations of the CNS 28 31, which is consistent with earlier observations made by using N-cadherin-blocking antibodies 32. For example, the neural retina in the mutant zebrafish is severely distorted, exhibiting a rosette-like cell rearrangement 30 (FIG. 2a). However, because of the importance of N-cadherin in the maintenance of the neuroepithelial adherens junction, it is likely that many of the defects in N-cadherin mutants originate from the disruption of the neuroepithelial architecture. Therefore, the precise roles of N-cadherin in neuronal interactions at later developmental stages remain less clear. Nevertheless, some fragmental information on the specific role of N-cadherin in axon migration is available: studies using blocking antibodies against N-cadherin showed that this molecule is required for the correct innervation of specific laminae in the chicken tectum by retinal optic nerves 33. Furthermore, in zebrafish N-cadherin mutants, retinal optic axons cannot migrate normally to their targets, showing some misrouting 30 (FIG. 2a). 12 JANUARY 2007 VOLUME 8

3 a Wild type N-cadherin mutant b Control Dominant-negative Retina Chiasma Optic tectum Figure 2 Effects of classic cadherin dysfunction on retinal morphogenesis and neurite extension. a Phenotypes of zebrafish N-cadherin mutants. Top panels show histological sections of embryonic eyes. Retinal laminar structures are disorganized. Bottom panels show retinal optic axons visualized by placing the lipophilic dyes DiI (red) and DiO (green) in the respective eyes. Misrouting of some axons is observed (arrows). b The effects of dominant-negative cadherin expression in Type III horizontal cells of the embryonic chicken retina. Their dendrites fail to extend when cadherin activities are blocked. The outlines of dendritic branches were visualized by co-expression of an enhanced green fluorescent protein construct. Top panels show a horizontal view; bottom panels show a vertical view. Panel a reproduced, with permission, from REF. 30 (2003) The Company of Biologists. Panel b reproduced, with permission, from REF. 40 (2006) The Company of Biologists. Ommatidium A unit of the compound eye of insects. Each ommatidium contains a cluster of photoreceptor cells and functionally provides the brain with one picture element. Lamina Neuropil structure that makes up part of the optic lobe of insects. Out of eight retinal axons, the R6 axons innervate L1 L5 neurons in the lamina. The lamina L1 L5 neurons relay R6 input to the medulla. Neuropil structure that makes up part of the optic lobe of insects. Out of eight retinal axons the R7 and R8 axons innervate the medulla, which also receives input from the lamina L1 L5 neurons. The roles of classic cadherins other than N-cadherin in neural development have been less well studied, although genetic approaches suggest that some of the type II cadherins are involved in axon sorting 34 and in the regulation of physiological functions of the brain, such as long-term potentiation in the hippocampus 35. Dominant-negative cadherin constructs have often been used as a tool to examine the roles of classic cadherins in vertebrate cells. Such constructs have been created by deleting parts of the extracellular domain of a classic cadherin (for example, N-cadherin), but leaving its cytoplasmic domain intact 36,37. When overexpressed in cells, these constructs compete with endogenous classic cadherins for interactions with catenins and other cytoskeletal proteins. As the activity of classic cadherins relies on such interactions 38, their depletion due to overexpression of the mutant cadherins causes downregulation of the endogenous cadherin activity. However, the cytoplasmic domain is conserved among the classic cadherins and these dominant-negative constructs can therefore nonspecifically block multiple cadherin subtypes. When a dominant-negative N-cadherin was expressed in the neural retina of Xenopus embryos, the extension of neurites from retinal ganglion cells was inhibited 39. A similar construct was able to block the radial extension of horizontal cell dendrites, as well as their synaptic connections with photoreceptor cells in the retina 40 (FIG. 2b). Such constructs were also used to show that cadherins are required for tangential migration of precerebellar neurons 41. In addition, mouse mutants of αn-catenin, one of the catenins supporting neuronal cadherin functions, show various defects in neural morphogenesis and axon extension 42. In each of these studies, however, it remained unclear whether N-cadherin or other cadherins were important for the observed effects, because of the nonspecific nature of the dominant-negative constructs and the general importance of αn-catenin for the functions of multiple cadherins. Nevertheless, these results provide evidence that classic cadherins have important roles in neural cell cell interactions and neurite extension in various systems. DN-cadherin in neuronal interactions In D. melanogaster, advanced genetic technologies have made it possible to analyse precisely the role of DN-cadherin, a putative D. melanogaster counterpart of the vertebrate N-cadherin, in neural morphogenesis. A null mutation of the DN-cadherin gene results in embryonic lethality 23. In the mutant embryos, subsets of longitudinal CNS axons show various aberrant trajectories, including errors in directional migration of growth cones. Flies with a hypomorphic mutation of DN-cadherin that allows them to grow to adulthood exhibit uncoordinated locomotion with disorganized intrabrain structures 43, suggesting that DN-cadherin is involved in wiring of the brain. This idea has been tested in more detail in visual and olfactory circuits. Visual system. In the D. melanogaster compound eye, each ommatidium contains eight photoreceptor cells (R cells), designated as R8. Of these, the R6 axons project to the lamina, whereas R7 and R8 axons innervate distinct layers of the medulla in the optic lobe (FIG. 3a,b). DN-cadherin is expressed by both the R cells and their target neurons. When DN-cadherin is absent from either of these cell types, the connectivity of R cells is severely disrupted 44. The wild-type lamina contains an array of neurons arranged in columns 45, each of which consists of five monopolar neurons, designated L1 L5 (FIG. 3b). From each ommatidium, a bundle of R6 axons reaches the lamina and associates with a specific column, where NATURE REVIEWS NEUROSCIENCE VOLUME 8 JANUARY

4 a b Eye Lamina Retina Lamina L1 L2 L3 L4 L5 Optic lobe c Wild type DN-cadherin mutant Flamingo mutant Figure 3 Effects of DN-cadherin or Flamingo mutation on axon projections in the lamina of the optic lobe. a Horizontal section of an adult Drosophila melanogaster head, visualized by autofluorescence. b Retinal axon projection patterns. Each ommatidium has eight photoreceptor cells ( R8; R8 is not shown here). R6 cells project to the lamina, whereas R7 and R8 project to the medulla. R6 cell axons of a single ommatidium form a fascicle, and this fascicle reaches a single column of L1 L5 cells in the lamina, where the axons defasciculate to connect with different columns which are organized into cartridges. c Summary diagram showing the effects of D. melanogaster N-cadherin (DN-cadherin) or Flamingo mutation on R cell axon innervation of the lamina. In the wild type, R6 axons extend to distinct specific cartridges. In the absence of DN-cadherin, these axons fail to extend to the target cartridges. Without Flamingo, the axons randomly innervate the cartridges. Image for panel a courtesy of Y. Iwai and T. Uemura, Kyoto University, Japan. Panel b modified, with permission, from Nature Neuroscience REF. 98 (2005) Macmillan Publishers Ltd. Fascicle A slender bundle of nerve fibres. Defasciculation The disentanglement of individual axon fibres from a bundle of fibres, called a fascicle or tract, which allows them to migrate in separate directions. Glomerulus In the nervous system, an anatomically discrete module that receives input from other neurons. the axons defasciculate. Each axon fibre extends away and innervates another specific column in an invariant pattern, forming cartridges. In each of these cartridges, R6 axon terminals derived from different ommatidia are synaptically connected with a cluster of mono polar neurons composing the column (FIG. 3c). When the DN-cadherin gene is selectively mutated in the R cells, their axons are able to terminate in the lamina but do not defasciculate and extend to target cartridges 46,47 (FIG. 3c). When the laminar neurons do not express DN-cadherin, the cartridges are severely disorganized and fail to receive R cell innervation 47. Single-cell resolution analysis has confirmed that DN-cadherin is required in the target cartridge for R cell extension, but not for the initial association with the lamina neurons. These results show that DN-cadherin specifically mediates the interactions between lamina neurons in the target cartridge and the axons of extending R cells. This function of DN-cadherin could not be replaced by DE-cadherin, indicating that these two cadherins have unique functions. Deletion of DN-cadherin from R cells also affects R7 axon targeting to the medulla 46,48. In wild-type embryos, R7 and R8 axons innervate distinct layers of the medulla, whereas in DN-cadherin mutants the R7 axon cannot reach its own target layer, and instead terminates around the R8 targeting zone (FIG. 4a), suggesting that DN-cadherin is required for targeting and stabilization of R7 growth cones at a specific layer of the medulla. The DN-cadherin gene locus has three alternately spliced exons, which can generate 12 isoforms. Although there is no evidence available to suggest that these isoforms have distinct biochemical functions, at least in the R cell axon targeting system, these isoforms do seem to be differentially utilized by subsets of retinal neurons 49. Olfactory system. Axons from olfactory receptor neurons (ORNs) in the antennae extend into the antennal lobe, which is comprised of multiple glomeruli, each containing dendritic processes of second-order projection neurons. Axons of ORNs expressing a common olfactory receptor converge onto a single glomerulus to form synapses. When DN-cadherin is deleted in ORNs, their axons can reach the antennal lobe but fail to converge onto a glomerulus. Furthermore, in these antennal lobes the glomerular organization itself is disrupted 50. When DN-cadherin is mutated in projection neurons, the dendrites correctly target a specific glomerulus. However, they are unable to restrict their arborizations to the target glomerulus and spread onto non-appropriate, neighbouring glomeruli 51. This spreading can be observed between DN-cadherin-positive and -negative glomeruli, suggesting that DN-cadherin homotypic interactions are important for dendritic refinement. The projection neuron dendritic refinement occurs even when ORN axons do not express DN-cadherin. These observations indicate that DN-cadherin mediates dendro-dendritic interactions. Classic cadherins: mechanisms of action The studies in the D. melanogaster visual and olfactory systems outlined above show that DN-cadherin is required for neurite interactions. The mechanisms by which DN-cadherin mediates these actions, however, are still not completely clear. Does DN-cadherin simply mediate the adhesive interactions between neurites, as anticipated from its known function? This is probably the case for retinal axon targeting to lamina neurons, and ORN axon innervation to glomeruli, as the formation of these connections was blocked by the absence of DN-cadherin. DN-cadherin is probably required for the stable contacts between these axons and target neurons; without it, the axons might retract, resulting in the phenotypes observed. In the olfactory glomeruli, DN-cadherindeficient projection neuron dendrites showed overshooting rather than retraction. The authors of the study argue that DN-cadherin is involved in mediating the adhesive interactions between dendrites, forming a glomerulus in order to 14 JANUARY 2007 VOLUME 8

5 a b Wild type R8 growth cones Lamina Overlapping R8 growth cones Lamina Wild type Wild type Flamingo (R cell mosaic) R8 zone R7 zone DN-cadherin mutant Flamingo mutant Overlapping R8 growth cones Lack of R7 axon terminals Lack of R8 axon terminals Lamina plexus Lamina plexus Figure 4 Effects of DN-cadherin or Flamingo mutation on retinal axon targeting in the medulla. Mutations in either Drosophila melanogaster N-cadherin (DN-cadherin) or Flamingo have been shown to result in abnormalities in axon targeting in the medulla 46,48,70,71. a DN-cadherin- (top) or Flamingo- (bottom) null mutation clones were generated in the eye, and their axon terminals were stained with antibodies against photoreceptor (R) cells. DN-cadherin and Flamingo mutant axons cannot reach the R7 and R8 target zone, respectively (arrows). b Schematic drawing showing the irregular spacing of Flamingo-null R8 axons 71. Image for panel a, top, courtesy of Y. Iwai and T. Uemura, Kyoto University, Japan. Image for panel a, bottom, courtesy of T. Usui, Kyoto University, Japan. confine them within this structure 51. However, the mechanisms by which DN-cadherin affects dendro-dendritic interactions in a glomerulus are unknown. Another possible interpretation of the DN-cadherindeficient glomerular phenotypes is that this cadherin functions as a repellent between individual dendrites belonging to neighbouring glomeruli, and that the loss of DN-cadherin results in the disruption of a signalling barrier between the glomeruli, leading to the overextension of dendritic arborizations. Classic cadherins have not been shown to have a repellent activity, at least in the vertebrate system. However, as noted above, DN-cadherin is not a simple orthologue of the vertebrate N-cadherin in terms of the primary structure, and so DN-cadherin might have acquired unique activities. Despite this possibility, the overall functions of neural classic cadherins seem to be conserved between the species. For example, misrouting of axon fibres was observed in both the developing CNS of DN-cadherin mutants 23 and the optic tract of N-cadherin-mutated zebrafish 30 (FIG. 2a). Neurite retraction was observed not only in the R6 axons that failed to connect with their targets in the absence of DN-cadherin, but also in the cadherin-blocked horizontal cell dendrites in the vertebrate retina (FIGS 2b,3c). Other outstanding questions include how DN-cadherin function can be restricted to specific sets of axon target connections, even though the visual and olfactory systems ubiquitously express this molecule. Several potential mechanisms can be considered. One possibility is that DN-cadherin activity is controlled spatiotemporally and functions only when necessary. This idea assumes the presence of regulators of cadherin activity, and candidates for this role have been identified. For example, leukocyte antigen-related-receptor protein tyrosine phosphatase (LAR) has been shown to bind the cadherin catenin complex and regulate its transportation to dendrites 52,53. If such enzymes are activated in specific neurons, enhanced DN-cadherin-mediated adhesion might occur only in these neurons. Supporting this notion, LAR is specifically required for R7 target selections, and its mutant phenotypes are similar to those of DN-cadherin mutants 54,55. Recent studies show that D. melanogaster mutants of liprin-α, a LAR-binding scaffold protein, show retinal axon targeting phenotypes similar to those of LAR and DN-cadherin mutants 56,57. Importantly, this protein acts only in the retinal axons and not in the target neurons. Axons might be able to control their own cadherin activity by utilizing such factors. If this is the case, R axons could maintain downregulation of cadherin activity during migration (note that DN-cadherin is not required for their migration to the target zone). On initial contact with the intended target neurons, signals might be generated that activate cadherin activity in the axons, and in turn such signals could act to stabilize cadherin-mediated connections. The putative cadherin regulators mentioned above might function in this activation process. Cadherins themselves could be used as receptors to generate the initial axon target contact-mediated signals, as cadherin cadherin interactions are known to be capable of activating cytoplasmic signalling molecules such as small Rho GTPases 58. Therefore, cadherins might operate not only as adhesion molecules but also as signalling molecules. As another possible mechanism to control cadherin actions during axon targeting, we can speculate that cadherin localization might be regulated by other molecules so that it accumulates at specific cell cell contact sites. NATURE REVIEWS NEUROSCIENCE VOLUME 8 JANUARY

6 Axon α β α α β β β As expected from the function of Flamingo, CELS has been shown to regulate PCP in inner ear hair cells 67. Flamingo and CELSR have also been shown to be important in several neuronal interactions. Dendrite Afadin Planar cell polarity (PCP). The property of epithelial cells polarizing along the plane of the epithelium. Hemisegment The animal body is segmented along the rostrocaudal axis, as seen in the insects. A hemisegment represents half of a symmetrical segment from either side of the body. β β β α α Cadherin Nectin 1 Nectin 3 α α-catenin α β β β-catenin Figure 5 Cooperation between the nectin and cadherin adhesion systems for establishing synaptic contacts. Nectin 1, preferentially localized in the axon, interacts with nectin 3, localized in the dendrite, which in turn promotes the recruitment of cadherins to synaptic contact sites. This recruitment is thought to be mediated by molecular interactions between afadin and α-catenin, which bind nectin and the cadherin β-catenin complex, respectively 99. Supporting this possibility, candidates for such molecules a small family of immunoglobulin-domain membrane proteins, called nectin 1 4 have recently been identified in vertebrate hippocampal neurons. Each nectin molecule can promote cell aggregation by homophilic interactions; these nectin interactions, in turn, promote the recruitment of cadherins to cell cell contact sites through uncharacterized mechanisms involving α-catenin 59. Nectins can also interact with other nectin subtypes in a heterophilic fashion, as in the combination of nectin 1 and nectin 3; these heterophilic interactions are much stronger than the homophilic interactions 60. Importantly, in hippocampal neurons, nectin 1 is localized in axons, whereas nectin 3 is distributed to both axons and dendrites 61. As a result, the interaction between nectin 1 and nectin 3 takes place preferentially between axons and dendrites, and this enhances the accumulation of cadherins to axodendritic interfaces (FIG. 5). In this way, cadherins can specifically stabilize synaptic contacts but not those involving other combinations of neurites, such as dendro-dendritic contacts. Cadherins alone cannot achieve such specialized localization. Rather, localized cadherin activity can be achieved by cooperation with the heterophilic nectin adhesion system. Other similar mechanisms might work for restricting cadherin activities to specific cell cell contact sites. However, such putative mechanisms have not yet been identified in D. melanogaster neurons. Non-classic cadherins: Flamingo/CELSRs D. melanogaster Flamingo is unique in its primary structure as it has a seven-pass transmembrane domain, with sequences that show similarity to those of the secretin receptor family of G protein-coupled receptors 62 (FIG. 1). Flamingo regulates planar cell polarity (PCP) in epithelial cell sheets by cooperating with other PCP-regulating elements such as Frizzled, a Wingless receptor 63. Three mammalian homologues of Flamingo have been identified, CELS, CELSR2 and CELSR3 (REFS 64 66). Flamingo in dendrite patterning of peripheral neurons. In D. melanogaster embryos, neurons of the PNS elaborate dendrites with stereotypical branching patterns, and define their own dendritic field. When dendrites from homologous neurons in the two hemisegments meet at the dorsal midline, they repel each other. The formation of normal dendritic fields and competition between dendrites of homologous neurons have been shown to require Flamingo 68 in its absence, subsets of dendrites from homologous neurons extend over the midline, disrupting the dendritic field. Single-cell-level analysis of flamingo mutants has revealed that Flamingo controls the initiation and extension of a particular population of dendritic branches, and in addition has the ability to promote axonal growth 69. Flamingo in axon target selection in the visual system. Flamingo is expressed in growth cones of R6 axons, and is required for them to select appropriate targets in the lamina. In the absence of Flamingo, R6 axons extend to, and form synapses with, incorrect targets 70 (FIG. 3c). As lamina neurons do not express Flamingo, it has been proposed that Flamingo mediates specific interactions between growth cones, which contributes to the sorting of R6 axons to appropriate targets 70. However, the molecular mechanisms underlying this Flamingomediated axon sorting remain to be determined. Its role in regulating PCP indicates that this molecule might provide order in the arrangement of axons in their fascicles, thereby directing individual axons to appropriate targets. Flamingo is also required for the appropriate sorting of R8 axons to specific targets in the medulla of the optic lobe 70,71. Without Flamingo, R8 axons cannot form stable contacts in their target region, and retract to more superficial layers (FIG. 4a). In contrast to the evenly spaced wild-type R8 growth cones, mutant growth cones are irregularly spaced, and the processes of individual growth cones often overlap (FIG. 4b). These observations have led to the conclusion that Flamingo facilitates competitive interactions between adjacent R8 growth cones, and through such processes promotes R8 axon target interactions. This putative function of Flamingo seems to be analogous to that in the tiling of dendrites of peripheral neurons. How Flamingo controls R8 axons, but not R7 axons remains a mystery, as is the case for N-cadherin, which controls only R7 axons. Vertebrate CELSRs in neuronal interactions. Among the three vertebrate homologues of Drosophila Flamingo, CELSR2 and CELSR3 are involved in neuronal morphogenesis. CELSR2 is expressed by several types of neurons, including cortical pyramidal neurons and cerebellar Purkinje cells 72,73. When Celsr2 function is suppressed by RNA interference (RNAi)-mediated knockdown in cortical or cerebellar slices of the rat brain, the complexity of their dendritic arborizations is significantly decreased JANUARY 2007 VOLUME 8

7 Stereocilia Mechanosensing organelles of hair cells. As hearing sensors, stereocilia are lined up in the Organ of Corti within the cochlea of the inner ear. (FIG. 6a). This effect of CELSR2 depletion seems to be due to the retraction of dendritic processes. As CELSR2 has the ability to interact homophilically, it has been proposed that this interaction takes place between neurites and has a role in maintaining the normal patterns of dendritic branching. Genetic ablation of Celsr3 results in other types of neuronal defects, such as the suppression of axon tract development 75. For example, thalamocortical or corticofugal axon projections do not form (FIG. 6b), and the corticospinal, spinocerebellar and pyramidal tracts are also abnormal. In these mutant brains, neuronal differentiation itself seems normal, and dendritic patterning is not affected. It has been noted that CELSR3 and frizzled 3 are co-expressed by neurons 75, suggesting that their actions are in synergy, as in the case of the action of Flamingo to regulate PCP. Curiously, blockade of Flamingo and CELSRs results in distinct phenotypes: for example, in flamingo mutants, dendrites overextend, whereas in the case of Celsr2 knockdown, they retract. It remains unclear whether these D. melanogaster and vertebrate homologues generate opposite signals or whether their mutant phenotypes will eventually be explained by common mechanisms that are as yet unidentified. Non classic cadherins: protocadherins The term protocadherin is often used to refer to all members of the cadherin superfamily other than classic and desmosomal cadherins. However, here we use it in a more narrow sense, excluding the Flamingo and FAT subfamilies. According to the recent classification based on phylogenetic analysis 8, the protocadherin subfamily can be further subdivided into three groups: the clustered protocadherins, comprising α-, β- and γ-protocadherins; δ-protocadherins; and others, many of which are solitary. The genes for α-, β- and γ-protocadherins are sequen tially organized, generating more than 50 transcripts from the three gene clusters. Each of the α- and γ-protocadherin gene clusters contain multiple variable exons as well as a set of constant exons. These exons are combined by cis-splicing of the mrna 76,77, thereby leading to the production of a large number of isoforms with various extracellular domain sequences. Singlecell PCR analysis has shown that individual variable exons are expressed in a monoallelic and combinatorial fashion 78. The δ-protocadherin subfamily genes also produce alternative splicing variants, but these genes encode no variable extracellular domains, resulting in only small variations in the gene products 8,79. The molecular functions of these protocadherins are still poorly understood. Many protocadherins have weak homophilic binding ability and can associate with characteristic cytoplasmic partners 8,79. However, it is unclear whether they function as adhesion molecules, like classic cadherins, or act only as signalling receptors. The functions of cadherin 23, a type of protocadherin, and protocadherin 15 have been better identified; they have been shown to be essential for the organization of stereocilia in the inner ear, and their loss causes deafness a Wild type Celsr2 knockdown b Wild type STR CX AC CC CC Celsr3 knockout CX STR Figure 6 Effects of CELSR deficiencies. a Rat Purkinje cells visualized by enhanced green fluorescent protein expression. Right panel shows an example of Purkinje cells in which Celsr2 expression was knocked down by RNA interference. CELSR2-depleted cells show less organized dendritic trees. b Coronal sections of wild-type and Celsr3-knockout newborn mouse brains, stained for neurofilaments at a rostral level. The anterior commissure (AC) is absent in the mutant. Thalamocortical and corticoefferent axons, and other fibres that cross the striatum, are all defective in the mutant. Instead, aberrant axons from the thalamus run in the marginal zone in the mutant basal forebrain (arrow). CC, corpus callosum; CX, cortex; STR, striatum. Panel a reproduced, with permission, from REF. 74 (2004) Elsevier Science. Panel b reproduced, with permission, from Nature Neuroscience REF. 75 (2005) Macmillan Publishers Ltd. in mammals 80. The distribution of cadherin 23 and protocadherin 15 suggests that these molecules function as physical ligands to link the stereocilia in an orderly fashion, and they have also been implicated in retinal cell interactions, such as those between photoreceptor outer segments and the microvilli of retinal pigment cells 80. As a whole, however, the protocadherin field is still in its infancy and awaits deeper functional analyses at both cellular and molecular levels. The large diversity of protocadherins suggests that they might have roles in establishing specific neuronal connections. For example, if each of the splicing variants of α- or γ-protocadherin has a unique cell binding specificity as seen in classic cadherin subfamily members, those molecules might function as a recognition cue during the synaptic associations between axons and their target neurons. Most of the protocadherins are, in fact, expressed in neurons, and some of them are localized in synapses. Individual transcripts of γ-protocadherins (PCDH-γ) are expressed by subsets of neurons and recruited to synaptic NATURE REVIEWS NEUROSCIENCE VOLUME 8 JANUARY

8 regions 81,82, although their distributions are not confined to synapses 83. As a first step to test the roles of this type of protocadherin, genetic studies have been conducted. In the spinal cord of mutant mice lacking the whole Pcdh-γ locus, early steps of neuronal migration, axon outgrowth and synapse formation occur normally. In the spinal cord, however, subsets of neurons begin to degenerate during later embryonic stages 83, suggesting that PCDH-γ is required for the survival of particular neuronal types. To investigate the specific roles of PCDH-γ in synaptogenesis, apoptosis can be minimized by removing BAX (B-cell leukaemia/lymphoma 2-associated protein X), or by using a hypomorphic allele of Pcdh-γ. In these mutant mice, the spinal cord shows decreased synaptic density; and the activity of the formed synapses is reduced, providing the first evidence for a role of this protocadherin in synaptic development 84. However, the specific roles of individual alternative splicing products of Pcdh-γ have not been investigated yet. PCDH-γ has recently been shown to be cleaved by the presenilin- and ADAM10- dependent proteolytic systems 85 87, but the biological role of this process remains unknown. α-protocadherin (PCDH-α) is localized in synapses 88, but only presynaptically in ciliary ganglionic neurons 89. Further functional analysis of PCDH-α as well as that of other protocadherins is expected. Concluding remarks Here I have discussed the roles of cadherin superfamily proteins, focusing on N-cadherin/DN-cadherin, Flamingo/CELSRs and a subgroup of protocadherins. D. melanogaster genetics has greatly advanced our understanding of the in vivo roles of the first two groups of cadherin, revealing that these molecules are required for either attractive or competitive/repulsive interactions between neuronal processes. Whether cadherin molecules can produce repulsive signals remains undeter mined at the molecular level and this idea should be tested in in vitro systems. Meanwhile, it is still unclear to what extent vertebrate and invertebrate homologues are equivalent in their functions, especially in the case of N-cadherin. D. melanogaster has no cadherin that can be considered to be the orthologue of vertebrate N-cadherin in terms of primary structure, and this is also the case for E-cadherin. Therefore, the results obtained from D. melanogaster studies might not be applicable to explain vertebrate phenomena. Technological advances in the fields of vertebrate research, such as single cell imaging and in vivo transfection, are enabling us to carry out more precise analyses of cadherin activities in the vertebrate nervous system. Studies using these approaches should resolve the above uncertainty. For most of the members of the protocadherin subfamily, we still lack sufficient information on their functions at both cellular and molecular levels. Solving this problem is an urgent issue in the protocadherin field. As this subfamily contains diverse members, detailed analysis of individual molecules is necessary. Gene knockout studies should also facilitate our understanding of their in vivo roles. FAT cadherins constitute another small subfamily of the cadherin superfamily 90. This subfamily was not included in this review because detailed results on its function in the nervous system are not available yet. Its members are, however, expressed in neurons ; in addition, one of the subfamily members, FAT1, shows intriguing biological activities at cell junctions 95. Fat1-knockout mice exhibit defects in the early forebrain 96, suggesting the involvement of FAT1 in neuronal development. One of the general interests of researchers studying the cadherin superfamily is whether the molecular diversity of its members and their binding specificities contribute to specific neuronal connections. Classic cadherins are differentially expressed in the vertebrate brain, and these expression patterns correlate with neuronal connectivity 18,19. Based on this observation, cadherin subtype-mediated selective cell adhesion has been proposed to have a role in specific synaptic connections 19 ; this idea was supported by the finding that artificial alterations of the cadherin subtypes expressed in the chicken tectal fibres by transgenic methods modified their target selectivities 34. The diversity of protocadherins is also suggested as a possible explanation for complex neuronal connectivity. However, evidence to support these hypotheses, particularly the one based on loss-of-function studies, is still not sufficient. So far, what is most clear is that cadherins are required as permissive adhesion molecules for synapse formation. In the D. melanogaster visual system, without assuming additional regulators, we cannot explain the role of DN-cadherin in specific axon targeting. Such a possible regulator exists in hippocampal neurons, as nectins instruct cadherin localization to synapses formed by these cells. Further tests are needed to determine whether cadherin or protocadherin molecules alone can function as instructive recognition molecules or if they always require cofactors. Assuming that they function only as permissive adhesion molecules, what is the reason for their diversity? Concerning classic cadherins, we can propose that each cadherin subtype is endowed with unique biochemical or physiological properties and that each neuron requires particular cadherin subtypes for specifying their synaptic functions. In fact, different cadherins are known to produce different degrees of adhesive strength 97. Their homophilic binding properties could be utilized for ensuring that a particular cadherin becomes localized in synapses formed between a specific pair of neurons. In summary, cadherin superfamily gene products are clearly important in mediating several aspects of neuronal interactions, including axon targeting and patterning of neurites. The precise molecular mechanisms underlying these cadherin actions still remain unclear for most of the members. Even for the most well-studied classic cadherins, the nature of their actions regarding neuronal behaviour is mysterious and controversial, as has been mentioned above. Moreover, we know too little about the cellular and molecular functions of protocadherins. If these problems are clarified, we might be able to gain deeper insights into the mechanisms underlying the formation of complex brain circuitries. 18 JANUARY 2007 VOLUME 8

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