Roles of cell-adhesion molecules nectin 1 and nectin 3 in ciliary body development
Maiko Inagaki1,
Kenji Irie1,
Hiroyoshi Ishizaki2,3,
Miki Tanaka-Okamoto3,
Koji Morimoto2,
Eiji Inoue2,
Toshihisa Ohtsuka2,
Jun Miyoshi3 and
Yoshimi Takai1,*
1 Department of Molecular Biology and Biochemistry, Osaka University Graduate
School of Medicine/Faculty of Medicine, Suita 565-0871, Japan
2 KAN Research Institute, 93 Chudoji-Awatamachi, Shimogyo-ku, Kyoto 600-8815,
Japan
3 Department of Molecular Biology, Osaka Medical Center for Cancer and
Cardiovascular Diseases, Osaka 537-8511, Japan
*
Author for correspondence (e-mail:
ytakai{at}molbio.med.osaka-u.ac.jp)
Accepted 14 January 2005
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SUMMARY
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Nectins are Ca2+-independent immunoglobulin-like
cell-cell-adhesion molecules consisting of four members. Nectins
homophilically and heterophilically trans-interact to form a variety of
cell-cell junctions, including cadherin-based adherens junctions in epithelial
cells and fibroblasts in culture, synaptic junctions in neurons, and Sertoli
cell-spermatid junctions in the testis, in cooperation with, or independently
of, cadherins. To further explore the function of nectins, we generated nectin
1/ and nectin 3/ mice. Both
nectin 1/ and nectin 3/ mice
showed a virtually identical ocular phenotype, microphthalmia, accompanied by
a separation of the apex-apex contact between the pigment and non-pigment cell
layers of the ciliary epithelia. Immunofluorescence and immunoelectron
microscopy revealed that nectin 1 and nectin 3, but not nectin 2, localized at
the apex-apex junctions between the pigment and non-pigment cell layers of the
ciliary epithelia. However, nectin 1/ and nectin
3/ mice showed no impairment of the apicolateral
junctions between the pigment epithelia where nectin 1, nectin 2 and nectin 3
localized, or of the apicolateral junctions between the non-pigment epithelia
where nectin 2 and nectin 3, but not nectin 1, localized. These results
indicate that the heterophilic trans-interaction between nectin 1 and nectin 3
plays a sentinel role in establishing the apex-apex adhesion between the
pigment and non-pigment cell layers of the ciliary epithelia that is essential
for the morphogenesis of the ciliary body.
Key words: Nectin, Afadin, Pigment epithelia, Non-pigment epithelia, Ciliary body, Pvrl1, Pvrl3, Mouse
 |
Introduction
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Cells in multicellular organisms form cell-cell junctions and contacts that
play essential roles in various cellular processes, including morphogenesis,
differentiation, proliferation and migration. Cell-cell junctions and contacts
are mediated by cell adhesion molecules (CAMs). The cadherin superfamily,
which consists of over 80 members, serves as key Ca2+-dependent
CAMs in a variety of cell-cell junctions
(Takeichi, 1995
;
Yagi and Takeichi, 2000
). The
immunoglobulin (Ig) superfamily also plays important roles as
Ca2+-independent CAMs
(Brummendorf and Lemmon, 2001
).
Recently, nectins (Pvrl Mouse Genome Informatics) have emerged as
Ig-like CAMs that play roles in a variety of cell-cell junctions and contacts
(Takai et al., 2003a
;
Takai et al., 2003b
;
Takai and Nakanishi, 2003
).
Nectins comprise a family of four members: nectin 1, nectin 2, nectin 3 and
nectin 4. Extracellular regions of all nectins form homo-cis-dimers and then
promote homophilic or heterophilic trans-interactions. In epithelial cells and
fibroblasts in culture, nectins initiate cell-cell adhesion and recruit
cadherins to the nectin-based cell-cell adhesion sites to cooperatively form
adherens junctions (AJs) (Takai et al.,
2003a
; Takai and Nakanishi,
2003
). Furthermore, nectins recruit proteins of tight junctions
(TJs), first junctional adhesion molecules (JAMs) and then claudins
(Tsukita et al., 1999
;
Tsukita et al., 2001
), to the
apical side of AJs in cooperation with cadherins. JAMs are
Ca2+-independent Ig-like CAMs that recruit the cell-polarity
protein complex, which consists of Par3, aPKC and Par6, by directly binding
Par3 (Ohno, 2001
). Nectin 1
and nectin 3, but not nectin 2, also directly bind Par3. In addition,
intracellular tails of nectins are associated with the actin cytoskeleton
through afadin (Mllt4 Mouse Genome Informatics), a nectin and actin
filament (F-actin)-binding protein. Furthermore, nectins induce activation of
Cdc42 and Rac small G proteins, which eventually enhances the formation of
cadherin-based AJs through the reorganization of the actin cytoskeleton. Cdc42
activated by nectins may also bind to Par6 and activate the polarity protein
complex (Ohno, 2001
;
Takai et al., 2003a
). Thus,
these mechanisms involving nectins are essential to form stable junctional
complexes and cell polarity.
Physiological roles of nectins have also been demonstrated in neurons. At
the synapses between the mossy fiber terminals and the pyramidal cell
dendrites in the CA3 area of hippocampus of the brain, both synaptic junctions
and puncta adherentia junctions are highly developed and actively remodeled in
an activity-dependent manner (Amaral and
Dent, 1981
). Nectin 1 and nectin 3 localize asymmetrically at the
presynaptic and postsynaptic sides of the puncta adherentia junctions,
respectively (Takai et al.,
2003b
). As N-cadherin symmetrically localizes at the synapse, both
nectins and N-cadherin are likely to cooperate in the formation of the puncta
adherentia junctions. At the contacts formed between commissural axons and the
processes of floor plate cells in the neural tube, nectin 1 and nectin 3
asymmetrically localize as key CAMs at the commissural axon side and the
floor-plate cell side, respectively (Okabe
et al., 2004b
). Nectin 1 and nectin 3 play an important role in
the trajectory of the commissural axons, whereas cadherins do not serve as
CAMs. Nectins have furthermore been demonstrated to play roles in the testis.
Nectin 2 and nectin 3 reside specifically in Sertoli cells and spermatids,
respectively (Takai and Nakanishi,
2003
). Nectins, but not cadherins, are at least major CAMs at the
Sertoli cell-spermatid junctions. Thus, evidence is accumulating that nectins
play important roles in a variety of cell-cell junctions and contacts.
During mouse development, nectins and afadin are expressed in polarized
epithelia, such as neuroepithelia, epithelial somites and facial primordia,
which are dynamically remodeling (Okabe et
al., 2004a
). To explore their roles in dynamic epithelial
remodeling during development, knockout studies on nectins and afadin are
essential. Thus far, afadin/ and nectin
2/ mice are available.
Afadin/ mice have revealed crucial roles of the whole
nectin-based cell-adhesion system by showing embryonic lethality with severe
defects in the formation of AJs and subsequent epithelial morphogenesis
(Ikeda et al., 1999
). They show
developmental defects at stages during and after gastrulation, including
disorganization of the ectoderm, impaired migration of the mesoderm, and loss
of somites and other structures derived from both the ectoderm and the
mesoderm (Ikeda et al., 1999
).
Nectin 2/ mice exhibit the male-specific infertility
phenotype and have defects in the later steps of sperm morphogenesis,
exhibiting distorted nuclei and abnormal distribution of mitochondria
(Bouchard et al., 2000
;
Mueller et al., 2003
;
Ozaki-Kuroda et al., 2002
). In
these mice, the structure of the Sertoli cell-spermatid junctions is severely
impaired, and the localization of nectin 3 and afadin is disorganized
(Ozaki-Kuroda et al., 2002
).
Thus, it is likely that the Sertoli cell-spermatid junctions rely largely on
nectins. In addition, spermatozoa of nectin 2/ mice
have a defect in binding to the zona pellucida and oocyte penetration
(Mueller et al., 2003
).
Physiological roles of other members of nectins in dynamic epithelial
remodeling during development, however, have not been established.
Specifically, knockout studies on nectin 1 and nectin 3 are likely to provide
some insights into mouse development because the most potent heterophilic
trans-interaction is detected between nectin 1 and nectin 3
(Takai et al., 2003a
;
Takai and Nakanishi, 2003
).
Here, we report an unexpected ocular phenotype of nectin
1/ and nectin 3/ mice,
demonstrating that the heterophilic trans-interaction between nectin 1 and
nectin 3 mediates the apex-apex adhesion between the pigment and non-pigment
cell layers of the ciliary epithelia, and that this nectin-mediated adhesion
is essential for the morphogenesis of the ciliary body.
 |
Materials and methods
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Animals and generation of nectin 1/ and nectin 3/ mice
C57BL/6 and BALB/cA mice were purchased from CLEA Japan (Tokyo, Japan). The
animals and procedures used in this study were in accordance with the
guidelines and approval of Osaka University Medical School Animal Care and Use
Committee. Targeting constructs of nectin 1 and nectin 3 were made to replace
the coding exon 2 (amino acids 26-143 of the nectin 1 protein) and the coding
exon 1 (amino acids 1-58 of the nectin 3 protein), respectively, with a
neo-resistance gene cassette (Fig. 1A, part
a; Fig. 1B, part
a). The neo-resistance gene cassette contained the exogenous
thymidine kinase promoter. RW4 embryonic stem (ES) cells were transfected with
these targeting vectors and selected as described previously
(Ikeda et al., 1999
).
Homologous recombinants were verified by Southern blotting using 5' and
3' external probes and the neo-resistance gene probe. Nectin
1+/ and nectin 3+/ ES cells were
microinjected into embryonic day 3.5 (E3.5) C57BL/6 blastocysts and
transferred to MCH pseudo-pregnant foster mothers to generate chimeras that
were mated with BDF1 mice for germline transmission. Genotyping was performed
by Southern blotting and PCR. For Southern blotting, the following fragments
were used: for the nectin 1 gene, a 1.2 kb EcoRI-HindIII
fragment (5'-probe) and a 0.4 kb KpnI fragment
(3'-probe); and for the nectin 3 gene, a 1.1 kb HindIII
fragment (5'-probe) and a 1.2 kb BamHI-EcoRV fragment
(3'-probe). For PCR, the following primer sets were used: for the nectin
1 gene, 5'-CCGTAAAGGTCAAGGGCAGAG-3' and
5'-GTGCCTGTCCCTTGTCCA-3'; for the nectin 3 gene,
5'-CTGCTGCTGCTGCTTATTCCC-3' and
5'-AACCTCAGCCTAGAAGTCCGC-3'; and for the neo gene,
5'-CTGTTGTGCCCAGTCATAGCC-3' and 5'-CACTGAAGCGGGAAGGGACTG
3'. Nectin 1/ and nectin
3/ albino mice were generated by a cross of nectin
1/ and nectin 3/ mice with
BALB/cA mice twice.

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Fig. 1. Targeted disruption of the nectin 1 and nectin 3 genes. (A) Targeted
disruption of the nectin 1 gene. (A) (a) The structure of the mouse nectin 1
gene with coding exons 2-5 is shown at the top. A targeting vector was
designed to remove the exon 2. The construct contained 2.8 kb 5'
flanking sequence and 4.0 kb 3' flanking sequence. (b) Southern blotting
of ES clones. HindIII-digested DNAs derived from ES cells were
hybridized with the 5'- or 3'-probe. WT, wild-type; +/,
nectin 1+/. (c) Genotyping by PCR of genomic DNA extracted
from littermate mice at 21 days of age. Wild-type (WT) and targeted alleles
give the bands of 639 bp and 459 bp, respectively. +/, nectin
1+/; /, nectin 1/.
(d) Western blotting of the extracts from the brains of wild-type and nectin
1/ mice with the anti-nectin 1 and anti-actin
antibodies. (B) Targeted disruption of the nectin 3 gene. (a) The structure of
the mouse nectin 3 gene with coding exon 1 is shown at the top. A targeting
vector was designed to remove the exon 1. The construct contained 6.5 kb
5' flanking sequence and 4.2 kb 3' flanking sequence. (b) Southern
blotting of ES clones. BamHI-digested DNAs derived from ES cells were
hybridized with the 5'- or 3'-probe. WT, wild-type; +/,
nectin 3+/. (c) Genotyping by PCR of genomic DNA extracted
from littermate mice at 21 days of age. Wild-type and targeted alleles give
the bands of 379 bp and 814 bp, respectively. WT, wild-type; +/, nectin
3+/; /, nectin 3/.
(d) Western blotting of the extracts from the brains of wild-type and nectin
3/ mice with the anti-nectin 3 and anti-actin
antibodies.
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Antibodies
Rabbit anti-nectin 1 and nectin 3 polyclonal antibodies, which recognize
the intracellular regions of nectin 1 and nectin 3, respectively, were
prepared as described (Takahashi et al.,
1999
; Satoh-Horikawa et al.,
2000
). Rat anti-nectin 1, nectin 2 and nectin 3 monoclonal
antibodies, which recognize the extracellular regions of nectin 1 (#48-12),
nectin 2 (#502-57) and nectin 3 (#103-A1), respectively, were prepared as
described (Mizoguchi et al.,
2002
; Satoh-Horikawa et al.,
2000
; Takahashi et al.,
1999
). A rabbit anti-afadin polyclonal antibody was prepared as
described (Mandai et al.,
1997
). A mouse anti-ZO-1 monoclonal antibody (AB01003; Sanko
Junyaku), a rat anti-P-cadherin monoclonal antibody (PCD-1; Takara Bio), a
mouse anti-actin monoclonal antibody (MAB1501; Chemicon), a mouse
anti-cadherin 11 monoclonal antibody (32-1700; Zymed) and a mouse
anti-collagen IX monoclonal antibody (#MS-344-P0; Lab Vision) were purchased
from commercial sources.
Histological analysis and immunofluorescence microscopy
For histological analysis, mouse embryos and tissues were dissected and
fixed with 10% formaldehyde at 4°C overnight, washed with 0.1 M
phosphate-buffered saline, pH 7.4 (PBS), dehydrated in graded alcohols,
embedded in paraffin wax, sectioned at 4 µm, and stained with Hematoxylin
and Eosin. For immunofluorescence microscopy, mouse embryos and tissues were
dissected and fixed with 2% paraformaldehyde in PBS at 4°C for 4 hours,
followed by being washed with PBS. After being placed into 10% sucrose
solution overnight and 25% sucrose solution for 4 hours, they were embedded in
OCT compound (Sakura Finetechnical), frozen in liquid nitrogen, and then
sectioned using a cryostat at 10 µm. The sections were mounted on glass
slides, air-dried and washed three times with PBS containing 0.05% saponin.
After being blocked in PBS containing 20% Block Ace (Dainippon Pharmaceutical)
and 0.05% saponin for 30 minutes, the samples were incubated at 4°C
overnight in PBS containing 5% Block Ace, 0.05% saponin and the primary
antibodies. The samples were washed with PBS containing 0.05% saponin three
times, and incubated at room temperature for 1 hour with the secondary
antibodies in PBS containing 5% Block Ace and 0.05% saponin. After being
washed for 5 minutes three times, the samples were mounted in 50% glycerol and
viewed with a Radiance 2100 confocal laser-scanning microscope (BioRad).
Immunoelectron microscopy
Immunoelectron microscopy was performed using the silver-enhancement
technique as described (Kinoshita et al.,
1998
). Adult mice were perfused with 4% paraformaldehyde in PBS
(pH 7.4). Tissue blocks were cut to 50 µm on a vibratome. The sections were
washed with 0.1 M phosphate buffer, pH 7.3 (PB) and then frozen and thawed in
isopentane cooled with liquid nitrogen and PB containing 25% sucrose, 10%
glycerol and 0.02% NaN2 at room temperature. The sections were then
incubated with 5 µg/ml of the primary antibodies in 50 mM Tris buffered
saline, pH 7.4 (TBS) containing 2% normal goat serum. After being washed with
TBS, the sections were incubated with the secondary antibodies coupled with
1.4-nm gold particles (Nanoprobes) and then reacted with HQSilver kit
(Nanoprobes) for 8 minutes. After osmification, the immunostained sections
were block stained with uranyl acetate, dehydrated and flat-embedded in Epon.
The ultrathin sections were then prepared and viewed with an electron
microscope (H-7500, HITACHI).
In situ hybridization
In situ hybridization was performed as described
(Takemoto et al., 2002
;
Okabe et al., 2004a
) with some
modifications. In brief, mRNA probes were labeled with digoxigenin-UTP by RNA
in vitro transcription according to the manufacturer's protocol (Roche
Diagnostics). Cryosections were prepared as described above. The sections were
air-dried, washed with water for 3 minutes and treated with 0.2 M HCl for 3
minutes. After being washed with water, the sections were treated with 0.1 M
triethanolamine (pH 8.0) and acetylated with 0.25% dehydrated acetic acid in
0.1 M triethanolamine for 10 minutes. The sections were washed with PBS for 3
minutes twice and incubated with a prehybridization solution [50% deionized
formamide, 5xSSPE (pH 7.5), 5% SDS and 1 mg/ml of yeast tRNA]. Each mRNA
probe was mixed with the prehybridization solution, boiled at 80°C for 5
minutes, and added to the sections. After the sections were incubated at
50°C overnight, the sections were washed with 5xSSC and then with
2xSSC containing 50% formamide at 50°C for 30 minutes three times.
After being blocked with 1.5% Blocking Reagent (Roche Diagnostics) in TNT
buffer [100 mM Tris-HCl (pH 7.5), 150 mM NaCl, and 0.1% Tween-20], the
sections were incubated with the anti-digoxigenin antibody conjugated to
alkaline phosphatase at room temperature for 2 hours. The sections were washed
with TNT buffer at room temperature for 30 minutes five times. Hybridized
probes were visualized by HNPP Fluorescent Detection Set (Roche Diagnostics)
and viewed with a Radiance 2100 confocal laser-scanning microscope.
Other procedures
Protein concentrations were determined with bovine serum albumin as a
reference protein (Bradford,
1976
). SDS-PAGE was carried out as described by Laemmli
(Laemmli, 1970
).
 |
Results
|
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Generation of nectin 1/ and nectin 3/ mice
We first generated nectin 1+/ and nectin
3+/ ES cells by homologous recombination using targeting
vectors designed to delete the exon 2 of the nectin 1 gene and the exon 1 of
the nectin 3 gene, respectively (Fig. 1A,
part a; Fig. 1B, part
a). When these targeting constructs were introduced into ES cells
by an electroporation, six G418-resistant colonies heterozygous for the nectin
1 gene and six G418-resistant colonies heterozygous for the nectin 3 gene were
obtained. The genotypes of the G418-resistant colonies were confirmed by
Southern blotting (Fig. 1A, part
b; Fig. 1B, part
b). These ES clones were used to generate chimeric mice and
successfully contributed to germ-line transmission. Nectin
1+/ and nectin 3+/ mice appeared normal
compared with the wild-type littermates (data not shown). Each nectin
1+/ or nectin 3+/ mice was intercrossed
and genotypes of the progeny were determined by PCR and Southern blotting
using the tail DNAs (Fig. 1A, part
c; Fig. 1B, part c;
data not shown). Nectin 1/ and nectin
3/ mice did not express the nectin 1 and nectin 3
proteins, respectively, as neither the nectin 1 nor the nectin 3 protein was
detected by the anti-nectin 1 or nectin 3 antibodies, which recognize the
intracellular regions of the nectin 1 or nectin 3 proteins, respectively
(Fig. 1A, part d;
Fig. 1B, part d). Therefore, we
referred these targeted alleles as null alleles. Nectin
1/ and nectin 3/ mice were
viable and apparently healthy under specific-pathogen-free conditions, except
that male nectin 3/ mice were infertile (data not
shown), suggesting a role for nectin 3 in spermatogenesis. The analysis on
this role of nectin 3 in spermatogenesis will be described elsewhere.
Microphthalmia in both nectin 1/ and nectin 3/ mice
Microphthalmia was the most striking feature observed in common between
nectin 1/ and nectin 3/ mice
(Fig. 2A,B). This finding was
unexpected because nectin 2/ mice did not show such
microphthalmia (Bouchard et al.,
2000
; Mueller et al.,
2003
; Ozaki-Kuroda et al., 2000). Wild-type mice are born with
both the eyes closed. However, about 30% of nectin 1/
pups (n=40), but not nectin 3/ pups
(n=30), were born with one or both of the eyes open (data not shown).
Nectin 1/ mice born with the eyes open tended to show
more severe defect in their eyes than nectin 1/
littermates with the eyes closed, although all mice born with the eyes closed
also showed microphthalmia. These results suggest that microphthalmia may be
caused by the same underlying mechanism involving nectin 1 and nectin 3, but
not nectin 2.

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Fig. 2. Microphthalmia in both nectin 1/ and nectin
3/ mice. (A) View of adult wild-type (WT) and nectin
1/ (/) mice. (B) View of adult
wild-type and nectin 3/ (/) mice. The
results shown are representative of nectin 1/ and
nectin 3/ mice.
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Separation of the apex-apex contact between the pigment and non-pigment cell layers of the ciliary epithelia in both nectin 1/ and nectin 3/ mice
To investigate the etiology of microphthalmia in nectin
1/ and nectin 3/ mice, we
histologically analyzed the eyes of adult wild-type, nectin
1/ and nectin 3/ mice. The
vitreous body was absent, the retinal layers were undulating, and the lenses
were somewhat deformed in the eyes of nectin 1/ and
nectin 3/ mice
(Fig. 3B, part a;
Fig. 3C, part a). In addition,
the ciliary processes were absent, and the ciliary epithelia adhered to the
lens in the eyes of nectin 1/ and nectin
3/ mice (Fig. 3B,
part b; Fig. 3C, part
b). Because the ciliary body secretes aqueous humor and
glycoproteins of the vitreous body
(Bertazolli Filho et al., 1996
;
Francis and Alvarado, 1997
;
Haddad et al., 1990
;
Zimmerman and Fine, 1964
), the
impairment of the ciliary body seemed to cause microphthalmia, the absence of
the vitreous body, the undulating retinal layers and the deformed lens in the
eyes of nectin 1/ and nectin
3/ mice. These results indicate that the ocular
phenotype of nectin 1/ mice is histologically
indistinguishable from that of nectin 3/ mice.

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Fig. 3. Histological analysis of the eyes of adult nectin
1/ and nectin 3/ mice. (A-C,
part a) Sections of the eyes of adult wild-type, nectin
1/ and nectin 3/ mice. (A-C,
part b) Higher magnifications. Arrows, ciliary processes; L, lens; V, vitreous
body. Scale bars: 1 mm in a; 100 µm in b.
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The eye development of nectin 1/ and nectin
3/ mice was prospectively examined during
embryogenesis. In mice, eyelid formation begins at embryonic day 13 (E13), and
from E14 to 16 the eyelids grow, flatten across the eye, and fuse tightly
(Harris and McLeod, 1982
). At
E15.5, the eyelids of wild-type, nectin 1/, and
nectin 3/ mice did not fuse and there was no obvious
difference between their eyes (Fig. 4A,
parts a, b; data not shown). At E16.5, the eyelids of wild-type
and nectin 3/ mice completely fused
(Fig. 4B, part a; data not
shown). By contrast, the eyelids of nectin 1/ mice
did not fuse at this stage (Fig. 4B, part
b). Furthermore, both nectin 1/ and
nectin 3/ mice showed a separation of the apex-apex
contact between the pigment and non-pigment cell layers of the ciliary
epithelia (Fig. 5B, parts b, c;
Fig. 5C, parts b, c). The
ciliary epithelia consist of two layers, the pigment and non-pigment
epithelia, and these two layers make the ciliary processes
(Raviola and Raviola, 1978
).
In normal eyes, the apices of the pigment and non-pigment epithelia were
apposed and contacted each other (Fig. 5A,
parts a-c). It has been reported that the apices of the pigment
and non-pigment epithelia adhere with each other by puncta adherentia
junctions, desmosomes and gap junctions
(Raviola and Raviola, 1978
).
However, in the eyes of nectin 1/ and nectin
3/ pups, the apices of the pigment and non-pigment
epithelia were separated at E16.5 and postnatal day 0 (P0)
(Fig. 5B, parts b, c;
Fig. 5C, parts b, c, see also
Fig. 8). The apices of the
pigment and non-pigment epithelia appeared to still contact at E15.5 in the
eyes of nectin 1/ and nectin
3/ pups, as well as in wild-type pups
(Fig. 5A, part a; Fig. 5B, part a;
Fig. 5C, part a). There was no
obvious difference in the vitreous body, the retinal layers or the lens
between wild-type, nectin 1/ and nectin
3/ mice at E16.5 and P0
(Fig. 4B, parts a, b;
Fig. 4C, parts a, b; Fig. 4D, parts a, b). These
results suggest that a separation of the apex-apex contact between the pigment
and non-pigment cell layers of the ciliary epithelia is a primary defect in
both nectin 1/ and nectin 3/
mice.

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Fig. 4. Developmental analysis of the eyes of nectin 1/
and nectin 3/ mice. (A-D) Sections of the eyes of
E15.5 (A), E16.5 (B) and P0 (C,D) mice. (A-D, part a) Wild-type embryos. (A-C,
part b) A nectin 1/ embryo. (D, part b) A nectin
3/ mouse. Scale bars: 500 µm.
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Fig. 5. Developmental analysis of the ciliary bodies of nectin
1/ and nectin 3/ mice. (A-C)
Wild-type (A), nectin 1/ (B) and nectin
3/ (C) mice. E15.5 (a), E16.5 (b) and P0 (c) mice.
PE, pigment epithelia; NPE, non-pigment epithelia; L, lens; R, retina. Scale
bars: 50 µm.
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Fig. 8. Developmental analysis of the localization of nectin 1, nectin 2, nectin 3
and afadin at the apex-apex junctions between the pigment and non-pigment cell
layers of the ciliary epithelia. E15.5 (left), E16.5 (middle) and P0 (right)
mice. The bottom row shows DIC microscopic images. PE, pigment epithelia; NPE,
non-pigment epithelia; L, lens; R, retina. Insets show higher magnifications
of the boxed areas. Scale bars, 50 µm.
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To examine whether differentiation of the pigment and non-pigment epithelia
of the ciliary body is impaired in nectin 1/ and
nectin 3/ mice, we next examined the expression of
the marker genes for the presumptive iris and ciliary epithelia in the ciliary
body of wild-type, nectin 1/ and nectin
3/ mice at P0
(Fig. 6). It has been reported
that collagen IX is expressed in the cells at optic cap margin, where the
presumptive iris and ciliary epithelia are located, between E14.5 and P2, and
that cadherin 11 is expressed there between E18.5 and P2
(Thut et al., 2001
). Both
collagen IX and cadherin 11 were similarly expressed in the ciliary body of
wild-type, nectin 1/ and nectin
3/ mice at P0. Thus, regardless of a separation of
the apex-apex contact between the pigment and non-pigment epithelia, the
development of the ciliary epithelial cells appears restored in nectin
1/ and nectin 3/ mice as
observed at least by the expression of these marker genes.

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Fig. 6. The expression of collagen IX (top row) and cadherin 11 (middle row) in the
ciliary body of nectin 1/ and nectin
3/ mice. Wild-type (left), nectin
1/ (middle) and nectin 3/
(right) P0 mice are shown. The bottom row shows DIC microscopic images. PE,
pigment epithelia; NPE, non-pigment epithelia; L, lens. Scale bars: 50
µm.
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Localization of nectin 1 and nectin 3 at the apex-apex junction between the pigment and non-pigment cell layers of the ciliary epithelia
We next examined the localization of nectin 1 and nectin 3 in the ciliary
body of adult wild-type mice. Because melanin in the pigment epithelia
inhibits transmission of laser, we used albino mice. The immunofluorescence
signals for P-cadherin and ZO-1 were concentrated at the contact sites between
the pigment and non-pigment cell layers of the ciliary epithelia
(Fig. 7A, parts a-d; Fig. 7E, parts a-d), consistent
with the previous observations
(Tserentsoodol et al., 1998
;
Wu et al., 2000
). The signal
for nectin 1 was also concentrated at the contact sites between the pigment
and non-pigment cell layers of the ciliary epithelia and colocalized with that
for ZO-1 (Fig. 7A, parts a-d). The signals for nectin 3 and afadin were also concentrated and colocalized
with those of nectin 1, ZO-1 and P-cadherin
(Fig. 7B, parts a-d; Fig. 7D, parts a-d;
Fig. 7E, parts a-d; data not
shown). Although the signal for nectin 2 was also observed
(Fig. 7C, part a), it appeared
as punctate spots and was clearly distinguished from those for nectin 1,
nectin 3, afadin, ZO-1 and P-cadherin, which appeared as lines
(Fig. 7A, parts a, b;
Fig. 7C, parts a, b; Fig. 7D, part a;
Fig. 7E, part a; data not
shown). This punctate signal for nectin 2 indicates that nectin 2 localizes
only at the apicolateral junctions, i.e. the most apical side of the lateral
membrane domain, but not at the entire apex-apex junctions. Thus, nectin 1 and
nectin 3, but not nectin 2, are likely to participate in adhesion at the
apex-apex junctions between the pigment and non-pigment cell layers of the
ciliary epithelia.

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Fig. 7. Localization of nectins at the apex-apex junctions between the pigment and
non-pigment cell layers of the ciliary epithelia in a wild-type adult albino
mouse. (A,B, part a) Nectin-1. (C, part a) Nectin-2. (D, part a) Nectin-3.
(Ea, part) P-Cadherin. (A, part b) ZO-1. (B-E, part b) Afadin. (A-E, part c)
Merge. (A-E, part d) DIC image. Insets show higher magnifications of the boxed
areas. Scale bars: 50 µm.
|
|
We next examined the localization of nectin 1, nectin 2, nectin 3 and
afadin in the ciliary body of wild-type pups at E15.5, E16.5, and P0. The
signals for nectin 1 and nectin 3, and afadin were concentrated as lines at
the contact sites between the pigment and non-pigment cell layers of the
ciliary epithelia at E15.5, E16.5, and P0
(Fig. 8). Although the signal
for nectin 2 was also observed there, it appeared as punctate spots and was
clearly different from those for nectin 1, nectin 3 and afadin, which appeared
as lines (Fig. 8). Taken
together with the observations that the apices of the pigment and non-pigment
epithelia became to be separated at E16.5 in the eyes of nectin
1/ and nectin 3/ pups
(Fig. 5B, parts b, c;
Fig. 5C, parts a, b) and that
such a separation of the apex-apex contact between the pigment and non-pigment
cell layers was not observed in the eyes of wild-type pups, these results
suggest that nectin 1, nectin 3 and afadin is likely to participate in
adhesion at the apex-apex junctions between the pigment and non-pigment cell
layers of the ciliary epithelia from the embryonic stage before E16.5.
However, it still remains to be defined whether the apex-apex junctions
between the pigment and non-pigment cell layers of the ciliary epithelia are
formed from the embryonic stage before E16.5, as apposition of two cell layers
does not immediately indicate cell adhesion. It also remains unknown whether
the residual apposed regions between the pigment and non-pigment cell layers
in nectin 1/ and nectin 3/
pups adhere or just contact with each other.
We then examined the precise localization of nectin 1 and nectin 3 at the
contact sites between the pigment and non-pigment epithelia by immunoelectron
microscopy (Fig. 9A, parts a,
b; Fig. 9B). For
this purpose, we used the anti-nectin 1 polyclonal antibody that recognized
the intracellular region of nectin 1, and the anti-nectin 3 monoclonal
antibody that recognized the extracellular region of nectin 3. Although the
immunoparticles for nectin 1 were detected at both the cytoplasmic faces of
the plasma membranes of the pigment and non-pigment epithelia, they were more
frequently detected on the side of the pigment epithelia than on the side of
non-pigment epithelia (Fig. 9A, parts a,
b, arrows). Consistently, the nectin 1 mRNA was expressed in both
the pigment and non-pigment epithelia, but it was more abundantly expressed in
the pigment epithelia (Fig. 9C, parts a,
b). The immunoparticles for nectin 3 were concentrated at the
junctions between the plasma membranes of both the pigment and non-pigment
epithelia (Fig. 9B, arrows).
The nectin 3 mRNA was expressed in both the pigment and non-pigment epithelia
(Fig. 9D, part a), suggesting
that nectin 3 localized at both the plasma membranes of the pigment and
non-pigmental epithelia. No signal for nectin 1 or nectin 3 was observed at
gap junctions (Fig. 9B,
arrowhead; data not shown). These results further indicate that nectin 1 and
nectin 3 localize at the apex-apex junctions between the pigment and
non-pigment cell layers of the ciliary epithelia.

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Fig. 9. Localization of nectin 1 and nectin 3 at the puncta adherentia junctions
between the pigment and non-pigment epithelia of the ciliary body. (A,B)
Localization of nectin 1 (A) and nectin 3 (B) at the ciliary body of the adult
wild-type eye in immunoelectron microscopy. PE, pigment epithelium; NPE,
non-pigment epithelium; M, melanin; N, nucleus. Arrows indicate immunogold
particles; arrowhead indicates a gap junction. Scale bars: 500 nm. (C,D) The
expression patterns of nectin 1 (C) and nectin 3 (D) mRNAs at the ciliary body
of the adult wild-type eye were analyzed by in situ hybridization. Nectin 1
(C, part a) and nectin 3 (D, part a) mRNA. (c) Sense controls for the nectin 1
and nectin 3 mRNAs. (b,d) Corresponding DIC images. Insets show higher
magnifications of the boxed areas. Scale bars: 50 µm.
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Localization of nectins at the apicolateral junctions of the pigment and non-pigment epithelia
We next examined the localization of nectin 1, nectin 2, nectin 3, afadin,
ZO-1 and P-cadherin at the apicolateral junctions of both the pigment and
non-pigment epithelia in the ciliary body. As the ciliary processes were
absent in adult nectin 1/ and nectin
3/ mice (see Fig.
3B, part b; Fig. 3C, part
b), we examined their localization in the ciliary body of nectin
1/ and nectin 3/ albino mice
at P0 when their ciliary body appeared to be abnormal
(Fig. 10). We first confirmed
that the nectin 1/ and nectin
3/ albino mice showed a separation of the apex-apex
contact between the pigment and non-pigment cell layers of the ciliary
epithelia at P0 (Fig. 10A).
The immunofluorescence signal for nectin 1 was concentrated at the contact
sites between the pigment and non-pigment cell layers of wild-type mice,
whereas that for nectin 1 was abrogated in nectin 1/
mice. The signal for nectin 1 remained at the contact sites between two
neighboring pigment cells, but not at those between two neighboring
non-pigment cells of nectin 3/ mice
(Fig. 10A,B). The signal for
nectin 2 was concentrated at the contact sites between the pigment and
non-pigment cell layers of wild-type mice as a punctate spots. The signal for
nectin 2 remained at the contact sites between two neighboring pigment cells
and those between two neighboring non-pigment cells of both nectin
1/ and nectin 3/ mice
(Fig. 10A). The signal for
nectin 3 was concentrated at the contact sites between the pigment and
non-pigment cell layers of wild-type mice, whereas that for nectin 3 was
abrogated in nectin 3/ mice. The signal for nectin 3
remained at the contact sites between two neighboring pigment cells and at
those between two neighboring non-pigment cells of nectin
1/ mice (Fig.
10A,C). The signals for afadin and ZO-1 were concentrated at the
contact sites between the pigment and non-pigment cell layers of wild-type
mice, and the signals remained at the contact sites between two neighboring
pigment cells and at those between two neighboring non-pigment cells of nectin
1/ and nectin 3/ mice
(Fig. 10A). The signal for
P-cadherin was concentrated at the contact sites between the pigment and
non-pigment cell layers of wild-type mice as punctate spots
(Fig. 10A). As the signal for
P-cadherin appeared as lines in adult mice (see
Fig. 7E, part a), P-cadherin
seemed to be recruited to the apex-apex junction between the pigment and
non-pigment epithelia at days later than P0. The signal for P-cadherin
remained at the contact sites between two neighboring pigment cells, but not
at those between two neighboring non-pigment cells of nectin
1/ and nectin 3/ mice
(Fig. 10). No signal for
E-cadherin or N-cadherin was observed at the contact sites between two
neighboring non-pigment cells (data not shown). Thus, another unknown
cadherin(s) might localize at the junctions between two neighboring
non-pigment cells. Taken together, nectin 1, nectin 2, nectin 3, afadin, ZO-1
and P-cadherin localized at the apicolateral junctions between the pigment
epithelia, whereas nectin 2, nectin 3, afadin and ZO-1, but not nectin 1 or
P-cadherin, localized at the apicolateral junctions between the non-pigment
epithelia.

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Fig. 10. Localization of nectins at the apicolateral boundaries of the pigment and
non-pigment epithelia. (A) Localization of nectin 1, nectin 2, nectin 3,
afadin, P-cadherin and ZO-1 at the apicolateral boundaries of the pigment and
non-pigment epithelia. Wild-type (left,) nectin 1/
(middle) and nectin 3/ (right) mice. (B) Localization
of nectin 1 at the apicolateral boundaries of the pigment epithelia, but not
at those of the non-pigment epithelia in nectin 3/
mice. (C) Localization of nectin 3 at the apicolateral boundaries of both the
pigment and non-pigment epithelia in nectin 1/ mice..
PE, pigment epithelia; NPE, non-pigment epithelia; R, retina. Insets show
higher magnifications of the boxed areas. Scale bars: 50 µm.
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|
 |
Discussion
|
---|
Nectin-based cell-cell adhesion plays important roles in morphogenesis of
multicellular organisms (Takai et al.,
2003a
; Takai and Nakanishi,
2003
). Implications of nectins have first been demonstrated in the
knockout study of mice lacking afadin, a major adaptor protein connecting
nectins to the actin cytoskeleton that binds all members of the nectin family
(Ikeda et al., 1999
). In the
absence of afadin, mouse embryos cease to develop at E8.5. However, knockout
studies on individual members of the nectin family have been proven to be more
informative. Although nectin 2/ mice show impaired
spermatogenesis (Ozaki-Kuroda et al., 2000), our present study on mice lacking
nectin 1 and nectin 3 show a virtually identical ocular phenotype:
microphthalmia. This work has demonstrated that the potent heterophilic
trans-interaction between nectin 1 and nectin 3, originally detected by an in
vitro study, actually promotes the apex-apex adhesion between the pigment and
non-pigment cell layers of the ciliary epithelia in mice. To our knowledge,
the apex-apex junction between the epithelia is unique to the ciliary body.
Impairment of the trans-interaction between nectin 1 and nectin 3 causes
separation of the pigment and non-pigment cell layers and disruption of the
ciliary body. The differentiation markers for the presumptive iris and ciliary
epithelia are similarly expressed in the ciliary body of wild-type, nectin
1/ and nectin 3/ mice. Thus,
the apex-apex adhesion mediated by nectin 1 and nectin 3 between the pigment
and non-pigment cell layers is unlikely to be involved in the ciliary
epithelial cell surviving and differentiating pathway, but is required for a
process of the ciliary body formation along with the subsequent folding of
these two cell layers. The ciliary body is known to produce both the aqueous
humor and some components of the vitreous body and is the source of the
zonules that support the lens (Bertazolli
Filho et al., 1996
; Francis and
Alvarado, 1997
; Haddad et al.,
1990
; Zimmerman and Fine,
1964
). Consistently, the vitreous body was absent, the retinal
layers were undulating and the lenses were deformed in adult nectin
1/ and nectin 3/ mice. Thus,
the apex-apex adhesion mediated by nectin 1 and nectin 3 between the pigment
and non-pigment cell layer are required for eye development. To our knowledge,
this phenotype of a separation between the pigment and non-pigment cell layer,
which is observed in nectin 1/ and nectin
3/ mice, has not been reported before. We have shown
that the primary defect of this trans-interaction is attributed to the
differential localization of nectins: nectin 1 and nectin 3 localize at both
the apex-apex and apicolateral junctions, whereas nectin 2 localizes at the
apicolateral junctions. These results are further supported by the observation
that mice lacking nectin 2 exhibit no microphthalmia. Thus, we conclude that
the ciliary epithelia consisting of two layers, the pigment and non-pigment
epithelia (Raviola and Raviola,
1978
), are apposed and adhered by puncta adherentia junctions and
gap junctions in which nectin 1 and nectin 3, together with P-cadherin and
ZO-1, play essential roles for CAMs (Fig.
11). It remains unknown how nectin 2 is excluded from the apical
surface of pigment and non-pigment epithelia. It might be due to a higher
affinity between nectin 1 and nectin 3 than between nectin 2 and nectin 3
(Takai et al., 2003a
).
Alternatively, nectin 2 might be sorted to the basolateral membrane, but not
to the apical membrane, in the pigment and non-pigment epithelia of the
ciliary body. The human nectin 1 mutations are responsible for cleft
lip/palate-ectodermal dysplasia, Margarita island ectodermal dysplasia and
Zlotogora-Ogür syndrome, which is characterized by cleft lip/palate,
syndactyly, mental retardation and ectodermal dysplasia
(Sozen et al., 2001
;
Suzuki et al., 2000
). Although
ocular involvement in individuals with this syndrome has not been reported,
the human nectin 1 mutations might potentially cause an ocular defect.

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Fig. 11. CAMs and their associated proteins at the apex-apex junctions between the
pigment and non-pigment epithelia, the apicolateral junctions between the
pigment epithelia and the apicolateral junctions between the non-pigment
epithelia in the ciliary body. (A) Schematic diagram of the eye. The area
surrounded by the broken red line is shown in B. PE, pigment epithelia; NPE,
non-pigment epithelia. (B) The localization of CAMs and their associated
proteins in the ciliary body. Nectin 1, nectin 3, P-cadherin, afadin and ZO-1
localize at the puncta adherentia junctions between the pigment and
non-pigment cell layers of the ciliary epithelia. Connexins localizes at the
gap junctions between the pigment and non-pigment cell layers of the ciliary
epithelia. Nectin 1, nectin 2 and nectin 3, afadin, ZO-1 and P-cadherin
localize at the AJs between the pigment epithelia. Nectin 2, nectin 3 and
afadin localize at the AJs between the non-pigment epithelia. Cadherin 11
and/or other unknown cadherins, different from P-, E- and N-cadherins,
associated with - and ß-catenins are likely to localize at these
AJs, as cadherin 11 and - and ß-catenins localize there. Claudins,
occludin and ZO-1 localize at the TJs between the non-pigment epithelia. PE,
pigment epithelia; NPE, non-pigment epithelia.
|
|
As P-cadherin localizes at this apex-apex junction
(Wu et al., 2000
), the roles
of the trans-interaction between nectin 1 and nectin 3 may be challenged by
the presence of P-cadherin. However, P-cadherin/ mice
have no ocular phenotype (Radice et al.,
1997
). Furthermore, the signal for P-cadherin at the apex-apex
junctions is observed in adult mice but not at P0, while those for nectin 1
and nectin 3 have already been observed at P0. Thus, nectin 1 and nectin 3,
but not P-cadherin, are essential for the formation of the apex-apex adhesion
between the pigment and non-pigment cell layers of the ciliary epithelia. The
force of the heterophilic trans-interaction between nectin 1 and nectin 3 has
been thought to be weaker than that of the homophilic trans-interaction of
E-cadherin. For example, whereas cadherins show the strong cell-cell adhesion
accompanied by compaction, maximization of adhesive contacts, the nectin 1-
and nectin 3-mediated adhesion is not accompanied by compaction
(Takai et al., 2003a
;
Takai and Nakanishi, 2003
). In
addition, force measurement analysis on paired cells has revealed that the
force of the heterophilic-trans-interaction between nectin 1 and nectin 3 is
much weaker than that of the homophilic-trans-interaction of E-cadherin
(Martinez-Rico et al., 2005
).
However, a recent single-molecule analysis with the purified proteins of
nectins and E-cadherin has remarkably shown that the force of the
heterophilic-trans-interaction between nectin 1 and nectin 3 is stronger than
that of the homophilic-trans-interaction of E-cadherin in a low loading rate
condition (less than 100 pN/s), while the force of the
heterophilic-trans-interaction between nectin 1 and nectin 3 is weaker than
that of the homophilic-trans-interaction of E-cadherin in a high loading rate
condition (more than 100 pN/s) (Y. Tsukazaki, K. Kitamura, K. Shimizu, A.
Iwane, Y.T. and T. Yanagida, unpublished). Thus, the force of the
heterophilic-trans-interaction between nectin 1 and nectin 3 itself should be
strong enough to establish the apex-apex adhesion between the pigment and
non-pigment epithelia of the ciliary body at P0, even when the signal for
P-cadherin is not observed there.
Another important insight learned from this study is that nectin 1 and
nectin 3 also play sentinel roles to form solid apex-apex junctional
structures between the pigment and non-pigment epithelia of the ciliary body
in adult mice. We have previously shown that nectins are initially involved in
the formation of AJs in cooperation with E-cadherin, and subsequently promote
the formation of TJs in epithelial cells in culture
(Takai et al., 2003a
;
Takai and Nakanishi, 2003
).
These findings may be extended to the formation of the ciliary body: the
heterophilic trans-interaction of nectin 1 and nectin 3 first forms the cell
adhesion, then recruits P-cadherin to the nectin 1- and nectin 3-based
adhesion sites, and finally ends up establishing the strong adhesion
undercoated with F-actin, mediated by afadin and ZO-1, at the apex-apex
junctions between the pigment and non-pigment cell layers of the ciliary
epithelia. Thus, the heterophilic trans-interaction between nectin 1 and
nectin 3 is required for the formation of three junctional structures in the
ciliary body: gap junctions, desmosomes and puncta adherentia junctions
(Raviola and Raviola,
1978
).
Phenotypes of mice lacking nectins are modified by functional redundancy
that depends on the localization of nectins and their heterophilic
trans-interaction. Afadin/ mice show embryonic
lethality with developmental defects because all of the combinations of
heterophilic trans-interactions between nectins are disrupted. By contrast,
nectin 1/, nectin 2/ and
nectin 3/ mice are viable and show no
life-threatening disorder. Neither nectin 1/ nor
nectin 3/ mice apparently show impaired organization
of AJs and TJs in most tissues where various types of nectins are expressed
(data not shown). There is no doubt that the apex-apex adhesion between the
pigment epithelia and non-pigment epithelia is one of the places with reduced
functional redundancy of nectins and thus vulnerable to the gene disruption
experiments. However, the apicolateral junctions between the pigment epithelia
or those between the non-pigment epithelia are preserved in the absence of
nectin 1 or nectin 3 because of the presence of nectin 2. The finding that
male nectin 3/ mice exhibit infertility is also
probably explained by the specific localization of nectin 2 and nectin 3 in
Sertoli cells and spermatids, respectively
(Ozaki-Kuroda et al., 2002
;
Takai and Nakanishi, 2003
), as
well as by the absence of nectin 1. Although about 30% of nectin
1/ pups are born with one or both eyes open, this
phenotype is not observed in nectin 3/ mice,
indicating an additional role specific to nectin 1. Consistently, nectin 1 has
recently shown to localize at the sites of eyelid fusion
(Okabe et al., 2004a
). Further
knockout studies would reveal additional roles of nectins in tissue
morphogenesis and organogenesis during mouse development.
 |
ACKNOWLEDGMENTS
|
---|
We thank Drs M. Takeichi (Center for Developmental Biology, RIKEN, Kobe,
Japan), N. Azuma (National Center for Child Health and Development, Tokyo,
Japan) and K. Takata (Gunma University, Gunma, Japan) for helpful discussions
and critical readings of the manuscript. The investigation at Osaka University
Medical School was supported by grants-in-aid for Scientific Research and for
Cancer Research from the Ministry of Education, Science, Sports, Culture and
Technology, Japan (2003, 2004).
 |
REFERENCES
|
---|
Amaral, D. G. and Dent, J. A. (1981).
Development of the mossy fibers of the dentate gyrus: I. A light and electron
microscopic study of the mossy fibers and their expansions. J.
Comp. Neurol. 195,51
-86.[CrossRef][Medline]
Bertazolli Filho, R., Laicine, E. M. and Haddad, A.
(1996). Biochemical studies on the secretion of glycoproteins by
isolated ciliary body of rabbits. Acta. Ophthalmol.
Scand. 74,343
-347.[Medline]
Bouchard, M. J., Dong, Y., McDermott, B. M., Jr, Lam, D. H.,
Brown, K. R., Shelanski, M., Bellve, A. R. and Racaniello, V. R.
(2000). Defects in nuclear and cytoskeletal morphology and
mitochondrial localization in spermatozoa of mice lacking nectin-2, a
component of cell-cell adherens junctions. Mol. Cell.
Biol. 20,2865
-2873.[Abstract/Free Full Text]
Bradford, M. M. (1976). A rapid and sensitive
method for the quantitation of microgram quantities of protein utilizing the
principle of protein-dye binding. Anal. Biochem.
72,248
-254.[CrossRef][Medline]
Brummendorf, T. and Lemmon, V. (2001).
Immunoglobulin superfamily receptors: cis-interactions, intracellular adapters
and alternative splicing regulate adhesion. Curr. Opin. Cell
Biol. 13,611
-618.[CrossRef][Medline]
Francis, B. A. and Alvarado, J. (1997). The
cellular basis of aqueous outflow regulation. Curr. Opin.
Ophthalmol. 8,19
-27.[Medline]
Haddad, A., de Almeida, J. C., Laicine, E. M., Fife, R. S. and
Pelletier, G. (1990). The origin of the intrinsic
glycoproteins of the rabbit vitreous body: an immunohistochemical and
autoradiographic study. Exp. Eye Res.
50,555
-561.[CrossRef][Medline]
Harris, M. J. and McLeod, M. J. (1982). Eyelid
growth and fusion in fetal mice. A scanning electron microscope study.
Anat. Embryol. 164,207
-220.[CrossRef][Medline]
Ikeda, W., Nakanishi, H., Miyoshi, J., Mandai, K., Ishizaki, H.,
Tanaka, M., Togawa, A., Takahashi, K., Nishioka, H., Yoshida, H. et
al. (1999). Afadin: A key molecule essential for structural
organization of cell-cell junctions of polarized epithelia during
embryogenesis. J. Cell Biol.
146,1117
-1132.[Abstract/Free Full Text]
Kinoshita, A., Shigemoto, R., Ohishi, H., van der Putten, H. and
Mizuno, N. (1998). Immunohistochemical localization of
metabotropic glutamate receptors, mGluR7a and mGluR7b, in the central nervous
system of the adult rat and mouse: a light and electron microscopic study.
J. Comp. Neurol. 393,332
-352.[CrossRef][Medline]
Laemmli, U. K. (1970). Cleavage of structural
proteins during the assembly of the head of bacteriophage T4.
Nature 227,680
-685.[Medline]
Mandai, K., Nakanishi, H., Satoh, A., Obaishi, H., Wada, M.,
Nishioka, H., Itoh, M., Mizoguchi, A., Aoki, T., Fujimoto, T., Matuda,
Y., Tsukita, S. and Takai, Y. (1997). Afadin: a novel actin
filament-binding protein with one PDZ domain localized at cadherin-based
cell-to-cell adherens junction. J. Cell Biol.
139,517
-528.[Abstract/Free Full Text]
Martinez-Rico, C., Pincet, F., Perez, E., Thiery, J. P.,
Shimizu, K., Takai, Y. and Dufour, S. (2005).
Separation force measurements reveal different types of modulation of
E-cadherin-based adhesion by nectin 1 and nectin 3. J. Biol.
Chem. 280,4753
-4760.[Abstract/Free Full Text]
Mizoguchi, A., Nakanishi, H., Kimura, K., Matsubara, K.,
Ozaki-Kuroda, K., Katata, T., Honda, T., Kiyohara, Y., Heo, K.,
Higashi, M., Tsutsumi, T., Sonoda, S., Ide, C. and Takai, Y.
(2002). Nectin: an adhesion molecule involved in formation of
synapses. J. Cell Biol.
156,555
-565.[Abstract/Free Full Text]
Mueller, S., Rosenquist, T. A., Takai, Y., Bronson, R. A. and
Wimmer, E. (2003). Loss of nectin-2 at Sertoli-spermatid
junctions leads to male infertility and correlates with severe spermatozoan
head and midpiece malformation, impaired binding to the zona pellucida, and
oocyte penetration. Biol. Reprod.
69,1330
-1340.[Abstract/Free Full Text]
Ohno, S. (2001). Intercellular junctions and
cellular polarity: the PAR-aPKC complex, a conserved core cassette playing
fundamental roles in cell polarity. Curr. Opin. Cell
Biol. 13,641
-648.[CrossRef][Medline]
Okabe, N., Ozaki-Kuroda, K., Nakanishi, H., Shimizu, K. and
Takai, Y. (2004a). Expression patterns of nectins and afadin
during epithelial remodeling in the mouse embryo. Dev.
Dyn. 230,174
-186.[CrossRef][Medline]
Okabe, N., Shimizu, K., Ozaki-Kuroda, K., Nakanishi, H.,
Morimoto, K., Takeuchi, M., Katsumaru, H., Murakami, F. and Takai,
Y. (2004b). Contacts between the commissural axons and the
floor plate cells are mediated by nectins. Dev. Biol.
273,244
-256.[CrossRef][Medline]
Ozaki-Kuroda, K., Nakanishi, H., Ohta, H., Tanaka, H., Kurihara,
H., Mueller, S., Irie, K., Ikeda, W., Sakai, T., Wimmer, E., Nishimune,
Y. and Takai, Y. (2002). Nectin couples cell-cell adhesion
and the actin scaffold at heterotypic testicular junctions. Curr.
Biol. 12,1145
-1150.[CrossRef][Medline]
Radice, G. L., Ferreira-Cornwell, M. C., Robinson, S. D.,
Rayburn, H., Chodosh, L. A., Takeichi, M. and Hynes, R. O.
(1997). Precocious mammary gland development in
P-cadherin-deficient mice. J. Cell Biol.
139,1025
-1032.[Abstract/Free Full Text]
Raviola, G. and Raviola, E. (1978).
Intercellular junctions in the ciliary epithelium. Invest.
Ophthalmol. Vis. Sci. 17,958
-981.[Abstract]
Satoh-Horikawa, K., Nakanishi, H., Takahashi, K., Miyahara,
M., Nishimura, M., Tachibana, K., Mizoguchi, A. and Takai, Y.
(2000). Nectin-3, a new member of immunoglobulin-like cell
adhesion molecules that shows homophilic and heterophilic cell-cell adhesion
activities. J. Biol. Chem.
275,10291
-10299.[Abstract/Free Full Text]
Sozen, M. A., Suzuki, K., Tolarova, M. M., Bustos, T., Fernandez
Iglesias, J. E. and Spritz, R. A. (2001). Mutation of
PVRL1 is associated with sporadic, non-syndromic cleft lip/palate in northern
Venezuela. Nat. Genet.
29,141
-142.[CrossRef][Medline]
Suzuki, K., Hu, D., Bustos, T., Zlotogora, J., Richieri-Costa,
A., Helms, J. A. and Spritz, R. A. (2000). Mutations
of PVRL1, encoding a cell-cell adhesion molecule/herpesvirus receptor, in
cleft lip/palate-ectodermal dysplasia. Nat. Genet.
25,427
-430.[CrossRef][Medline]
Takahashi, K., Nakanishi, H., Miyahara, M., Mandai, K., Satoh,
K., Satoh, A., Nishioka, H., Aoki, J., Nomoto, A., Mizoguchi, A. and
Takai, Y. (1999). Nectin/PRR: an immunoglobulin-like cell
adhesion molecule recruited to cadherin-based adherens junctions through
interaction with Afadin, a PDZ domain-containing protein. J. Cell
Biol. 145,539
-549.[Abstract/Free Full Text]
Takai, Y. and Nakanishi, H. (2003). Nectin and
afadin: novel organizers of intercellular junctions. J. Cell
Sci. 116,17
-27.[Abstract/Free Full Text]
Takai, Y., Irie, K., Shimizu, K., Sakisaka, T. and Ikeda, W.
(2003a). Nectins and nectin-like molecules: roles in cell
adhesion, migration, and polarization. Cancer Sci.
94,655
-667.[Medline]
Takai, Y., Shimizu, K. and Ohtsuka, T. (2003b).
The roles of cadherins and nectins in interneuronal synapse formation.
Curr. Opin. Neurobiol.
13,520
-526.[CrossRef][Medline]
Takeichi, M. (1995). Morphogenetic roles of
classic cadherins. Curr. Opin. Cell Biol.
7, 619-627.[CrossRef][Medline]
Takemoto, M., Fukuda, T., Sonoda, R., Murakami, F., Tanaka, H.
and Yamamoto, N. (2002). Ephrin-B3-EphA4 interactions
regulate the growth of specific thalamocortical axon populations in vitro.
Eur. J. Neurosci. 16,1168
-1172.[CrossRef][Medline]
Thut, C. J., Rountree, R. B., Hwa, M. and Kingsley, D. M.
(2001). A large-scale in situ screen provides molecular evidence
for the induction of eye anterior segment structures by the developing lens.
Dev. Biol. 231,63
-76.[CrossRef][Medline]
Tserentsoodol, N., Shin, B. C., Suzuki, T. and Takata, K.
(1998). Colocalization of tight junction proteins, occludin and
ZO-1, and glucose transporter GLUT1 in cells of the blood-ocular barrier in
the mouse eye. Histochem. Cell Biol.
110,543
-551.[CrossRef][Medline]
Tsukita, S., Furuse, M. and Itoh, M. (1999).
Structural and signalling molecules come together at tight junctions.
Curr. Opin. Cell Biol.
11,628
-633.[CrossRef][Medline]
Tsukita, S., Furuse, M. and Itoh, M. (2001).
Multifunctional strands in tight junctions. Nat. Rev. Mol. Cell
Biol. 2,285
-293.[CrossRef][Medline]
Wu, P., Gong, H., Richman, R. and Freddo, T. F.
(2000). Localization of occludin, ZO-1, and pan-cadherin in
rabbit ciliary epithelium and iris vascular endothelium. Histochem.
Cell Biol. 114,303
-310.[Medline]
Yagi, T. and Takeichi, M. (2000). Cadherin
superfamily genes: functions, genomic organization, and neurologic diversity.
Genes Dev. 14,1169
-1180.[Free Full Text]
Zimmerman, L. E. and Fine, B. S. (1964).
Production of hyaluronic acid by cysts and tumors of the ciliary body.
Arch. Ophthalmol. 72,365
-379.[Medline]