RESEARCH ARTICLE 1315
Development 137, 1315-1325 (2010) doi:10.1242/dev.043190
© 2010. Published by The Company of Biologists Ltd
Nectin-2 and N-cadherin interact through extracellular
domains and induce apical accumulation of F-actin in apical
constriction of Xenopus neural tube morphogenesis
Hitoshi Morita1,2, Sumeda Nandadasa3, Takamasa S. Yamamoto2, Chie Terasaka-Iioka2, Christopher Wylie3 and
Naoto Ueno1,2,*
SUMMARY
Neural tube formation is one of the most dynamic morphogenetic processes of vertebrate development. However, the molecules
regulating its initiation are mostly unknown. Here, we demonstrated that nectin-2, an immunoglobulin-like cell adhesion molecule,
is involved in the neurulation of Xenopus embryos in cooperation with N-cadherin. First, we found that, at the beginning of
neurulation, nectin-2 was strongly expressed in the superficial cells of neuroepithelium. The knockdown of nectin-2 impaired neural
fold formation by attenuating F-actin accumulation and apical constriction, a cell-shape change that is required for neural tube
folding. Conversely, the overexpression of nectin-2 in non-neural ectoderm induced ectopic apical constrictions with accumulated Factin. However, experiments with domain-deleted nectin-2 revealed that the intracellular afadin-binding motif, which links nectin-2
and F-actin, was not required for the generation of the ectopic apical constriction. Furthermore, we found that nectin-2 physically
interacts with N-cadherin through extracellular domains, and they cooperatively enhanced apical constriction by driving the
accumulation of F-actin at the apical cell surface. Interestingly, the accumulation of N-cadherin at the apical surface of
neuroepithelium was dependent on the presence of nectin-2, but that of nectin-2 was not affected by depletion of N-cadherin. We
propose a novel mechanism of neural tube morphogenesis regulated by the two types of cell adhesion molecules.
INTRODUCTION
Neural tube formation is an essential and characteristic
morphogenetic event of vertebrate development, involving the
dynamic rearrangement of cells, including mediolateral intercalation
and apical constriction (Davidson and Keller, 1999; Colas and
Schoenwolf, 2001). Apical constriction is seen in neural tube
formation in all vertebrates except for teleosts. It is a morphological
change in which the epithelial cells undergo acute contraction in
their apical surface, accompanied by cell elongation in the
apicobasal axis, thus taking on a wedge-like shape. This cell-shape
change is followed by the invagination of the neuroepithelium,
which forms a neural groove along the dorsal midline flanked by
neural folds: bulges of neural ectoderm. Eventually, the folds fuse at
the midline, completing the formation of the neural tube (Colas and
Schoenwolf, 2001).
Apical constriction is observed not only in vertebrates, but also in
invertebrates in circumstances such as invagination of the mesoderm
and dorsal closure in Drosophila melanogaster (Lecuit and Lenne,
2007). To the extent that the molecular mechanisms have been
elucidated, similar molecular pathways apparently govern this
process in both phyla. Small GTPases, including Rap1 for
1
Department of Basic Biology, School of Life Science, The Graduate University for
Advanced Studies (SOKENDAI), Shonan Village, Hayama, Kanagawa 240-0193,
Japan. 2Division of Morphogenesis, National Institute for Basic Biology, Nishigonaka
38, Myodaiji, Okazaki, Aichi 444-8585, Japan. 3Division of Developmental Biology,
Cincinnati Children’s Hospital Research Foundation, 3333 Burnet Ave, Cincinnati,
OH 45229, USA.
*Author for correspondence ([email protected])
Accepted 11 February 2010
vertebrates (Haigo et al., 2003), and RhoGEF2, Rap1 and Rho1 for
invertebrates (Barrett et al., 1997; Häcker, 1998; Sawyer et al.,
2009), play important roles. As a common downstream target of
these pathways the apically localized actomyosin tensile system is
activated via Rho kinases and generates a force to narrow the cell
surface, ensuring apical constriction (Wei et al., 2001; Dawes-Hoang
et al., 2005; Nishimura and Takeichi, 2008). Recent studies have
shown that the Shroom family of actin-binding molecules is
involved in the apical constriction of both cultured cells and
Xenopus embryos and may facilitate the apical accumulation of actin
filaments (F-actin) (Haigo et al., 2003; Hildebrand, 2005; Fairbank
et al., 2006; Lee et al., 2007; Nishimura and Takeichi, 2008).
Although the apical actin bundle is required for apical constriction
(Burnside, 1971; Karfunkel, 1972; Colas and Schoenwolf, 2001;
Corrigall et al., 2007; Lecuit and Lenne, 2007; Lee and Harland,
2007; Kinoshita et al., 2008), the detailed mechanism of apical
accumulation of F-actin is just beginning to be elucidated. Here we
show that nectin-2, a transmembrane cell adhesion molecule, is
required for the apical constriction of neuroepithelial cells during
Xenopus neural tube formation, by facilitating the apical
accumulation of F-actin.
Nectin is a member of the immunoglobulin (Ig) superfamily, with
four isoforms in mammals, and contains three extracellular Ig-like
domains, a single-pass transmembrane region and four conserved
amino acids of a binding motif for afadin, an F-actin binding protein
that connects nectin to the F-actin in the intracellular region in its Cterminus (Takai and Nakanishi, 2003; Takai et al., 2008). Nectin is
known to mediate cell adhesion and is implicated in signal
transduction with platelet-derived growth factor (PDGF) receptor
and integrins (Takai and Nakanishi, 2003; Takai et al., 2008).
Immunohistochemical studies of MDCK cells and the mouse small
DEVELOPMENT
KEY WORDS: Nectin-2, N-cadherin, Xenopus, Neural tube closure, Apical constriction, F-actin
1316 RESEARCH ARTICLE
MATERIALS AND METHODS
Database search and electronic northern
We searched Xenopus nectin in the NCBI BLAST server
(http://blast.ncbi.nlm.nih.gov/Blast.cgi) and JGI X. tropicalis genome
database v4.1 (http://genome.jgi-psf.org/Xentr4/Xentr4.home.html) against
amino acid sequences of mouse nectins and identified UniGene cluster
numbers for putative Xenopus nectin genes: Xenopus laevis nectin-1
(Xl.49696), nectin-2 (Xl.27064), nectin-3 (Xl.59402) and nectin-4
(Xl.73379); Xenopus tropicalis nectin-1 (Str.73359), nectin-2 (Str.47530),
nectin-3 (Str.44628) and nectin-4 (Str.5750). Electronic northern was
performed by calculating the ratio of expressed sequence tag (EST)
expression levels of each gene from the oocyte to the tailbud stages, using
the EST Profile in the UniGene database.
Embryo culture and manipulation
Xenopus embryos were obtained by standard methods, fertilized in vitro,
dejellied in 3% cysteine solution (pH 7.8) and cultured at 13°C.
Microinjection was performed in 0.1⫻ Steinberg’s solution containing 3%
Ficoll PM400 (Amersham), and the injected embryos were cultured until
they reached the desired stage. Embryos were injected at the four-cell stage
into the dorsal-left-side blastomere in knockdown experiments or into both
of the ventral blastomeres in overexpression experiments. As needed,
membrane-targeted green fluorescent protein (memGFP) or red fluorescent
protein (memRFP) was co-injected as a tracer. Embryos were staged
according to Nieuwkoop and Faber (Nieuwkoop and Faber, 1994). The
aberrant surface phenotype was visually judged and defined by a rough
surface and ectopic pigmentation.
In situ hybridization and b-galactosidase staining
Whole-mount in situ hybridization (WISH) was performed as described
previously (Harland, 1991; Goda et al., 2009). b-Galactosidase staining was
performed in advance of the WISH process as described previously (Chung
et al., 2005).
RT-PCR
RT-PCR analysis for nectin-2 expression was performed as described
previously (Chung et al., 2005) with the following primers: forward, 5⬘TAGGATCCGTGAAAGCTGATCCAAAAAAC-3⬘; reverse, 5⬘-GGAATTCTTACGTCTCTTTAGAGTCGTAG-3⬘.
DNA constructs
We obtained Xenopus laevis nectin-2, afadin and N-cadherin cDNA clones
from an XDB3 cDNA database (http://xenopus.nibb.ac.jp/): clone numbers
XL170j24 (for nectin-2a; GenBank accession number GU290315),
XL219e23 (nectin-2b; GU290316), XL293e12ex (afadin) and XL289n05ex
(N-cadherin). nectin-2 rescue construct was generated by changing
nucleotides at the morpholino target site of nectin-2a as follows, indicated
in small letters: (–16) 5⬘-AAtAttATatcGGTAAATG-3⬘ (+3). The nectin-2
deletion constructs were generated by removing the sequences of nectin-2a
that correspond to the following amino acids: nec2-DC4 (A467-V470),
nec2-DIC (R353-V470), nec2-DEC (L37-L313). For afadin cDNA, as the
XL239e12ex sequence was partially lost, we referred to the sequence of
Xenopus tropicalis afadin in the genome database (JGI Protein ID: 153941)
to reconstruct Xenopus laevis afadin (GenBank accession number
GU290317). The coding sequence of N-cadherin was subcloned from
XL289n05ex using PCR with the following primers: forward 5⬘CACCATGTGCCGGAAAGAGCCCTT-3⬘ and reverse 5⬘-AGTTCAGTCGTCGCTCCCTCCGTACAT-3⬘. Xenopus laevis E-cadherin was
obtained from a stage 10 cDNA library, prepared as described previously
(Chung et al., 2005), using PCR with the following primers: forward
5⬘-TAGATATCACCATGGGGTTGAAGAGGCCCT-3⬘ and reverse 5⬘GCGATATCTTAATCCTCATCACCTCCATAC-3⬘. Xenopus laevis Ccadherin construct was as previously described (Lee and Gumbiner, 1995).
For the extracellular domain constructs, the following amino acid sequences
were used: nec2-ex-GST/-FLAG (M1-N325), Ncad-ex-FLAG (M1-T722),
Ecad-ex-FLAG (M1-G696), Ccad-ex-FLAG (M1-D703). A b-cateninbinding domain deleted N-cadherin (Ncad-Db) was generated by deleting
the sequence that corresponds to the amino acids G839-D906. Rescue
constructs of N-cadherin and Ncad-Db were generated by changing
nucleotides at morpholino target site of FLAG-tagged N-cadherin (NcadFLAG) or Ncad-Db (Ncad-Db-FLAG), respectively, as follows indicated in
small letters: (–4) 5⬘-CACCATGTGtaGaAAgGAaCCaTTt-3⬘ (+21).
Morpholino oligonucleotides and mRNA
Antisense morpholino oligonucleotides (MOs) were purchased from Gene
Tools, LLC (OR), and consisted of the following sequences: nec2-MO, 5⬘CACCATTTACCCCGATCCTCTTATC-3⬘; Ncad-MO, 5⬘-GAAGGGCTCTTTCCGGCACATGGTG-3⬘ (Nandadasa et al., 2009); control MO, 5⬘CCTCTTACCTCAGTTACAATTTATA-3⬘. mRNAs were synthesized
using mMESSAGE mMACHINE SP6 (Ambion) and purified on a NICK
column (GE Healthcare).
Western blot, GST pull-down assay and co-immunoprecipitation
For western blot, embryos or explants at desired stages were lysed in lysis
buffer [50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 50 mM NaF, 5 mM
EDTA, 0.5% NP-40] with protease inhibitors. The supernatant of the lysate
was sampled and denatured by the same volume of 2⫻ SDS sample buffer
(0.5 M Tris-HCl pH 6.8, 10% SDS, 50% glycerine, 2-mercaptoethanol).
After boiling for 5 minutes, the samples were processed in SDS-PAGE,
DEVELOPMENT
intestine showed that nectin was preferentially localized to adherens
junctions (AJs), which the F-actin bundle underlies intracellularly
(Takeichi, 1988; Dejana et al., 1995; Takahashi et al., 1999; SatohHorikawa et al., 2000). Mouse nectin is also expressed in embryonic
epithelial cells, including the neuroepithelium and developing
cranial nerve ganglia (Okabe et al., 2004). These expression patterns
suggest that nectin might play roles not only in the maintenance of
adult tissues but also in developmental processes, such as formation
of the neural tissue. However, even though the nectins are expressed
from the early embryonic stages, no obvious defects in
embryogenesis have been seen in knockout mice for the individual
nectin genes (nectin-1, -2 or -3) (Bouchard et al., 2000; Inagaki et
al., 2005). This lack of phenotype could be due to redundant
functioning among the nectin genes.
In Drosophila, Echinoid (Ed), which shares some similar
properties with vertebrate nectin, has been implicated in the
embryonic morphogenesis (Wei et al., 2005; Laplante and Nilson,
2006; Lin et al., 2007). Studies suggested that Ed controls apical
constriction by assembling actin cables at the interface of juxtaposed
cells expressing and lacking Ed. Therefore, structural similarities
between nectin and Ed cause us to speculate that vertebrate nectin
may function in embryonic morphogenesis.
In this study, we found that Xenopus nectin-2 induced apical
constriction in cooperation with N-cadherin during neurulation, and
that the interacting point of these two molecules lay in the
extracellular domains. We first found that nectin-2 was strongly
expressed in the neuroepithelium throughout neurulation. Depletion
of nectin-2 caused a closure defect of the neural tube accompanied
by impaired apical constriction. Conversely, nectin-2 overexpression
in non-neural ectoderm induced ectopic apical constriction with
apical F-actin accumulation. However, this effect of nectin-2 did not
require the known molecular linkage between nectin-2 and F-actin,
via afadin. As N-cadherin has been shown by us previously to be
required for neural tube folding (Nandadasa et al., 2009), we tested
whether the action of nectin is also associated with N-cadherin. We
found that N-cadherin and nectin-2 bind through their extracellular
domains and that they cooperatively promote apical constriction.
Furthermore, we found that the role of nectin in F-actin
accumulation may be mediated by the intracellular domain of Ncadherin through b-catenin. Our findings provide the first
mechanistic evidence of the function of nectin in vertebrates and the
regulatory mechanism by which the neuroepithelial cells undergo
apical constriction.
Development 137 (8)
blotted onto the PVDF membrane (Bio-Rad), labeled with the primary and
secondary antibodies and detected using an ECL Plus kit (GE Healthcare).
For GST pull-down assays, 293T cells were transiently transfected with the
indicated constructs by Lipofectamine (Invitrogen). Forty hours after
transfection, the cells transfected with GST were lysed in the lysis buffer
containing 0.17% NP-40 with protease inhibitors and incubated with
Glutathione Sepharose 4B (Pharmacia). For other constructs, conditioned
media were used in the pull-down process. The precipitates were then
washed with the lysis buffer and subjected to western blot. For coimmunoprecipitation of endogenous b-catenin, Ncad-FLAG, Ncad-DbFLAG or FLAG-tagged nectin-2 (nec2-FLAG) was injected into the animal
pole of four-cell-stage embryos at 2 ng for Ncad-FLAG and Ncad-Db-FLAG
and 1 ng for nec2-FLAG. The embryos were cultured until they reached
stage 9, and their animal caps were dissected and lysed in the lysis buffer,
followed by centrifugation at 15,000 rpm (17,400 g) for 30 minutes. The
supernatant was subjected to co-immunoprecipitation reaction with Protein
A Sepharose beads (GE Healthcare) using mouse anti-FLAG antibody
(Sigma). The beads were washed with the lysis buffer, and subsequently
analyzed by western blot.
Immunohistochemistry and F-actin staining
For immunohistochemistry, embryos were fixed in MEMFA (0.1 M MOPS,
pH 7.4, 2 mM EGTA, 1 mM MgSO4, 3.7% formalin) for 1 hour at room
temperature (RT) and then placed in PBST (PBS with 0.1% Tween 20). As
needed, they were sliced using a Microslicer (DTK-3000W; DSK, Kyoto,
Japan). After incubation in blocking solution [10% fetal bovine serum (FBS)
in PBST] for 1 hour at RT, the samples were incubated with primary
antibodies overnight at 4°C. After washing three times with PBST, they were
incubated with secondary antibodies overnight at 4°C and then washed
extensively with PBST before imaging. For F-actin staining, samples were
prepared as above and an aliquot of 4 U/ml Alexa Fluor 546 phalloidin
(Molecular Probes) was added to the secondary antibodies. For the Ncadherin staining, embryos were fixed in Dent’s fixative (Dent et al., 1989)
and, after being blocked in 10% FBS, were incubated with primary and
secondary antibodies, which were diluted in Can Get Signal immunostain
solution A (Toyobo, Osaka, Japan).
Antibodies
For western blot, the following antibodies were used at the indicated
dilutions. The primary antibodies were rabbit anti-GFP (1:2000; Molecular
Probes), mouse anti-GST (1:1000; Santa Cruz), rabbit anti-FLAG (1:1000;
Sigma), rabbit anti-N-cadherin (1:100; Abcam) and mouse anti-b-catenin
(1:1000; Sigma). The secondary antibodies were sheep anti-mouse IgG
HRP-conjugated (1:10,000; GE Healthcare) and donkey anti-rabbit IgG
HRP-conjugated (1:10,000; GE Healthcare). Anti-nectin-2 antibody was
produced from the Xenopus nectin-2 cDNA fragment corresponding to
amino acids V128-T216, which was fused to pGEX-6P-1 (GE Healthcare).
This protein was purified, cleaved from the GST by a protease and used to
immunize mice. The antiserum was used for western blot after its specificity
was checked (see Fig. S5A in the supplementary material).
For immunohistochemistry, the following antibodies were used at the
indicated dilutions. The primary antibodies were rabbit anti-GFP (1:200;
MBL, Nagoya, Japan), mouse anti-a-tubulin (1:500; Sigma), rabbit antiFLAG (1:200; Sigma), mouse anti-FLAG (1:200; Sigma) and mouse antiN-cadherin (1:200; Abnova). The secondary antibodies were Alexa Fluor
488 goat anti-rabbit IgG (1:500; Molecular Probes) and Cy5-conjugated goat
anti-mouse IgG (1:500; Jackson ImmunoResearch).
Image acquisition, processing and statistical analysis
Images of fluorescently labeled samples were obtained using LSM 510
META (Zeiss) with a Plan-Neofluar 20⫻/0.5 numerical aperture objective,
a C-Apochromat 40⫻/1.2 numerical aperture water-immersion objective,
an argon laser (488 nm) and helium-neon lasers (543 nm and 633 nm).
Images were obtained as 512⫻512 or 1024⫻1024 pixels with z-series,
processed with LSM 510 software (Zeiss) for the maximum projection of zseries and with Photoshop 7.0 (Adobe Systems) for tilting and trimming, and
analyzed by using ImageJ v1.42. All statistical analyses were performed
with the paired Student’s t-test.
RESEARCH ARTICLE 1317
RESULTS
nectin-2 is strongly expressed in the superficial
layer of the neuroepithelium of Xenopus embryos
We found putative orthologs for all four mouse nectins in both
Xenopus laevis and Xenopus tropicalis (see Fig. S1A in the
supplementary material; see Materials and methods). Their
estimated expression levels, based on the EST database (i.e.
electronic northern), and RT-PCR analysis showed that nectin-2 is
the predominant form expressed in early Xenopus embryos (see Fig.
S1B,C in the supplementary material). Therefore, we focused on
nectin-2 in subsequent experiments.
We found that Xenopus has two paralogs of nectin-2: nectin-2a
and nectin-2b (62.6% identical). Compared with mouse nectin-2,
they showed good conservation of the extracellular region (41.5%
identical to Xenopus nectin-2a; 41.9% to Xenopus nectin-2b), and
complete conservation of the C-terminal afadin-binding motif (see
Fig. S1D in the supplementary material). In addition, nectin-2
tagged with venus (vns), a variant of yellow fluorescent protein
(Nagai et al., 2002), (nec2-vns) was localized to the plasma
membrane (see Fig. S1E in the supplementary material), as reported
for mouse nectin-2 (Takahashi et al., 1999).
We next performed WISH to see the spatial expression of
nectin-2. At the onset of gastrulation, nectin-2a was uniformly
expressed in the ectodermal layer (data not shown). From the
beginning of neurulation, nectin-2a expression became more
prominent in the superficial layer of the neural plate, along the
entire anteroposterior axis, as well as continuing to be weakly
expressed in non-neural ectoderm (Fig. 1A,B). This pattern was
maintained throughout neurulation (Fig. 1C-F). In the neural
plate, nectin-2a was expressed as two lines along the
anteroposterior axis, which we thought probably corresponded to
the hinge points at which apical constriction is most prominent
(Davidson and Keller, 1999; Wallingford and Harland, 2002; Lee
et al., 2007). The staining specificity was confirmed by the lack
of staining with a sense probe for nectin-2a (Fig. 1G,H). The
expression pattern of nectin-2b was almost identical to that of
nectin-2a (data not shown). These spatiotemporally regulated
expression patterns of nectin-2 during neurulation imply its
involvement in neural development.
Depletion of nectin-2 disrupts the formation of
the neural folds
To investigate the roles of nectin-2 in early development, we next
performed a knockdown experiment by using an MO against
nectin-2 (nec2-MO), which effectively depleted the two variants of
nectin-2 (Fig. 2A,B). Although the nec2-MO-injected embryos did
not show any abnormalities until the early neurula stage, at the
beginning of neural tube formation they failed to form complete
neural fold and neural ridge (Fig. 2F), which is one of the hallmarks
of neurulation and appears as a pair of pigmented lines along the
dorsal side (Fig. 2D), on the MO-injected side. The pigmented
neural fold lines were completely absent in severely affected
embryos. Transverse sections through the trunk region clearly
showed the normally raised morphology of neural ridges in an
uninjected embryo (Fig. 2E) and the much flatter morphology in a
nec2-MO-injected morphant embryo (Fig. 2G). This effect of nec2MO was partially rescued in a dose-dependent manner (Fig. 2H-J)
by co-injection with a rescue construct (nec2-res; Fig. 2A,C),
confirming that the neural tube defect may be due to the lack of
nectin-2. We also confirmed, by WISH, that the nec2-MO injection
did not affect the expression patterns of neural differentiation marker
genes (see Fig. S2 in the supplementary material). These results
DEVELOPMENT
Nectin-2 and Ncad cooperate to effect NT closure
1318 RESEARCH ARTICLE
Development 137 (8)
injected embryos, however, this accumulation was severely
impaired in the MO-containing cells (Fig. 3F,G,J,K). Thus, nectin2 is required cell-autonomously for F-actin to accumulate properly
at the apical side of the Xenopus neuroepithelium.
showed that nectin-2 is required for neural fold formation and thus
for proper neural tube closure, without affecting the differentiation
of the neural tissue.
Nectin-2 is required for the apical constriction of
neuroepithelial cells
To identify how the neural folding process was affected by the nec2MO, we next investigated the morphant phenotype at the cellular
level by sectioning the MO-injected embryos. On the uninjected
side, the apical surface of the superficial cells was constricted,
showing the characteristic wedge-like cell morphology (Fig. 3A,A⬘,
arrow). By contrast, the nec2-MO-injected cells did not show apical
constriction (Fig. 3A,A⬘, asterisk). Quantifications of the apicalsurface length and cell height showed that apical constriction and
cell elongation were suppressed on the nec2-MO-injected side, and
these effects of nec2-MO were partially rescued by nec2-res (Fig.
3B,C). Together, these results indicate that nectin-2 is required for
the apical constriction of neuroepithelium during neurulation.
We further examined the F-actin localization in nectin-2
knockdown embryos. Control embryos showed the accumulation of
apical F-actin in neuroepithelium (Fig. 3D,E,H,I). In nec2-MO-
Fig. 2. Knockdown of nectin-2 disrupts
neural fold formation. (A) Nucleotide
sequences of nectin-2 constructs and nec2MO. Asterisks: nucleotides identical in each
construct and nec2-MO. (B) Depletion of
nectin-2 by nec2-MO. Lysates from injected
embryos were subjected to western blot.
(C) Resistance of nec2-res to nec2-MO.
Lysates from injected embryos were
subjected to western blot. (D-I) Phenotypes
of developing neural fold in uninjected (D,E),
nec2-MO-injected (F,G) or partially rescued
(H,I) embryos. Asterisks: injected side.
Arrowheads: neural ridges. (J) Summary of
phenotypes. The MOs were injected at 0.25
pmol (B,C,F-J).
DEVELOPMENT
Fig. 1. Expression pattern of nectin-2 during neurulation.
(A-F) Expression pattern of nectin-2a in stages of early neural fold (stage
15; A,B), mid-neural fold (stage 16; C,D) and late neural fold (stage 18;
E,F). (G,H) Negative control with sense probe (stage 15). Dorsal surface
(A,C,E,G) and transverse sections through the trunk region (B,D,F,H) were
observed. Arrowheads: nectin-2a expression in the superficial layer of the
neuroepithelium. Dotted lines: borders between ectoderm (outer layer),
notochord and prospective somites. no, notochord; so, somites.
Nectin-2 induces ectopic apical constriction with
F-actin accumulation
To further assess the function of nectin-2, we carried out an
ectopic overexpression experiment by injecting 100 pg of nectin2 mRNA into the region that gives rise to non-neural ectoderm,
which does not normally undergo apical constriction (control
embryos; Fig. 4A,B,E,F). In contrast to the control, nectin-2injected embryos exhibited abnormal pigmentation and a rough
epithelial surface (Fig. 4C,D). In addition, F-actin staining
revealed acute shrinkage of the apical cell surface accompanied
by the ectopic accumulation of F-actin in these cells (Fig. 4G,H;
Table 1). This effect became more pronounced as the amount of
injected nectin-2 mRNA was increased (Fig. 4M). When
sectioned, some cells in the nectin-2-injected embryo showed
ectopic wedge shape with constricted apical surfaces, increased
cell height and, importantly, apically accumulated F-actin (Fig.
4K,L) in contrast to the cuboidal shape of normal ventral
epithelial cells (Fig. 4I,J). The surface length and apicobasal
height of these ectopically constricted cells were similar to those
of neuroepithelial cells (Fig. 4N,O; compare with Fig. 3B,C).
Thus, these data all support the idea that nectin-2 could induce
ectopic apical constriction, which is an apparently similar
constriction movement to that seen in the neural plate (Fig. 4P),
accompanied by F-actin accumulation at the apical side of the
cells. Finally, we confirmed that nectin-2 was preferentially
localized to the apical cell membrane when ectopically expressed
in Xenopus embryos (Fig. 4Q,R), as previously reported for
cultured cells and mouse tissues (Takai and Nakanishi, 2003;
Okabe et al., 2004), indicating that nectin-2 is expressed at, and
functions in, the very place that apical constriction occurs.
Interestingly, the ectopic apical constriction was not observed in
all the daughters of the injected cells; rather, the constricted cells
were scattered throughout the ectoderm. One possibility for this is
that the distribution of overexpressed nectin-2 might be uneven in
the tissue, and the cells with higher concentration of nectin-2 could
undergo constriction.
Fig. 3. Apical constriction of neuroepithelium is inhibited by
nec2-MO. (A,A⬘) Section through an embryo given a unilateral
injection of nec2-MO. Arrow: cells on the uninjected side undergoing
apical constriction. Asterisk: nec2-MO-injected side. Tubulin staining
was used to trace the cortices of superficial cells (A⬘). (B,C) Ratio of
apical surface length to perimeter (B) and cell height along the
apicobasal axis (C). Each datum was obtained from 15 cells of three
embryos. Nec2-res mRNA was injected at 12.5 pg. Error bars: mean ±
s.e.m. (D-G) Dorsal surface views of the trunk region in control MO
(D,E) and nec2-MO (F,G) injected embryos stained by phalloidin. White
brackets: MO-injected side. (H-K) Sections of control MO (H,I) and
nec2-MO-injected (J,K) embryos stained by phalloidin. Arrows: MOcontaining superficial cells. MOs were injected at 0.25 pmol. Scale bars:
20 mm in A,H,J; 50 mm in D,F.
Ectopic apical constriction is induced by nectin-2
but not through its known F-actin linkage
Nectin is connected to F-actin via afadin (Takai and Nakanishi,
2003; Takai et al., 2008). Therefore, we hypothesized that nectin-2
might induce apical constriction through its intracellular interaction
with F-actin, by promoting the accumulation of F-actin at the apical
region. To test this hypothesis, we prepared three deletion constructs
of nectin-2 (Fig. 5A): nec2-DC4 (which lacks the four afadinbinding amino acids of the C-terminal region), nec2-DIC (which
lacks the entire intracellular region) and nec2-DEC (which lacks the
three extracellular Ig-like domains). As expected from the previous
study (Takahashi et al., 1999), full-length nectin-2 and nec2-DEC
bound to afadin, whereas nec2-DC4 did not (see Fig. S3 in the
supplementary material), supporting the evidence of physical
linkages from nectin-2 to F-actin through afadin.
We then used these deletion constructs in overexpression studies.
Unexpectedly, 100 pg of nec2-DC4 and -DIC mRNA induced the
aberrant epithelial surface morphology in the non-neural ectoderm,
with ectopic apical constriction and characteristic accumulation of
F-actin (Fig. 5B-F,I-L; Table 1). These effects were comparable to
that of full-length nectin-2 (100 pg). By contrast, the overexpression
of 100 pg of nec2-DEC mRNA showed less impact on the surface
morphology (Fig. 5B,G,H), even though there was a weak effect
RESEARCH ARTICLE 1319
Fig. 4. Overexpression of nectin-2 induces ectopic apical
constriction. (A-D) Surface views of non-neural ectoderm of control
(A,B) and nectin-2 mRNA-injected (C,D) embryos at stages 13-14 (early
neurulae). B and D are magnified views of white boxes in A and C,
respectively. (E-H) Non-neural ectoderm of control (E,F) and nectin-2injected (G,H) embryos. Arrows: apically constricted cells. (I-L) Sections
showing non-neural ectoderm of control (I,J) and nectin-2-injected (K,L)
embryos. Arrows: apically constricted cells. (M) Aberrant surface
phenotype in control (memGFP-injected) and nectin-2-injected
embryos. Error bars: mean ± s.e.m. (N,O) Ratio of apical surface to
perimeter (N) and cell height along the apicobasal axis (O). Error bars:
mean ± s.e.m. (P) F-actin staining of normal neuroepithelium at stage
16 (mid-neurula). (Q,R) Subcellular localization of nec2-FLAG in nonneural ectoderm. Arrows: apically localized nectin-2. 100 pg of each
mRNA was used, except for nectin-2 in M (100 pg or 200 pg) and
nec2-FLAG in Q,R (20 pg). Scale bars: 50 mm.
compared with uninjected control, and its cell shape and F-actin
distribution were almost indistinguishable from those of the control
embryos (Fig. 5M,N; Table 1). Transverse sections showed ectopic
wedge-like cell morphology in embryos overexpressed with nec2DC4 and -DIC (Fig. 5O-R) but not nec2-DEC (Fig. 5S,T). These
results indicate that the extracellular domain of nectin-2, rather than
the intracellular afadin-binding motif, is required for it to induce
apical constriction and F-actin accumulation. We further examined
whether the extracellular domain is sufficient for the apical
constriction in neuroepithelium by rescuing the defective neural fold
phenotype. Co-injections of nec2-DC4 or -DIC with nec2-MO
partially rescued the morphant phenotype, whereas the effect of
nec2-DEC was not significant (Fig. 5U), suggesting that the
extracellular domain of nectin-2 may be sufficient for neural fold
formation through the apical constriction.
DEVELOPMENT
Nectin-2 and Ncad cooperate to effect NT closure
1320 RESEARCH ARTICLE
Development 137 (8)
Table 1. Number of apically constricted cells in mRNA-injected embryos
Injected mRNA
Number of samples analyzed
Apically constricted cells (%)*
P-value
Control (memGFP)
nectin-2 (100 pg)
nec2-DC4 (100 pg)
nec2-DIC (100 pg)
nec2-DEC (100 pg)
12 embryos, 6019 cells
6 embryos, 2332 cells
12 embryos, 3951 cells
5 embryos, 1407 cells
9 embryos, 3738 cells
0.02
3.37
6.00
2.00
0.06
–
<0.01†
<0.01†
<0.01†
0.35†
nectin-2 (50 pg)
N-cadherin (50 pg)
nec2 (50 pg) + Ncad (50 pg)
nec2 (50 pg)/Ncad (50 pg)§
nec2 (50 pg) + Ecad (50 pg)
nec2 (50 pg) + Ccad (50 pg)
10 embryos, 4616 cells
10 embryos, 4664 cells
9 embryos, 2702 cells
38 embryos, 8032 cells
10 embryos, 3059 cells
10 embryos, 3371 cells
0.91
0.02
2.90
0.47
1.67
1.89
–
–
<0.01‡
<0.01¶
<0.05‡
<0.05‡
To rule out effects from inappropriate expression of nectin-2
constructs, we examined their expression levels and locations in the
non-neural ectoderm. We confirmed by immunohistochemistry that
all of the constructs were appropriately localized to the plasma
membrane and showed similar expression levels (see Fig. S4A in the
supplementary material). Further examination by western blot
revealed that the protein levels of nec2-vns and nec2-DEC-vns were
slightly lower than those of the other two constructs (see Fig. S4B
in the supplementary material). As nec2-DEC-vns was expressed at
about the same level as nec2-vns, which efficiently induced ectopic
apical constriction, the results indicate that nec2-DEC lacks the
ability to induce ectopic constriction compared with the full-length
protein.
Nectin-2 interacts with N-cadherin, enhancing
apical constriction
The findings that apical constriction is independent of the known
nectin-F-actin linkage but requires the extracellular domain of
nectin-2 indicated that some sort of extracellular machinery was
required to effect this morphological change. Published studies
indicate that the extracellular domain of homodimerized nectin-2
binds a nectin dimer on neighboring cells to promote cell adhesion
(Takai and Nakanishi, 2003; Takai et al., 2008). Therefore, we next
examined the binding between extracellular domains of nectin-2 by
a pull-down assay, using a C-terminally truncated form of nectin-2
that consisted of only the extracellular (ex) domain (nec2-ex).
Unexpectedly, we could not detect a homophilic interaction between
the nectin-2 extracellular domains (Fig. 6A), suggesting that nectin2 is unlikely to promote apical constriction by itself.
We then sought other molecules that might interact with nectin-2
extracellularly and associate with F-actin intracellularly. The most
probable candidate was cadherin, because it is one of the most
commonly expressed transmembrane proteins localized to AJs, and
importantly, interacts with F-actin (Wheelock and Johnson, 2003;
Gumbiner, 2005). The cadherin family is composed of many
subfamilies, which are expressed in specific tissues (Halbleib and
Nelson, 2006). During Xenopus neurulation, N-cadherin is
preferentially expressed in the neural tube from the early neurula
stage (Detrick et al., 1990; Fujimori et al., 1990), and has been
shown previously to be required for neurulation movements
(Nandadasa et al., 2009). Therefore, we tested whether nec2-ex
could interact with the extracellular domain of N-cadherin (Ncad-
ex). In contrast to the lack of a detectable homophilic interaction in
nectin-2, Ncad-ex clearly co-precipitated with nec2-ex (Fig. 6A),
indicating that these molecules may interact through direct and
heterophilic binding in the extracellular space.
We next examined whether this nectin-N-cadherin interaction
could induce apical constriction, by overexpressing either nectin-2
or N-cadherin or both. At a low dosage of mRNA (50 pg), neither
nectin-2 nor N-cadherin alone caused obvious changes in the nonneural ectoderm (Fig. 6B-E; Table 1). However, their co-expression,
at the same dose, effectively induced marked ectopic apical
constriction and F-actin accumulation (Fig. 6F-H; Table 1).
Moreover, to assess whether nectin-2 and N-cadherin associate
either in cis or trans interaction, we separately injected them into
adjacent blastomeres of four-cell-stage embryos and found that cells
at the border between nectin-2-expressing and N-cadherinexpressing areas constricted in a significantly lower frequency
compared with the co-injection of them (Fig. 6I; Table 1). These
results indicate that nectin-2 may function cooperatively with Ncadherin to effect apical constriction in the cis interaction.
As nectin-2 (100 pg) alone induced ectopic apical constriction in
the non-neural ectoderm, which expresses E-cadherin and Ccadherin but not N-cadherin (Choi and Gumbiner, 1989; Ginsberg
et al., 1991; Levi et al., 1991), we examined, by pull-down assays,
whether E- or C-cadherin had a similar interaction with nectin-2.
However, we could not detect an interaction between the
extracellular domain of nectin-2 with that of E- or C-cadherin (Ecadex or Ccad-ex; Fig. 6A), which highlighted the specificity of the
interaction between nectin-2 and N-cadherin. Although we could not
prove a biochemical interaction between nectin-2 and E- or Ccadherin, in overexpression experiments, they enhanced the aberrant
surface morphology (Fig. 6H) and the ectopic apical constriction,
albeit at lower frequencies than induced by the nectin-2 (50 pg)N-cadherin pair (Fig. 6J-M; Table 1). Therefore, although the
interaction between nectin-2 and N-cadherin was stronger and more
effective for inducing apical constriction than the interaction
between nectin-2 and E- or C-cadherin, the fact that some apical
constriction occurred suggests that E- and/or C-cadherin interact
with nectin-2 at a level sufficient to induce ectopic apical
constriction, which may account for the phenotype induced by the
overexpression of nectin-2 mRNA (100 pg) alone in non-neural
ectoderm. We also found that the expression level of endogenous
nectin-2 protein was lower in the ventral side than the dorsal side of
DEVELOPMENT
*Constricted cells were defined as cells that apparently reduced their apical surface area (<40% area compared with the mean area of uninjected cells).
†
Compared with control embryos.
‡
Compared with nectin-2 (50 pg)-injected embryos.
§
Each mRNA was injected into the distinct ventral blastomeres of four-cell stage embryos. Cells adjacent to the border between nectin-2-expressing (memGFP) and Ncadherin-expressing (memRFP) areas were analyzed.
¶
Compared with embryos injected with N-cadherin (50 pg) and nectin-2 (50 pg) into the same blastomeres [Ncad (50 pg) + nec2 (50 pg)].
Nectin-2 and Ncad cooperate to effect NT closure
RESEARCH ARTICLE 1321
the neurula embryos (see Fig. S5B in the supplementary material),
suggesting that the ventral expression level of nectin-2 may not be
enough to induce apical constriction with E- or C-cadherin.
Nectin-2 and N-cadherin functionally associate to
promote apical constriction in the neural tube
The cooperative effect of nectin-2 and N-cadherin on ectopic apical
constriction implies that N-cadherin has an in vivo role in
neuroepithelial apical constriction. N-cadherin has been implicated
in neural tube morphogenesis (Nandadasa et al., 2009), and we
found that, by using N-cadherin MO (Ncad-MO), the N-cadherindepleted cells failed to undergo apical constriction, and their apical
accumulation of F-actin was inhibited (Fig. 7A,B), resembling the
nectin-2 knockdown phenotype (Fig. 3F,J). This similarity supports
our hypothesis that they play a cooperative role in neural tube
closure. In addition, N-cadherin protein is apically located in neural
plate cells normally and in the epidermis when ectopically expressed
(Nandadasa et al., 2009). These results led us to hypothesize that
nectin-2 is required for the function of N-cadherin in the neural
ectoderm, and for its localization to the apical cytoplasm.
We first tested this hypothesis by assaying the synergy between
the two proteins. When a low dose of either nec2-MO or NcadMO was injected into the dorsal side of the embryos, the neural
fold formation was weakly affected (Fig. 7C). When combined,
however, these MOs more severely disrupted the neural fold
formation (Fig. 7C), indicating that the molecular interaction
between nectin-2 and N-cadherin may play a crucial role in
neuroepithelial folding. We next assessed the interdependence of
these proteins for their subcellular localization. As reported
(Nandadasa et al., 2009), N-cadherin was strictly localized to the
apical portion of the neuroepithelial cells in control embryos (Fig.
7D,E). However, this localization was abrogated by the depletion
of nectin-2 (Fig. 7F,G), without affecting the level of the Ncadherin protein (see Fig. S6 in the supplementary material).
Nectin-2 was also apically located in the Xenopus
neuroepithelium (Fig. 7H,I). However, its localization was not
affected by the knockdown of N-cadherin (Fig. 7J,K). These
results suggest that N-cadherin requires nectin-2 for its apical
localization in the neuroepithelial cells, probably through its
extracellular domain.
If N-cadherin is responsible for the apical accumulation of Factin, associated proteins must mediate the linkage between them in
the cytoplasm (Gumbiner, 2005). Although which molecules
mediate this linkage is a matter of controversy (Drees et al., 2005;
Yamada et al., 2005), one of the key molecules that may mediate it
is b-catenin (Gates and Peifer, 2005). As cadherins possess a wellconserved b-catenin-binding region at the C-terminus (Ozawa et al.,
1990; Pokutta and Weis, 2007), we designed a mutant N-cadherin
construct lacking the putative b-catenin-binding region (Ncad-Db),
which did not bind to the endogenous Xenopus b-catenin (see Fig.
S7A in the supplementary material).
To assess the requirement for b-catenin to mediate the linkage
between F-actin and N-cadherin during neural tube closure, we
injected Ncad-Db mRNA into the dorsal side of four-cell-stage
DEVELOPMENT
Fig. 5. The extracellular domain of
nectin-2 is required for apical
constriction. (A) Schematic drawing of
nectin-2 deletion constructs. (B) Aberrant
surface phenotype in uninjected control
and nec2-DC4-, -DIC- or -DEC-injected
embryos. Error bars: mean ± s.e.m.
(C-H) Surface views of non-neural
ectoderm of embryos injected with nec2DC4 (C,D), nec2-DIC (E,F) or nec2-DEC
(G,H), observed at stages 13-14 (early
neurulae). D, F and H are magnified views
of the areas enclosed by white boxes in
C, E and G, respectively. (I-N) Cell shape
and F-actin staining of embryos injected
with nec2-DC4 (I,J), -DIC (K,L), -DEC
(M,N). Arrows: apically constricted cells.
(O-T) Sections through embryos injected
with nec2-DC4 (O,P), -DIC (Q,R) or -DEC
(S,T). Arrows: apically constricted cells.
(U) Rescue experiments for nectin-2
depletion with the nectin-2-deletion
constructs. mRNAs were injected at 12.5
pg, and nec2-MO was 0.25 pmol. Error
bars: mean ± s.e.m. *P<0.05, n.s.: not
significant, t-test. 100 pg of each mRNA
was injected (B-T). Scale bars: 50 mm.
1322 RESEARCH ARTICLE
Development 137 (8)
Fig. 6. Nectin-2 interacts with Ncadherin, enhancing apical
constriction. (A) A GST pull-down
assay with nec2-ex and Ncad-ex,
Ecad-ex or Ccad-ex performed using
the 293T cell line.
(B-G) Overexpression of nectin-2 or
N-cadherin in non-neural ectoderm.
Low doses of nectin-2 (50 pg; B,C)
or N-cadherin (50 pg; D,E) alone and
in combination (F,G) were injected
and observed at stages 13-14 (early
neurulae). Arrows: apically
constricted cells. (H) Embryos with
aberrant surface in the
overexpression experiments with 50
pg of nectin-2 and 50 pg of N-, E- or
C-cadherin. Error bars: mean ±
s.e.m. (I) Nectin-2 (50 pg) and Ncadherin (50 pg) were separately
injected into adjacent blastomeres of
four-cell-stage embryos with
memGFP and memRFP, respectively.
The injected embryos were observed
at the early neurula stage.
(J-M) Overexpression of E- (J,K) or
C-cadherin (L,M) with or without 50
pg of nectin-2. Arrows: apically
constricted cells. Scale bars: 50 mm.
DISCUSSION
In early vertebrate development, neural tube closure is one of the
essential, and most dynamic, morphological processes. In this study,
we found that nectin-2 plays a crucial role in this process by
regulating apical constriction in collaboration with N-cadherin.
Embryonic expression of nectins
Among the four types of nectin, only nectin-2 mRNA was present at
significant levels during early Xenopus embryogenesis, although
whether other nectins are contributed maternally as proteins remains
to be clarified. As Xenopus tropicalis also has these four nectins, like
other vertebrates including humans, the four nectin genes are
unlikely to reflect the tetraploidization of the Xenopus laevis
genome, but seem to have evolved as independent genes.
Interestingly, a recent study showed that zebrafish nectin-1 is
expressed in the anterior neural keel of the early neurula and in the
eye and brain of the larva (Helvik et al., 2009), implying a conserved
function for nectins in the regulation of developmental
morphogenesis.
Identification of the nectin-2 domain required for
apical constriction
In this study, we identified a novel function of nectin-2 in vertebrates
as a component of the neural apical constriction machinery. Our
results indicate that apical constriction seems to be induced by the
extracellular domain of nectin-2 but not by its intracellular afadinbinding motif linked to F-actin. This suggests that the F-actin
linkage through afadin may not be strong enough to cause F-actin
accumulation, although afadin might still serve as a mediator for cell
adhesion and for the maintenance of tissue integrity. Our finding also
highlights some functional differences from the previous findings in
Drosophila, which include the necessity of the intracellular domain
of Ed to regulate cell morphology (Lin et al., 2007). This difference
implies an evolutionally diverged function of these structurally
related adhesion molecules for the same cell-morphological change.
Overexpression experiments show that it is unlikely that the
ectopic apical constriction caused by overexpressed nectin-2, nec2DC4 or -DIC was owing to the formation of cis dimers with
endogenous nectin-2, and exerted through their intact afadin-F-actin
linkage, because the extracellular domain of nectin-2 did not show
detectable homophilic binding. In addition, the transmembrane
region may not be responsible for this phenotype, because nec2DEC, which contains this region, did not induce ectopic apical
constriction. Thus, the effect caused by overexpression of nectin-2,
nec2-DC4 or -DIC may not result from cis interaction with
endogenous nectin-2.
DEVELOPMENT
embryos. At the mid-neurula stage, Ncad-Db-injected embryos
showed significantly abnormal neural fold formation (Fig. 7L), and
phalloidin staining revealed a partial loss of apically located F-actin
(Fig. 7P,Q), compared with control embryos (Fig. 7N,O). This may
result from a competition between endogenous and mutant Ncadherin for the extracellular binding to nectin-2. Furthermore, we
performed a rescue experiment for the N-cadherin morphants using
rescue constructs of full-length N-cadherin (Ncad-res) and Ncad-Db
(Ncad-res-Db; see Fig. S7B,C in the supplementary material). The
defective neural fold phenotype of Ncad-MO-injected embryos was
rescued by Ncad-res but not by Ncad-res-Db (Fig. 7M). Together,
these results indicate that nectin-2 facilitates the apical localization
of N-cadherin, followed by the accumulation of F-actin probably
through the b-catenin-mediated linkage, which leads to apical
constriction and proper neural tube closure.
Nectin-2 and Ncad cooperate to effect NT closure
RESEARCH ARTICLE 1323
Fig. 7. N-cadherin is functionally associated with
nectin-2 in the apical constriction of the
neuroepithelium. (A,B) F-actin staining of Ncad-MOinjected embryos. Dorsal (A) and section (B) views.
White brackets: MO-injected side. Arrows: MOcontaining superficial cells. Ncad-MO was injected at 1.2
pmol. (C) Embryos with defective neural fold in the
knockdown with either nec2-MO or Ncad-MO, or both.
Control MO and nec2-MO were injected at 0.13 pmol;
Ncad-MO was 0.6 pmol. Error bars: mean ± s.e.m.
*P<0.05, ***P<0.001, t-test. (D-G) Localization of Ncadherin in the neuroepithelium of control (D,E) and
nec2-MO-injected (F,G) embryos. MOs were injected at
0.25 pmol. (H-K) Localization of nec2-FLAG in the
neuroepithelium of control (H,I) and Ncad-MO-injected
(J,K) embryos. Nec2-FLAG mRNA was injected at 20 pg,
and Ncad-MO was 1.2 pmol. (L) Defective neural fold
phenotype in embryos injected with 50 pg of full-length
N-cadherin or Ncad-Db mRNA. Error bars: mean ± s.e.m.
*P<0.05, t-test. (M) A rescue experiment for N-cadherin
depletion with Ncad-res or Ncad-res-Db. mRNAs were
injected at 50 pg and Ncad-MO was 1.2 pmol. Error
bars: mean ± s.e.m. *P<0.05, t-test. (N-Q) F-actin
staining of neural ectoderm in control (N,O) and NcadDb-injected (P,Q) embryos. Ncad-Db mRNA was injected
at 50 pg. Scale bars: 20 mm, except for 100 mm in A.
2009) showed that N-cadherin and E-cadherin are specifically
required for the assembly of cortical F-actin in the neural plate
and in the non-neural ectoderm, respectively, suggesting that Nand E-cadherin have different functional specificities in the
different tissues. Thus, the identification of nectin-2 as a
functional partner of N-cadherin may also provide a clue to
understanding the molecular basis of the divergent functions of
certain cadherin family proteins in vivo. For the future, it is
intriguing to narrow down interacting domains between nectin-2
and N-cadherin.
Molecular and cellular mechanisms of the nectin2-N-cadherin system for apical constriction
Taking our results together with those of previous studies, we
propose the following model for the apical constriction of the
neuroepithelium during Xenopus neurulation. At the start of
neurulation, the expression of nectin-2 is upregulated in the
superficial layer of the neuroepithelium, where N-cadherin is also
expressed. The nectin-2 protein preferentially localizes to the AJs
by an unknown mechanism and binds to N-cadherin through its
extracellular domain, facilitating the apical accumulation of Ncadherin. The accumulated N-cadherin promotes the translocation
of F-actin, which is indirectly associated with the cytoplasmic
domain of N-cadherin, to the apical cytoplasm. Finally, the apical Factin bundle contracts via the force of activated myosin, which may
be phosphorylated by Shroom and ROCK activity (Lee et al., 2007;
Nishimura and Takeichi, 2008). In this model, the nectin-2-Ncadherin system serves as pre-constriction machinery to confer cells
DEVELOPMENT
Strong interaction between nectin-2 and
N-cadherin
Relationships between nectin family molecules and cadherins have
been reported as a co-localization pattern (Takahashi et al., 1999;
Miyahara et al., 2000; Okabe et al., 2004) and as an indirect
connection mediated by associated intracellular proteins (Tachibana
et al., 2000; Pokutta et al., 2002). However, no direct interaction
between nectins and cadherins has been shown previously.
Moreover, we found that N-cadherin was dependent on nectin-2 for
its apical accumulation in the neuroepithelium, which may be
required for apical constriction. These findings serve as the first
evidence for the direct interaction and functional relationship
between these adhesion molecules. Although we cannot rule out the
possibility that nectin-2 may also function for cell adhesion in
neuroepithelium independently of cadherins (Takahashi et al., 1999),
the clearer interaction of nectin-2 and N-cadherin than that of nectin2 homodimer suggests a functional importance of this heterophilic
interaction in vivo.
We also found that the interaction between nectin-2 and
cadherin is subtype specific: i.e. nectin-2 interacted with Ncadherin more strongly than with E- and/or C-cadherin. In
addition, N-cadherin is predominantly expressed in the
neuroepithelium from the beginning of neurulation (Detrick et al.,
1990; Fujimori et al., 1990), where nectin-2 is also expressed
at a high level. Thus, the strong, specific interaction and
spatiotemporally concurrent expression of nectin-2 and Ncadherin appear to be crucial for apical constriction and folding
of the neural tube. Recently, Nandadasa et al. (Nandadasa et al.,
with the competence to undergo apical constriction. To understand
this process further, it will be important to analyze the relationship
between the nectin-cadherin machinery and the other regulatory
systems such as the Shroom-ROCK pathway in apical constriction.
Acknowledgements
We thank Dr B. M. Gumbiner for the C-cadherin construct, Drs R. Mayor, Y.
Mimori-Kiyosue and S. Koshida for helpful discussions and comments, and
members of the Division of Morphogenesis, NIBB, for technical assistance,
discussions, and comments. This work was supported by grants from the
Ministry of Education, Science, Sports, and Culture of Japan to N.U. and NIH
(RO1HD044764-06) to S.N. and C.W. Deposited in PMC for release after 12
months.
Competing interests statement
The authors declare no competing financial interests.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/lookup/suppl/doi:10.1242/dev.043190/-/DC1
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Nectin-2 and Ncad cooperate to effect NT closure