COMMEMORATIVE LECTURE
Mechanisms of morphogenetic cell assembly
Masatoshi Takeichi
Department of Cell and Developmental Biology, Graduate School of Biostudies, Kyoto University, Kyoto, Japan(Received for publication on March 27, 2002)

Key words: cadherin, cell adhesion, morphogenesis, synapse

Animal cells isolated from tissues can reaggregate
in vitro. Within the aggregates, cells reconstruct tissue
structures, in which ‘‘cell sorting’’ is an essential process. It is thought that this self-assembling ability is abasic property of animal cells. More than 25 years ago, I
became attracted to this phenomenon, and began toidentify molecules involved in the tissue reconstruction
processes. Yesterday, I explained the early story of mystudies, and so I am going to skip over this part of thestory today, and focus on our recent progress.
Through the studies from 1974 into the 1980’s, weidentiﬁed a group of adhesion molecules designated as
the cadherins.1 Cadherin is a so-called ‘‘homophilic’’adhesion molecule, connecting cells via a like-like molecular interaction (Fig. 1). Without cadherin, cells intissues tend to come apart, although they generally still
maintain weak associations, because many other adhesion proteins are also present on the cell surface. To-day, in the initial part of my talk, I would like to summarize the general properties of the cadherin molecule,and in the rest of the talk, tell you about our recent
ﬁndings on the role of cadherin in synaptic junction andneural network formation.
The functions of cadherin molecules can be divided
into two categories. First, these molecules are essential
for tight cell-cell associations. Second, cadherins areinvolved in speciﬁc cell adhesion or sorting. Both functions are regulated through the actin-based cytoskeleton. The next slide indicates how cadherins are important for tight cell-cell adhesion. These are early mouseembryos (Fig. 2). During development, they undergoso-called ‘‘compaction.’’ At the early 8-cell stage, thecells are round, showing clear boundaries between
themselves, but their boundaries gradually become
invisible during the late 8-cell stage of development.
However, when the cadherin system is blocked in em-Cadherin molecules are essential for tight cell-cell association.

Fig. 2 Compaction of early mouse embryos is blocked by antibodies
to E-cadherin.

Fig. 1

Presented at the commemorative lecture of the award ceremony of the 2001 Keio Medical Science Prize, November 29, 2001.
Reprint requests to: Dr. Masatoshi Takeichi, RIKEN Center for Developmental Biology, 2-2-3 Minatojima-Minamimachi, Chuo-ku, Kobe
650-0047, Japan
Present afﬁliation: RIKEN Center for Developmental Biology, Kobe, Japan72

Keio J Med 2002; 51 (2): 72–76

Fig. 3 The classic cadherin subfamily. Each cadherin undergoes atype-speciﬁc homophilic interaction.

bryos at this late stage, individual cells again becomeround, indicating that their associations have become
very loose. One can do similar experiments using moredifferentiated tissues, such as embryonic retinal fragments. If retinal fragments collected from early embryos
are cultured in the presence of anti-cadherin antibodies,
these fragments become disassociated into small cellclusters or single cells. Such experiments clearly demonstrate the importance of cadherins in maintaining
cells together in a variety of cellular systems.
Next, I should emphasize that there are a numberof different cadherin molecules, such as E-cadherin(E-cad), N-cadherin (N-cad), and M-cadherin (Fig. 3).
Some cadherins are called only by numbers, such as
cadherin-6, -8 and -11. More than 15 cadherins havebeen identiﬁed from each vertebrate species including
human beings, and are now called ‘‘classic cadherins.’’
In vitro, cells expressing different cadherins are sorted
from each other based on their properties of homophilic interaction.
Then, a question arises as to the role of this varietyof cadherins in vivo. When we look at the expressionof a given cadherin during development, we noticethat its pattern dynamically changes. Interestingly, suchchanges, in general, correlate with morphogenetic
events. A typical example is seen during neuronal tube
formation and neural crest differentiation. In the earlychicken embryo, the overlying ectoderm expresses LCAM, a chicken homologue of mammalian E-cad, butwhen the neural tube forms, L-CAM gradually disappears from the forming tube, and instead N-cad begins
to appear. Another cadherin, cadherin 6B (cad6B), isalso added to the dorsal portion of the neural tube.
When neural crest cells begin to migrate, these cells
stop expressing N-cad and cadh6B, and instead start toexpress cadherin-7 (cad7). Migrating crest cells seem toexpress only cad7.

73

Fig. 4 Cadherin type changes during neural crest emigration fromthe neural tube.

What is the biological role of these changes? Let me
focus on the neural crest-forming region. Dorsal neuraltube cells use N-cad and cad6B for their mutual connections. Crest cells leaving the neural tube, however,lose these two cadherins, and instead begin to express
cad7 (Fig. 4). To understand what kind of cell behavior
is controlled by these changes, we did experiments toperturb their expression pattern by overexpressing Ncad or cad7 at the dorsal neural tube. What we found
was that, when either N-cad or cad7 had been overexpressed in the dorsal neural tube, crest cells expressing this ectopic molecule became unable to migrateout of the neural tube.2 This result suggests that theswitching of cadherin types during neural crest development is essential for the crest cells to leave thetube. Under the overexpression of N-cad or cad7, presumably, crest and tube cells stick together, being unable to separate from one another. These observations
are an example of how the differential expression ofmultiple cadherins is important for morphogenetic processes.
Now I would like to discuss the regulatory aspectsof the cadherin system. The activities of cadherins areregulated through cytoplasmic components. In epithelial cells, the cadherins organize so-called ‘‘adherens
junctions,’’ part of the junction complex, to which a
number of cytoplasmic proteins, including ‘‘catenins’’,
become directly or indirectly associated. The cytoplasmic domain of cadherin can be subdivided into two
portions, the N-terminal ‘‘juxtamembrane (JM)’’ and Cterminal half domains (Fig. 5). To the latter, b-cateninbinds directly, and a-catenin binds to this b-catenin; thismolecular complex further associates with the actin ﬁlaments via the a-catenin. To the JM domain, anothercatenin, p120-catenin, binds.3 What are the roles ofthese catenins? The b-catenin/a-catenin complex is wellknown to be essential for the cadherins to function as

74

Takeichi M: Cadherin-mediated morphogenesis

Fig. 5

A cadherin-catenin complex.
Fig. 7 Colo205 cells transfected with the control full-length Ncadherin or mutant N-cadherin in which the JM domain had been
deleted. Left, phase-contrast micrographs; right, immunoﬂuorescencestaining for the introduced molecules. (Prepared by Shinji Aono)

Fig. 6 PC12 cells before and after transfection with the a-catenincDNA.

adhesion molecules. Here is a nice example. PC12 is a
carcinoma cell line derived from a lung tumor. Thesecells lose their a-catenin gene, and grow as single cells.
However, once a-catenin cDNA has been introduced
into these cells, they begin to aggregate strongly,organizing epithelioid spheres (Fig. 6).4 Thus, the introduction of a-catenin was sufﬁcient to restore epithelial cell-cell junctions in these mutant cells, indicatingthat the C-terminal half domain, as the b-catenin/
a-catenin complex binding site, plays a critical role injunction formation.
What about the JM domain? We have evidence that
the JM domain is a very important regulatory portion of
the cadherin molecules. This idea came from the analysis of the adhesive properties of another tumor cellline, called Colo205, derived from a colon carcinoma.
These cells grow just like the PC12 cells; they nevershow tight associations, and grow as loose aggregates.
However, very interestingly, this cell line, different
from PC12, expresses all the important components
for the cadherin system, E-cadherin, a-catenin, and bcatenin.5 Nevertheless, they cannot normally aggregate.
This was a very curious phenomenon for us, prompting
us to determine why their cadherin system could notwork.

In the course of various series of experiments, wefound that, when a mutant cadherin, in which the JM
domain had been artiﬁcially removed, was introduced
into Colo205 cells, their cadherin-dependent adhesion
was restored (Fig. 7), indicating that cadherin molecules
can normally function in this cell line, if they lack theJM domain. To explain this interesting phenomenon,
we hypothesized that the JM domain has some inhibitory signals to suppress the cadherin function, assumingthat in normal cells, these hypothetical inhibitory signals are inactive, whereas in Colo205 cells, the inhibitory system is abnormally activated. If such inhibitory
signals indeed exist, these signals might be used forthe regulation of cell adhesion, e.g., for promoting celldetachment necessary for cell relocation or rearrangement.
To test this idea, we chose somite development as amodel system. Somite development involves a numberof cell rearrangement processes, and among them wefocused on myotome morphogenesis.6 Myotome cells
arise from the expanding dermomyotome, and are rearranged to form the myotome cell sheets. We injected
the JM domain-deﬁcient cadherin into somites using
adenoviral expression vectors, and found that myotome
cells expressing these mutant molecules could not undergo normal relocation; they became stuck at a lateralposition of the body forming clusters, never expanding
to organize two dimensional cell sheets (Fig. 8). Thisphenomenon indicated that without the JM domain,cell-cell adhesion could not be regulated, causing cells
to clump. The JM domain is thus indeed necessary forthe regulation of cell relocation; it perhaps temporarily
inhibits cadherin-mediated cell adhesion to allow cells
to relocate. We do not know what kind of inhibitors areworking with the JM domain, and identiﬁcation of themis an important future goal.
Now I would like to move to the second story. How
the neural network is formed is a central question inneural biology. Neural network formation can be considered as a problem of cell-cell adhesion, as the net-

Keio J Med 2002; 51 (2): 72–76

75Fig. 9 Processes of synapse formation, and the effect of cadherin
blockade.

Fig. 8 Effects of the ectopic expression of the control full-length Ncadherin (cN) or JM-domain-deleted mutant N-cadherin (cN JM (-))
in somites of chicken embryos. cDNAs encoding these molecules andb-galactosidase were used for co-transfection, and cells expressing thelatter were stained. Myotomes are visualized by this staining. The
images in the right panels are a larger magniﬁcation of the enclosedportions of the left panels. (Prepared by Kazuki Horikawa)work is established by a sequential connection of neurons. If we could understand how neurons recognize
each other and become connected with one another, weshould be able to answer important questions concerning the mechanisms of neural network formation.
The synapse is the site for interneuron connections.
There are two types of synapses, excitatory and inhibitory. Many of the excitatory synapses are formed bycontacts between dendritic spines and axons, and others,by direct contacts of the axons with the dendrtic shaft.
In textbooks of neural biology, you see receptors, channels, and transmitters in the illustrations of synapses,but may not see any molecules connecting the synaptic
plasma membranes in apposition. This omission meansthat this aspect of the synapse has been poorly studied.
We became interested in uncovering the mechanisms
for the recognition and connection between the plasma
membranes constituting synapses.
Several years ago, we found that cadherin and catenins were localized in synaptic junctions, although thesemolecules avoided the transmitter release zones, suggesting that the cadherin system may be involved ininterneuronal synaptic connections in some speciﬁcways.7 We are testing this idea using various approaches.
Today I want to tell you about some of our recent
results obtained from experiments using in vitro culturesof hippocampal neurons. Hippocampal neurons canform synapses in vitro. The cellular processes of excitatory synapse formation are the following: Neurons initially extend dendrites and axons. From the dendrites,
ﬁne ﬁlopodia protrude, and when the ﬁlopodia meet anaxon, their tips attach to it, swelling at the contact por-tion (Fig. 9). Subsequently, synaptic vesicles start toaccumulate in the presynaptic sites, and postsynaptic
density proteins also accumulate, leading to the maturation of synapses.
Using such cultured neurons, we attempted to inhibit cadherin by two different methods, expression ofa dominant-negative cadherin construct,8 and genetic
knockout of the aN-catenin gene, whose presence isessential for cadherin function.4 Now I show you theresults of the ﬁrst approach (manuscript in preparation). When the dominant negative cadherin was overexpressed in neurons, their dendritic morphology was
dramatically altered (Fig. 9). Many of the dendritic
spines were transformed into ﬁlopodium-like processes,
and others were irregularly deformed, losing the typicalmushroom shape, although they tended to maintaincontacts with axons. Next, we studied the intracellularorganization in these treated neurons. Synapsin, a synaptic vesicle protein, shows a punctate distribution innormal neurons, each punctum representing a clusterof synaptic vesicles. When the dominant-negative cadherin was expressed, their distribution became diffuse,suggesting that synaptic vesicle accumulation had beenblocked. Consistently, the uptake of FM-64, a ﬂuorescent dye, into synapses was greatly reduced, indicating
the suppression of synaptic vesicle recycling in cadherinblocked neurons. Furthermore, we examined the distribution of PSD-95, a postsynaptic density protein, andagain found that its accumulation into synapses had
been perturbed.
As another approach, we collected hippocampal
neurons from aN-catenin KO mice, and cultured themin vitro. The results were consistent with the observations I just mentioned. Dendritic spines elongated in
the mutant neurons. Thus, both experiments, genetic
and in vitro overexpression experiments, showed similar effects of cadherin blockade on synapse formation.
To summarize, when cadherin-mediated adhesion isblocked, synaptic contacts destabilize, and dendritic
spine shape is altered (Fig. 9). Intracellularly, the preand post-synaptic protein organization became dis-76

Takeichi M: Cadherin-mediated morphogenesis

synaptic cell contacts. Thus, a common adhesion mechanism is operating over a wide variety of spectrum
ranging from ﬁbroblastic adhesion to synaptic neuronalcontacts, suggesting that the principles for the controlof cell-cell adhesion may be identical throughout such awide variety of adhesion systems. It would be an intriguing future subject to understand how such variations
in cell-cell contact have been generated during theevolution of complex multicellular organisms.
References
Fig. 10 LTP in cadherin-11 knockout mice. (The photograph wasprepared by Osamu Chisaka.)rupted. These ﬁndings strongly suggest that cadherin
plays a crucial role in synaptic junction formation.
Finally, we are asking whether cadherin is also important for the physiological functions of synapses. Thespine elongation phenotype is reminiscent of that seenin the Fragile-X syndrome causing mental retardationand other neurological defects. It should also be stressedthat synapse morphology is dynamically changed during
the establishment of long-term potentiation (LTP). As I
have shown you, cadherin blockade affects spine shape.
We can, therefore, speculate that this adhesion molecule could be an important regulator for synaptic
physiological activities. In fact, we previously foundthat in the hippocampus of cadherin-11 KO mice, LTP
was enhanced,9 suggesting that synapses becomes more
plastic when this cadherin was deleted from the hippocampus (Fig. 10). This ﬁnding together with the otherobservations suggests that cadherin may act as a modulator of synapse physiology by altering synaptic contact structures.
In sum, cadherin was identiﬁed as a molecule essential for the adhesion of ﬁbroblasts and epithelial cells,
and found to be important for the regulation of morphogenetic cell behavior. Recent studies have now
shown that this molecular family is also involved in

1. Takeichi M: Cadherin cell adhesion receptors as a morphogeneticregulator. Science 1991; 251: 1451–1455
2. Nakagawa S, Takeichi M: Neural crest emigration from the neuraltube depends on regulated cadherin expression. Development
1998; 125: 2963–2971
3. Shibamoto S, Hayakawa M, Takeuchi K, Hori T, Miyazawa K,Kitamura N, Johnson KR, Wheelock MJ, Matsuyoshi N, TakeichiM, et al: Association of p120, a tyrosine kinase substrate, with Ecadherin/catenin complexes. J Cell Biol 1995; 128: 949–995
4. Hirano S, Kimoto N, Shimoyama Y, Hirohashi S, Takeichi M:
Identiﬁcation of a neural alpha-catenin as a key regulator of cadherin function and multicellular organization. Cell 1992; 70: 293–301
5. Aono S, Nakagawa S, Reynolds AB, Takeichi M: p120 ctn acts as
an inhibitory regulator of cadherin function in colon carcinoma
cells. J Cell Biol 1999; 145: 551–562
6. Horikawa K, Takeichi M: Requirement of the juxtamembrane
domain of the cadherin cytoplasmic tail for morphogenetic cell
rearrangement during myotome development. J Cell Biol 2001;
155: 1297–1306
7. Uchida N, Honjo Y, Johnson KR, Wheelock MJ, Takeichi M: The
catenin/cadherin adhesion system is localized in synaptic junctions
bordering transmitter release zones. J Cell Biol 1996; 135: 767–779
8. Fujimori T, Takeichi M: Disruption of epithelial cell-cell adhesion
by exogenous expression of a mutated nonfunctional N-cadherin.
Mol Biol Cell 1993; 4: 37–47
9. Manabe T, Togashi H, Uchida N, Suzuki SC, Hayakawa Y,Yamamoto M, Yoda H, Miyakawa T, Takeichi M, Chisaka O:Loss of cadherin-11 adhesion receptor enhances plastic changes inhippocampal synapses and modiﬁes behavioral responses. MolCell Neurosci 2000; 15: 534–546