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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, 2-2-3
Minatojima-Minamimachi,
Chuo-ku, Kobe 650-0047,
Japan.
e-mail: [email protected]
doi:10.1038/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 neurons1. Such arborizations contribute
to the formation of dendritic fields, which are specific for
each neuronal type. Under the guidance of attractive or
repulsive factors2, axonal growth cones migrate towards
their target neurons and eventually make contact with
them to form synapses3, 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 adhesion4–6.
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 FAT7,8. The biological functions
of the family members seem to have diverged: some of
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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
binding9,10 (FIG. 1). The number of these repeats varies
greatly among the members, from 1–34. 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
recently11, and so is not discussed in detail here.
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Plasma membrane
Vertebrate classic cadherins
(e.g. N-cadherin, E-cadherin)
Drosophila E-cadherin
Drosophila N-cadherin
Mammalian CELSR1
Drosophila Flamingo
Mammalian α-protocadherin
Mammalian γ-protocadherin
Extracellular Intracellular
Cadherin repeat
Non-chordate-specific domain
EGF-like domain
HormR domain
Laminin globular-like domain
GPS domain
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.
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.
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 organs12. 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, bind7,13. The cadherin-coupled β-catenin
further associates with α-catenin, which is known to be
an actin-binding protein14,15. 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 centre13. A given cadherin typically
interacts with the same cadherin subtype, exhibiting
homophilic interactions, although it can also bind other
limited subtypes heterophilically16,17. 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
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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
contacts18,19.
Invertebrates have similar classic cadherin-like
molecules that, through their cytoplasmic domain, can
bind catenins7,20–22. However, these molecules differ
from their vertebrate counterparts in terms of primary
structure, in particular by having larger extracellular
domains23 (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 cells25.
Conventional knockout of the mouse N-cadherin gene
causes early embryonic lethality, mainly because of heart
defects26,27. Zebrafish N-cadherin mutants, however,
survive longer, exhibiting various malformations of the
CNS28–31, which is consistent with earlier observations
made by using N-cadherin-blocking antibodies32. For
example, the neural retina in the mutant zebrafish is
severely distorted, exhibiting a rosette-like cell rearrangement30 (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
nerves33. Furthermore, in zebrafish N-cadherin mutants,
retinal optic axons cannot migrate normally to their
targets, showing some misrouting30 (FIG. 2a).
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a
Wild type
N-cadherin mutant
b
Control
Dominant-negative
Chiasma
Retina
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 R1–R6 axons
innervate L1–L5 neurons in the
lamina. The lamina L1–L5
neurons relay R1–R6 input to
the medulla.
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 sorting34 and in
the regulation of physiological functions of the brain,
such as long-term potentiation in the hippocampus35.
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 intact36,37. 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 interactions38, 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 inhibited39.
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 retina40 (FIG. 2b).
Such constructs were also used to show that cadherins
are required for tangential migration of precerebellar
neurons41. In addition, mouse mutants of αN-catenin,
one of the catenins supporting neuronal cadherin functions, show various defects in neural morphogenesis
and axon extension42. 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
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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 lethality23. 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 structures43, 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 R1–R8. Of these, the R1–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 disrupted44.
The wild-type lamina contains an array of neurons
arranged in columns45, each of which consists of five
monopolar neurons, designated L1–L5 (FIG. 3b). From
each ommatidium, a bundle of R1–R6 axons reaches
the lamina and associates with a specific column, where
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c
a
Wild type
Eye
Medulla
Lamina
Optic lobe
DN-cadherin mutant
b
R3 R4
R5
R2 R7
R6
R1
Retina
R3 R4
R5
R2 R7
R6
R1
R3 R4
R5
R2 R7
R6
R1
R3 R4
R5
R2 R7
R6
R1
R3 R4
R5
R2 R7
R6
R1
R3 R4
R5
R2 R7
R6
R1
Flamingo mutant
Lamina
L1
L2
L3
L4
L5
Medulla
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 (R1–R8; R8 is not shown here). R1–R6 cells
project to the lamina, whereas R7 and R8 project to the medulla. R1–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, R1–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,
R1–R6 axon terminals derived from different ommatidia
are synaptically connected with a cluster of monopolar
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 cartridges46,47 (FIG. 3c).
When the laminar neurons do not express DN-cadherin,
the cartridges are severely disorganized and fail to receive
R cell innervation47. 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
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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 medulla46,48. 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 neurons49.
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 disrupted50. 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 glomeruli51. 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
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a
R8 zone
R7 zone
Lamina
Lack of
R7 axon
terminals
Medulla
DN-cadherin mutant
Wild type
Lamina
Lack of
R8 axon
terminals
Medulla
Wild type
b
Flamingo mutant
Wild type
Lamina plexus
Medulla
R8 growth
cones
Flamingo (R cell mosaic)
Lamina plexus
Medulla
Overlapping
R8 growth cones
Overlapping R8 growth cones
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
medulla46,48,70,71. 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 axons71. 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 structure51. 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
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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 mutants23 and the optic
tract of N-cadherin-mutated zebrafish30 (FIG. 2a). Neurite
retraction was observed not only in the R1–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 dendrites52,53. 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 mutants54,55. 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 mutants56,57.
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
GTPases58. 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.
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α
Axon
Dendrite
β
α
Nectin 1
Cadherin
α
Afadin
α-catenin
α
β
β
β
β
β
α
As expected from the function of Flamingo, CELSR1
has been shown to regulate PCP in inner ear hair
cells67. Flamingo and CELSR have also been shown to
be important in several neuronal interactions.
α
β
β
α
Nectin 3
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, respectively99.
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-catenin59. 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 interactions60.
Importantly, in hippocampal neurons, nectin 1 is localized in axons, whereas nectin 3 is distributed to both
axons and dendrites61. 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.
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.
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 receptors62 (FIG. 1).
Flamingo regulates planar cell polarity (PCP) in epithelial
cell sheets by cooperating with other PCP-regulating
elements such as Frizzled, a Wingless receptor63. Three
mammalian homologues of Flamingo have been identified, CELSR1, CELSR2 and CELSR3 (REFS 64–66).
16 | JANUARY 2007 | VOLUME 8
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 Flamingo68 — 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 growth69.
Flamingo in axon target selection in the visual system.
Flamingo is expressed in growth cones of R1–R6 axons,
and is required for them to select appropriate targets in
the lamina. In the absence of Flamingo, R1–R6 axons
extend to, and form synapses with, incorrect targets70
(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 R1–R6 axons to appropriate targets70. 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 lobe70,71. 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 cells72,73. 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 decreased74
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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
development75. 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 neurons75, 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 analysis8, 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 mRNA76,77, 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
fashion78. 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 products8,79.
The molecular functions of these protocadherins
are still poorly understood. Many protocadherins have
weak homophilic binding ability and can associate with
characteristic cytoplasmic partners8,79. 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
NATURE REVIEWS | NEUROSCIENCE
a
Wild type
Celsr2 knockdown
b
Wild type
Celsr3 knockout
CX
CX
CC CC
STR
STR
AC
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 mammals80. 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 cells80.
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
VOLUME 8 | JANUARY 2007 | 17
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REVIEWS
regions81,82, although their distributions are not confined
to synapses83. 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 stages83, 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 development84. 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 ADAM10dependent proteolytic systems85–87, but the biological
role of this process remains unknown. α-protocadherin
(PCDH-α) is localized in synapses88, but only presynaptically in ciliary ganglionic neurons89. 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 undetermined 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.
18 | JANUARY 2007 | VOLUME 8
FAT cadherins constitute another small subfamily
of the cadherin superfamily90. 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 neurons91–94; in addition, one of the subfamily members, FAT1, shows intriguing biological activities at cell junctions95. Fat1-knockout
mice exhibit defects in the early forebrain96, 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 connectivity18,19. Based on this observation,
cadherin subtype-mediated selective cell adhesion has
been proposed to have a role in specific synaptic connections19; 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 selectivities34. 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 strength97. 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.
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Acknowledgements
I thank T. R. Clandinin, C. Desplan, H. Togashi and T. Usui for
providing the original drawings for schematic illustrations; A.
Goffinet, Y. Iwai, I. Masai, K. Tanabe and T. Uemura for providing photographs; S. Hirano for data analysis; and S. Ito for
her help in preparing figures. Work in the laboratory was supported by the program Grants-in-Aid for Specially Promoted
Research of the Ministry of Education, Science, Sports, and
Culture of Japan.
Competing interests statement
The author declares no competing financial interests.
DATABASES
The following terms in this article are linked online to:
Entrez Gene: http://www.ncbi.nlm.nih.gov/entrez/query.
fcgi?db=gene
CELSR1 | CELSR2 | CELSR3 | FAT1 | Flamingo | LAR
Access to this links box is available online.
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