Download Diversification of epithelial adherens junctions with independent

EVOLUTION & DEVELOPMENT
7:5, 376 –389 (2005)
Diversification of epithelial adherens junctions with independent
reductive changes in cadherin form: identification of potential
molecular synapomorphies among bilaterians
Hiroki Oda,a,b, Kunifumi Tagawa,c,1 and Yasuko Akiyama-Odaa,b,d
a
JT Biohistory Research Hall, 1-1 Murasaki-cho, Takatsuki, Osaka 569-1125, Japan
Tsukita Cell Axis Project, ERATO, Japan Science and Technology Corporation, Kyoto, Japan
c
Kewalo Marine Laboratory, Pacific Biomedical Research Center, University of Hawaii, 41 Ahui Street,
Honolulu, HI 96813-5511, USA
d
PRESTO, Japan Science and Technology Agency, Saitama, Japan
b
Author for correspondence (email: [email protected])
1
Present address: Marine Biological Laboratory, Graduate School of Science, Hiroshima University, 2445 Mukaishima, Onomichi, Hiroshima 722-0073, Japan.
SUMMARY The adherens junction (AJ) is the most
universal junction found in bilaterian epithelia and may
represent one of the earliest types of cell–cell junctions. The
adhesion molecules responsible for forming AJs are the
classic cadherins (referred to simply as cadherins), whose
extracellular domain organization displays marked variety
among species examined so far. In this study, we attempted to
reconstruct the evolution of cadherin by analyzing new data
from several arthropods (two insects, one noninsect hexapod,
three crustaceans, and one chelicerate) and previously
published sequences for Drosophila melanogaster and other
animals. The results of comparative analyses using the
BLAST tool and immunohistochemical analyses revealed
that the extracellular domain organizations of a decapod, an
isopod, a spider, and a starfish cadherin, which are present at
AJs in the embryonic epithelia are homologous. Independent
reductive changes from the ancestral state were evident
in the epithelia of hexapods1branchiopod, vertebrates1
urochordates, and a cephalochordate. The form of cadherins
in hexapods is more closely related to that of a branchiopod
than to that of malacostracan crustaceans, and one of those of
vertebrates is more closely related to that of urochordates
than to that of a cephalochordate. Although the sampling of
taxa is limited at this stage of research, we hypothesize that
the reductive events in cadherin structure related to AJ
formation in the epithelia may possess information about
bilaterian relationships as molecular synapomorphies.
INTRODUCTION
indicate members of the classic cadherin family. The extracellular structure of cadherin bridges the extracellular space
between neighboring cells via cis- and trans-interactions
(Gooding et al. 2004), whereas the intracellular structure provides an anchor for scaffolding the actin cytoskeleton via the
catenins (Gumbiner 2000). Cadherin functions are essential
for a variety of morphogenetic processes in bilaterian development (Takeichi 1995; Tepass et al. 2000).
Despite the conservation of the CP domains, the extracellular structures of cadherins display substantial variation
(Oda et al. 2002). The extracellular regions of mouse mE- and
mN-cadherins (see Table 1 for cadherin name abbreviations)
consist of five extracellular cadherin (EC) domains aligned in
tandem, and this form is common among vertebrates. However, it is not universal among bilaterians. In the extracellular
regions of known nonchordate cadherins, three different domain types in addition to the EC domain have been identified:
Adherens junctions (AJs) are the type of cell–cell junction
found universally in bilaterian epithelia (Lane et al. 1994). The
similarities in molecular composition between the vertebrates,
Drosophila and Caenorhabditis elegans (Knust and Bossinger
2002) suggest that the AJs are homologous across bilateria.
Members of the classic cadherin family, consisting of singlepass transmembrane proteins with homotypic cell–cell adhesion activity, play a central role in the formation and function
of the AJs (Takeichi 1995; Gumbiner 2000; Tepass et al.
2000). The members of this family are distinguished from
other members of the cadherin superfamily in that they have a
highly conserved cytoplasmic (CP) domain that interacts with
catenins (Nollet et al. 2000; Yagi and Takeichi 2000). Genes
encoding classic cadherins have been reported only for
bilaterian metazoans. The term ‘‘cadherin’’ is used herein to
376
& BLACKWELL PUBLISHING, INC.
Oda et al.
Evolution of adherens junction cadherin
Table 1. List of classic cadherins used in this study
Species
Name of
cadherin
Arthropoda, Hexapoda, Diptera
Drosophila melanogaster
DE-cadherin
DN-cadherin
Arthropoda, Hexapoda, Lepidoptera
Bombyx mori
Bm1-cadherin1
Bm2-cadherin1
Arthropoda, Hexapoda, Orthoptera
Gryllus bimaculatus
Gb1-cadherin
Gb2-cadherin1
Arthropoda, Hexapoda, Collembola
Folsomia candida
Fc1-cadherin
Fc2-cadherin1
Arthropoda, Crustacea, Branchiopoda
Artemia franciscana
Af1-cadherin
Af2-cadherin
Arthropoda, Crustacea, Decapoda
Caridina japonica
Cj-cadherin
Arthropoda, Crustacea, Isopoda
Ligia exotica
Le-cadherin
Arthropoda, Chelicerata, Arachnida
Achaearanea tepidariorum At-cadherin
Mollusca, Bivalvia
Saccostrea echinata
Se-cadherin1
Echinodermata, Echinoidea
Lytechinus variegatus
LvG-cadherin
Echinodermata, Asteroidea
Asterina pectinifera
Ap-cadherin
Hemichordata, Enteropneusta
Ptychodera flava
Pf1-cadherin
Pf2-cadherin1
Chordata, Cephalochordata
Branchiostoma belcheri
Bb1-cadherin
Bb2-cadherin
Chordata, Urochordata, Ascidiacea
Ciona intestinalis
Ci1-cadherin
Ciona savignyi
Cs2-cadherin
Botryllus schlosseri
BS-cadherin
Chordata, Vertebrata, Teleostei
Danio rerio
Dr1-cadherin
Danio rerio
Dr2-cadherin
Chordata, Vertebrata, Mammalia
Mus musculus
mE-cadherin
mN-cadherin
m6-cadherin
m8-cadherin
m10-cadherin
m11-cadherin
Chordata, Vertebrata, Aves
Gallus gallus
cHz-cadherin
1
Form
Accession
no.
A1
A2
BAA05942
T00021
ND
ND
AB1902943
AB1902933
A1
ND2
AB1902953
AB1902963
A1
ND2
AB1902973
AB1902983
A1
A2
AB1902993
AB1903003
A2
AB1903013
A2
AB1903023
A2
AB1903033
ND2
AB075367
E1
U34823
E2
AB075365
H
ND2
AB075368
AB075369
C
C
AB075366
AB120427
V1
V1
V1
AB031540
AB057736
U61755
V1
V1
NP571895
AAN61915
V1
V1
V1
V1
V1
V1
X06115
M31131
NP031692
NP031693
AAL67951
D31963
V2
AY312363
Only partial cDNA information is available.
The partial cDNA information indicates that an A2/E2-type PCCD
complex is present.
3
Identified in this study.
ND, not determined; PCCD, primitive classic cadherin domain.
2
377
the nonchordate-specific (NC) domain, the cysteine-rich
EGF-like (CE) domain, and the laminin globular-like (LG)
domain (Oda and Tsukita 1999). All known nonchordate
cadherins and chick cHz-cadherin have a domain complex
called the primitive classic cadherin domain (PCCD) complex
(Oda and Tsukita 1999; Oda et al. 2002) which consists of
NC, CE, and LG domains sandwiched between the last EC
and the transmembrane (TM) domain. Among the nonchordate and chick cadherins, structural variations are observed in
the number of EC domains and the organization of the
PCCD complex. For the cephalochordate amphioxus, two
classic cadherin-related molecules with no EC domains have
been identified (Oda et al. 2002, 2004). These amphioxus
cadherins, as well as mouse E- and N-cadherins, Drosophila
DE-cadherin, sea urchin LvG-cadherin and C. elegans HMR1 cadherin, are localized to the epithelial AJs (Takeichi 1988;
Oda et al. 1994, 2002, 2004; Miller and McClay 1997; Costa et
al. 1998). Different forms of cadherins are involved in AJ
formation in the epithelia of different animals. How this diversity arose is not known.
In this study, we attempted to reconstruct the evolution of
cadherin by analyzing new data from several arthropods in
combination with publicly available data. We performed comparative analysis of the domain organizations of cadherin
molecules by using the BLAST tool. This allowed us to deduce
which forms are ancestral and to detect independent reductive
changes from the ancestral state in different animal lineages,
which may account for the variety of forms of cadherins. Based
on these results combined with immunohistochemical data, we
propose a hypothesis in which the reductive events in cadherin
structure related to AJ formation in the epithelia may contain
phylogenetic information as molecular synapomorphies.
MATERIALS AND METHODS
Animals
Bombyx mori, Gryllus bimaculatus, and Asterina pectinifera
were kindly provided by N. Sumida (Kyoto Institute of Tech.,
Kyoto, Japan), K. Kimura (Hokkaido University of Education, Iwamizawa, Japan), and E. Shoguchi (Kyoto University,
Kyoto, Japan), respectively. Ligia exotica was collected near
the Seto marine biological laboratory (Wakayama, Japan).
Achaearanea tepidariorum was collected at the Kyoto University. Artemia franciscana (Tetra), Caridina japonica (Masuda,
Kyoto, Japan), and Folsomia candida (Spheroaqua, Hamamatsu, Japan) were purchased from suppliers. For other
animals included in this analysis, cadherin sequences were
obtained from the public database (DDBJ/EMBL/GENBANK).
cDNA library construction
mRNA was isolated from each animal species using a QuickPrep Micro mRNA purification kit (Amersham Biosciences,
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EVOLUTION & DEVELOPMENT
Vol. 7, No. 5, September^October 2005
Piscataway, NJ, USA). cDNA was synthesized using a TimeSaver cDNA synthesis kit (Amersham Biosciences, Carlsbad,
CA, USA), a SuperScript lambda system (Invitrogen), or a
SuperScript lambda choice system (Invitrogen). The cDNAs
were ligated to lgt11 (Stratagene), lZAP (Stratagene, La
Jolla, CA, USA), or lZipLox (Invitrogen) to construct libraries. The cDNA libraries were used for cDNA cloning.
cDNA cloning
The polymerase chain reaction (PCR) with degenerate
primers was performed to amplify cDNA fragments related
to classic cadherin. The primer sequences used were as follows: IN1, 50 -ATHAAYTAYGAIGAIGARGGIGG-30 ; KL2,
50 -CCRTACATRTCIGCIARYTT-3 0 ; HY2, 50 -CCRTCICCYTCRTAIGCRTARTG-3 0 ; and NYA1, 50 -AAYTAYGCNTAYGARGG-30 (where H 5 A, C, or T; Y 5 C or T; I 5
inosine; R 5 A or G; and N 5 A, C, T, or G). The cDNAs
described above were used as the templates for PCR. The
PCR conditions were as follows: 1 cycle of 951C for 5 min; 1
cycle of 941C for 40 sec, 551C for 2 min 30 sec, 721C for 40 sec;
40 cycles of 941C for 40 sec, 551C for 40 sec, 721C for 40 sec; 1
cycle of 721C, for 7 min; and then a 41C soak. Three of the
primer combinations worked effectively: IN1 and KL2, IN1
and HY2, and NYA1 and KL2. The cloned fragments were
then used to screen appropriate cDNA libraries. For library
screening, digoxigenin (DIG)-labeled probes were made using the PCR DIG probe synthesis kit (Roche Diagnostics,
Mannheim, Germany), and the resulting signals were visualized by means of the anti-DIG-peroxidase antibody (Roche
Diagnostics) and the ECL Western Blot Detection System
(Amersham Biosciences). Overlapping clones for each cDNA
were obtained through several rounds of library screening.
Both strands of representative cDNA clones were sequenced.
The compiled data were deposited in the DNA database.
Amino acid sequence analysis
To define the domains present in each cadherin, the amino
acid sequences were analyzed using the PROSITE scanning
tool at http://www.expasy.org/tools/scanprosite/and the twosequence alignment BLAST tool at http://www.ncbi.nlm.nih.
gov/blast/bl2seq/bl2.html. The defined domains and their
names are shown in Fig. 1.
Molecular phylogenetic trees were constructed by the
neighbor-joining method (Saitou and Nei 1987) using PHYLIP version 3.5 (Felsenstein 1993) and by the maximum parsimony method using PAUP version 4.0b (Swofford 2001).
Amino acid sequences were aligned manually. Ninety-three
amino acid sites from the CP domains of the cadherins and
732 amino acid sites from the extracellular regions of the
cadherins were used. Heuristic searches to find the maximum
parsimony trees were performed using tree-bisection-recom-
Fig. 1. The domain organizations of classic cadherins from arthropods (A1, A2), echinoderms (E1, E2), a hemichordate (H), a
cephalochordate (C), urochordates, and vertebrates (V1, V2) are
diagrammed. Repeated domains are numbered from the N-terminus. The domain types are indicated at the bottom: NT, N-terminal
domain; EC, extracellular cadherin domain; NC, nonchordate-specific domain; CE, cysteine-rich EGF-like domain; LG, laminin
globular-like domain; TM, transmembrane domain; CP, cytoplasmic domain.
bination branch-swapping, and confidence in the phylogenies
was assessed by bootstrap resampling of the data.
The domain organizations were compared by means of the
two sequence alignment tools described above. The amino
acid sequence of each domain in one cadherin was blasted
against the entire amino acid sequence of another cadherin.
Plotting of the expected values (E-values) on the matrix was
used to detect collinear similarities indicative of conservation.
Antibody production
For antibody production, seven fusion proteins containing
parts of different cadherins were constructed using the
Oda et al.
pMAL-p2 vector (New England Biolabs, Beverly, MA, USA).
The polypeptides contained in the fusion proteins were as
follows: Gb1-cadherin, aa119–aa693; Af1-cadherin, aa175–
aa774; Af2, aa169–aa743; Le-cadherin, aa193–aa792; At-cadherin, aa171–aa751; Ap-cadherin, aa187–aa761; and Pf1cadherin, aa180–aa788. The fusion proteins were expressed in
Escherichia coli BL21 (DE3), separated by SDS-PAGE, and
electroeluted from the gel. Rats or guinea-pigs were immunized with the purified proteins. Antisera were directly used
at dilutions of 1:200 to 1:500 for Western blot analysis and
immunohistochemical staining. To test antibody specificity,
six fusion proteins containing parts of different cadherins were
constructed by using the pGEX vectors (Amersham Biosciences). The polypeptides contained in the fusion proteins were
as follows: Le-cadherin, aa193–aa792, aa957–aa1555; Cjcadherin, aa170–aa785; DN-cadherin, aa263–aa868; DEcadherin, aa149–aa717; and Gb1-cadherin, aa119–aa693.
For the Western blot analysis presented in Fig. 6(C), an
anti-GST antibody (Amersham Biosciences) was used.
Evolution of adherens junction cadherin
379
RESULTS
Designations of the cadherin domain
organizations
The cadherins used in this study are listed in Table 1, and the
defined domains and their names are shown in Fig. 1. In
addition to the EC, NC, CE, LG, TM, and CP domains
described in our previous studies (Oda and Tsukita 1999; Oda
et al. 2002), we designate an N-terminal (NT) domain that
BLAST analysis found weakly similar in DN-, Ap-, LvG-,
and Pf1-cadherin (see Figs. 3, 4, 8). For convenience, the
different forms of cadherin were designated by a letter indicating the phylum or subphylum (e.g., A for Arthropoda) and
a number (Fig. 1). The form represented by mE- and mNcadherins was designated V1, that by cHz-cadherin was V2,
that by Bb1- and Bb2-cadherins was C, that by DE-cadherin
was A1, that by DN-cadherin was A2, that by LvG-cadherin
was E1, that by Ap-cadherin was E2, and that by Pf1-cadherin was H.
Antibody staining
The states of the A1 and A2 forms were
evolutionarily stable in arthropods
Gryllus, Caridina, and Ligia embryos were dissected in CGBS
(55-mM NaCl, 40-mM KCl, 10-mM Tricine, pH 6.9) and then
fixed with 3.7% formaldehyde in CGBS. Artemia larvae were
fixed with 3.7% formaldehyde in NH (0.5-M NaCl, 0.1-M
HEPES, pH 7.5) after brief sonication. Achaearanea embryos
were fixed in a two-phase solution of heptane and 5.5% formaldehyde in PEM (100-mM PIPES, 1-mM EDTA, 2-mM
MgSO4, pH 6.9), followed by removal of the vitelline membrane. Asterina and Ptychodera gastrulae were fixed with
3.7% formaldehyde in NH. After fixation, the samples were
washed with phosphate-buffered saline (PBS) with 0.1%
Tween-20 (PBS-T) followed by gradual replacement with ethanol. They were then stored at 201C until use. For antibody staining, samples were dehydrated, blocked with 5%
skim milk in PBS-T and then incubated with primary antibody overnight at 41C. The anti-cadherin antisera were used
at a dilution of 1:200 or 1:400, and secondary antibodies
labeled with Cy3 or fluorescein isothiocyanate (Chemicon,
Temecula, CA, USA) were used at a dilution of 1:200.
YOYO-1 iodide (Molecular Probes, Eugene, OR, USA) was
used to stain the nuclei in Asterina gastrulae. This staining
was performed as follows. After antibody staining, embryos
were treated with RNaseA (1 mg/ml) in PBS-T for 30 min at
room temperature, washed with PBS three times, and incubated with YOYO-1 iodide in PBST at a concentration of
0.1 mM. To stain Ptychodera b-catenin, a commercially available rabbit antiserum raised against the C-terminal site of
human and mouse b-catenin (C2206; Sigma, St. Louis, MO,
USA) was used. Stained samples were examined using a Zeiss
Axiophot II equipped with a Bio-Rad laser confocal system
(MRC1024).
To reconstruct the evolution of the cadherin form, our analysis initially focused on the phylum Arthropoda. We obtained
new data from three hexapod, three crustacean, and one chelicerate species, and combined these data with publicly available data for other species (Table 1). From each of the
hexapods and a branchiopod, two types of cadherin cDNA
were cloned. From the isopod, decapod, and chelicerate, only
one type of cadherin cDNA was cloned. The amino acid sequences of the CP domains were then analyzed using the
neighbor-joining method (Fig. 2A) and the maximum parsimony method (Fig. 2B). All the arthropod sequences were
grouped separately from other animal sequences and were
further divided into two groups: one containing DE-cadherin
and the other DN-cadherin. To examine the correlation between the patterns of amino acid substitutions in the CP domains and the extracellular domain organizations (A1 or A2),
the entire amino acid sequences of Gb1-, Fc1-, Af1-, Af2-, Le, Cj-, and At-cadherin were determined. Gb1-, Fc1-, and Af1cadherin, whose CP domains bear DE-type sequences (Fig. 2,
A and B), have A1-form extracellular domain organization,
while Af2-, Le-, Cj-, and At-cadherin, whose CP domains
bear DN-type sequences (Fig. 2, A and B), have A2-form
domain organization. Although only partial cDNA information was obtained for Bm1-, Gb2-, and Fc2-cadherin, it was
made sure that Bm1-cadherin (DE-type; Fig. 2, A and B) has
an A1-type PCCD complex, and that Gb2- and Fc2-cadherin
(DN-type; Fig. 2, A and B) have an A2-type PCCD complex.
Moreover, BLAST-based domain comparisons confirmed the
conservation of each domain organization (Fig. 3). These results suggest that the states of the A1 and A2 forms were
evolutionarily stable in arthropods.
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EVOLUTION & DEVELOPMENT
Vol. 7, No. 5, September^October 2005
Fig. 2. Molecular phylogenetic analyses of the cadherins using the neighbor-joining (A, C) and maximum parsimony (B, D) methods.
Unrooted trees based on the cytoplasmic (CP) domains of cadherins (A, B) and the extracellular regions of the arthropod A2-form
cadherins (C, D). The numbers at nodes indicate bootstrap values (%).
The change from the A2/E2- to the A1-form can be
explained by a loss of specific domains
Next, to examine which form is ancestral, A1 or A2, we
compared the arthropod cadherins with the cadherins that
were present outside the phylum Arthropoda. The A2 form
displayed almost the same configuration as the echinoderm
E2 form, as exemplified by the starfish sequence (Oda et al.
2002). To determine whether this resemblance is the result of
conservation or convergence, we performed BLAST-based
domain comparison analysis between the A2- and the E2form cadherins (Fig. 4, A,B). This analysis revealed that
domains at the same positions tended to be more similar to
each other than to those at other positions. These collinear
Oda et al.
Evolution of adherens junction cadherin
A
381
B
C
D
E
Fig. 3. BLAST-based domain comparisons of the arthropod cadherins. Comparisons are shown between the following pairs of cadherins:
Gb1- and DE-cadherin (A), Af1- and DE-cadherin (B), Cj- and DN-cadherin (C), Af2- and Cj-cadherin (D), and At- and Cj-cadherin (E).
The amino acid sequence of each domain in the extracellular region of one cadherin (SEQ1) was blasted against the entire amino acid
sequence of another cadherin (SEQ2). The E-values (Eo20) are plotted on the matrix. The lowest and second lowest E-values in each row
indicating the most significant matches between domains are highlighted with solid and dotted lines, respectively.
382
EVOLUTION & DEVELOPMENT
A
SEQ1
Cj
B
C
D
SEQ2
Ap
NT EC1 EC2 EC3 EC4 EC5 EC6 EC7 EC8 EC9
NT 0.20
EC1
6.4
3.7 0.12
EC2
8.3
EC3
EC4
2e-07
EC5
6.4 3.7 3e-06 19
0.018 0.26
EC6
19
8e-06 2.2 3e-04
EC7
0.26 7e-07 0.003
EC8
0.014
0.008 1.7
7e-15
EC9
1e-04
4.9 4.9
0.023
EC10
EC11
0.98
0.068
EC12
19
0.58 0.068 7e-05
EC13
1.7 0.34 3e-07
EC14
0.068
0.34 0.011
EC15
0.014
EC16
0.26 11
1e-05
EC17
NC
CE1
LG1
CE2
LG2
CE3
SEQ2
SEQ1
At
Vol. 7, No. 5, September^October 2005
NT
NT
EC1
EC2
EC3
EC4
EC5
EC6
EC7
EC8
EC9
EC10
EC11
EC12
EC13
EC14
EC15
EC16
EC17
NC
CE1
LG1
CE2
LG2
CE3
EC10 EC11 EC12 EC13 EC14 EC15 EC16 EC17 NC
6.4
0.052 3e-04
CE1 LG1 CE2 LG2 CE3
3.7
4.9
0.15
5e-06 2e-06 0.018 0.003 14
0.34
0.001 7e-04
11
2.2 0.12 0.014 2.9
1e-05 0.44 3e-08 3e-08 5e-06 5e-07 0.089
0.005 0.04 0.089
3e-05
3e-09
2.2
0.34 0.008 3e-04
2e-09 0.001 0.031 2e-05
0.26
7e-05 0.58 1e-17 6e-04 2e-04 1e-08 2.9
0.15 2e-04 1e-13 0.34 0.15 0.15
0.26 1e-04 0.018 0.031 3e-10
0.008
0.005
0.98
0.15
0.068 0.005 0.15 9e-10
2.9 8e-09
6.4
8e-27
2e-13
8e-06
7e-31
0.34
5e-10
0.04
Ap
EC1 EC2 EC3 EC4 EC5 EC6 EC7 EC8 EC9 EC10 EC11 EC12 EC13 EC14 EC15 EC16 EC17 NC
4.9
1e-13
CE1 LG1 CE2 LG2 CE3
2e-05 1.6
18
5e-05
8e-05
0.038
0.93
0.002 13
18
18
0.42 10
1e-04
0.084
13
2e-05 0.19
3e-12
2.1 0.003
10
10
1e-08
0.049
0.24
5e-05 0.013 2e-05
0.064
8e-05
1e-04
0.14
0.24
8e-10 0.71
0.084
18
0.24 0.029
0.01 0.32 1e-15
0.038 9e-07
0.006
0.049 0.008
2.1
3e-04
4.6 0.01
2e-10 4.6 4.6
3.5
18
6e-05
10
0.71 3e-08
2.7 3e-07
4e-29
3e-13
0.002
3e-31
0.005
2e-16
0.014
Ap
NT EC1 EC2 EC3 EC4 EC5 EC6 EC7 EC8 EC9
NT 0.001
EC1
0.005
EC2
3e-13
6.3
EC3
2e-16
EC4
8e-14
EC5
2e-04
2e-06
7e-05 8.3
EC6
0.052
11
0.002 5e-10 0.010
EC7
0.014
0.068 1.7
4e-15 1.3
LvG EC8
EC9
0.20
EC10
0.20 0.003 1.3 4e-05
EC11
0.12 0.010 11
EC12
2e-05
0.15 6e-06
EC13
18
18
18
0.75 0.34 2.9
EC14
18
0.002
0.98
EC15
11
EC16
NC
0.63
CE1
LG1
CE2
LG2
CE3
5e-15
SEQ2
SEQ1
SEQ2
SEQ1
Ap
EC1
NT
EC1 0.18
EC2
EC3
EC4
EC5
EC6
EC7
EC8
EC9
EC10
EC11
EC12
EC13
EC14 17
EC15
EC16
EC17
NC
CE1
LG1
CE2
LG2
CE3
EC10 EC11 EC12 EC13 EC14 EC15 EC16 EC17 NC
2.8
14
0.004
14
2.8
0.023 0.12
1e-06 0.002
11
0.75 1.3 11
2.2 0.052 0.023 9e-07 0.001 0.018 1e-09
8.3
7e-07
0.98 0.004
4e-04
4e-05 0.34 1e-05 2e-05
1e-10 0.34
0.004
0.34 2e-04 2.2 2e-14 3e-04 0.34 0.089 2.2
0.12 0.018 0.15 0.010
0.26
0.26
2e-14 1e-05
0.003
6.4 11
1e-06
3.7 3e-06
3e-52
3e-20
0.009 0.002
0.002
5e-46
2.4
4e-04
5e-46
6e-04
cHz
EC2 EC3 EC4 EC5 EC6 EC7 EC8 EC9 EC10 EC11 EC12 EC13 EC14 EC15 NC
7.4
CE1 LG1 CE2 LG2 CE3
1.7
0.012 3.3
9.7
6e-04
0.20
2e-18
CE1 LG1 CE2 LG2 CE3
0.079
6e-07 17
0.004
5e-04
0.10
0.016 0.51
0.10 6e-04
1.5
17
2e-06
0.18 2.6
0.18 0.88 0.021
0.10 1e-05 0.67
0.67 0.016 0.079 5.7 17
0.002 0.10 3e-11 0.012 0.047 7e-09 3e-04 3e-07 0.14 0.021 3e-05
0.14
0.67
3e-08 0.14
0.88
9e-06 5.7
3e-10 0.0022e-07 0.027
0.016 0.51 0.39 0.007 0.14
0.002 3e-12 0.88 0.036
3e-07
7.4
1.5
5e-09
0.51 2.0
1e-04 2.6 1.1 3e-05 1e-04 8e-05 1e-10 0.10 2e-06
4.4 8e-05
1e-05
2.0 0.88 4e-06 0.036
4e-04 4e-05
0.001 7.4 7e-06
0.009 8e-04
0.30 0.10 0.88 3e-12 0.18
0.18
2.0
0.30
4e-05
7e-26
8e-05
0.18
2e-26
0.88
3e-14
0.002
8e-12
Fig. 4. BLAST-based domain comparisons of the arthropod, echinoderm and vertebrate cadherins. Comparisons are shown between the
following pairs of cadherins: Cj- and Ap-cadherin (A), At- and Ap-cadherin (B), LvG- and Ap-cadherin (C), and Ap- and cHz-cadherin
(D). Data are shown in the same manner as those in Fig. 3.
Oda et al.
similarities suggest that the resemblance between the A2 and
E2 form is the result of conservation rather than convergence.
In addition, the A2/E2 form displays strong similarities to the
sea urchin E1 form (Miller and McClay 1997) and the chick
V2 form (Tanabe et al. 2004). BLAST-based domain comparisons confirmed that these similarities are also because of
conservation (Fig. 4, C and D). The E1 form differed from the
A2/E2 form only in that the E1 form lacked one EC domain
corresponding to the EC2 domain of the A2/E2 form (Fig.
4C). The domains corresponding to the NT, EC2, and EC3
domains of the A2/E2 form were not detected in the V2 form
(Fig. 4D). Moreover, cadherins bearing A2/E2-type PCCD
complexes also exist in a mollusk and a hemichordate (Table
1; Oda et al. 2002). Thus, the A2/E2-related characters are
widely distributed among bilaterians. As revealed in the
neighbor-joining and maximum parsimony trees based on the
CP domains (Fig. 2, A and B), the phylogenetic occurrences
of the cadherins bearing the A2/E2-related characters are not
necessarily correlated with the patterns of amino acid substitutions. Together, these conditions strongly suggest that the
A2/E2 form has an origin close to or possibly prior to the
origin of bilateria.
In contrast to the scattered distribution of the A2/E2 form
and its closely related forms, the A1 form is restricted to a
subset of Arthropoda, indicating a more recent origin. To
elucidate the origin of the A1-form domain organization, we
performed BLAST-based domain comparison analysis between the A1- and the A2/E2-form cadherins (Fig. 5, A and
B). This revealed that the EC1–EC6 domains, the EC7 domain, and the NC, CE, and LG domains of the A1 form are
homologous to the EC8–EC13 domains, the EC17 domain,
and the NC, CE1, and LG1 domains of the A2 form, respectively. Assuming that the A2/E2 form is ancestral and
that the A1 form is derived, the change from A2/E2 to A1 can
be explained by a simple and likely event in which the three
separate extracellular parts of the A2/E2 form (i.e., the NT
domain, various EC domains, and the C-terminal domains of
the PCCD complex) were lost. In contrast, domain duplica-
Evolution of adherens junction cadherin
383
tions that might explain the change from A1 to A2/E2 are not
supported (Fig. 5, A and B).
The two distinct cadherin forms and their
expression modes define the ‘‘ancestral’’ and
‘‘derived’’ groups of arthropods
To investigate the polarity of cadherin evolution from a functional perspective, we raised antibodies against some of the
arthropod A1- and A2/E2-form cadherins as shown in Fig. 6
and its legend denoting the specificity of the antibodies, and
determined which cadherins are involved in AJ formation in
the epithelia of developing embryos. Similar to that reported
for DE-cadherin (Oda et al. 1994), Gb1- and Af1-cadherins
were exclusively expressed in epithelial tissues, where they
were concentrated at the AJs (Fig. 7, A and C). In contrast,
Gb2- and Af2-cadherins, like DN-cadherin (Iwai et al. 1997),
were not expressed in epithelial tissues but were found in
mesenchymal tissues (Fig. 7, B and D). The expression patterns in the cricket and Artemia were similar to that of Drosophila. Cj-, Le- and At-cadherins were expressed in epithelial
tissues, where the proteins were localized to the AJs (Fig. 7, E,
F, and H). The same cadherins were also expressed in mesenchymal tissues (Fig. 7, G and I). The expression patterns in
the malacostracan crustaceans and the chelicerate were different from those of the hexapods and branchiopod, but were
similar to those of the echinoderm sea urchin and starfish. It
was reported that the E1-form LvG-cadherin is expressed in
all epithelial cells of the sea urchin gastrula and is localized at
the AJs, and that its transcripts are present in all cells of the
embryo throughout gastrulation including both primary and
secondary mesenchyme (Miller and McClay 1997). An antiserum that we raised against the A2/E2-form Ap-cadherin
stained the AJs in all ectodermal and endodermal epithelial
cells of the starfish gastrula (Fig. 7J), although no concentrated signals were detected in mesenchymal cells of the same
gastrula (Fig. 7, J and K; arrows). Whether these mesenchymal cells expressed Ap-cadherin at weak levels was
A
B
Fig. 5. BLAST-based
domain
comparisons of the arthropod
A1- and A2-form cadherins. Comparisons are shown between the
following pairs of cadherins: Gb1and Cj-cadherin (A), and Af1- and
At-cadherin (B). Data are shown
in the same manner as those in
Fig. 3.
384
EVOLUTION & DEVELOPMENT
Vol. 7, No. 5, September^October 2005
Fig. 6. Tests for antibody specificity. (A) Western blot analysis. The embryonic or larval lysates and the antibody used to probe the lysate
are indicated at the top of each lane. The antibodies recognized proteins with sizes similar to DE- or DN-cadherin. The antibody raised
against Le-cadherin reacted with a protein in the lysate of Caridina, which was likely to be Cj-cadherin. (B) Schematic illustrations showing
the cadherin regions used for the construction of six GST fusion proteins (boxes 1–6). Box 1 is the same as that used for the production of
the anti-Le-cadherin antibody. (C) The six GST-fusion proteins (lanes 1–6) were separated by SDS-PAGE, blotted onto nitrocellulose, and
probed with an antibody against GST (top panel) or the anti-Le-cadherin antibody (bottom panel). The anti-Le-cadherin antibody crossreacted with the GST fusion proteins for Cj- and DN-cadherins. (D, E) Drosophila embryos stained with the anti-Le-cadherin antibody.
Patterns of expression characteristic of DN-cadherin were observed, indicating that the anti-Le-cadherin antibody cross-reacted with DNcadherin in situ. The data indicate that the anti-Le-cadherin antibody recognizes a wide range of crustacean and hexapod A2-form
cadherins with no reaction with the A1-form cadherins.
unclear. Taken together, these observations suggest that the
ubiquitous or epithelial expression of A2/E2-form cadherin
is ancestral, whereas the mesenchymal expression of A2/E2form cadherin is derived. In agreement with this suggestion,
the A2/E2-form cadherins with the ancestral mode of expression and those with the derived mode of expression were
separated in the phylogenetic trees based on amino acid substitutions in the CP domains (Fig. 2, A and B) and the ex"
Fig. 7. Expression of various cadherins in the epithelial and/or mesenchymal tissues of developing animals. (A, B) Embryos of Gryllus
bimaculatus were stained with anti-Gb1-cadherin (A) or anti-Le-cadherin antibody (B). The anti-Le-cadherin antibody reacted with Cj- and
DN-cadherins (see Fig. 6) and possibly with Gb2-cadherin. The staining patterns are consistent with expression patterns determined by
whole mount in situ hybridization with probes prepared for Gb1- and Gb2-cadherin transcripts (data not shown). (C, D) Larvae of Artemia
franciscana were stained with anti-Af1-cadherin (C) or anti-Af2-cadherin (D) antibody. (E–G) An embryo of Ligia exotica (E) and embryos
of Caridina japonica (F, G) were stained with anti-Le-cadherin antibody. (H, I) An embryo of Achaearanea tepidariorum stained with antiAt-cadherin antibody is shown with the focal planes adjusted to the ectodermal layer (H) and to the mesodermal layer (I). (J, K) A gastrula
of Asterina pectinifera was double-stained with anti-Ap-cadherin antibody (J) and YOYO-1 iodide (K). Small arrowheads indicate mesenchymal cells located between the ectoderm and endoderm layers. (L–O) A gastrula of Ptychodera flava was double-stained with anti-Pf1cadherin (L, N) and b-catenin (M, O) antibodies. Surface (L, M) and sagittal (N, O) views of the gastrula are shown. Arrowheads indicate
the epithelial cells enclosing the protocoel (asterisks). CNS, central nervous system; ect, ectoderm; mes, mesoderm; end, endoderm; bp,
blastopore; m, future mouth; a, future anus; h, hydropore. Scale bars 20 mm.
Oda et al.
tracellular regions (Fig. 2, C and D). For the animals in which
the A2/E2-form cadherins show the mesenchymal expression,
A1-form cadherins were found to be present and involved in
AJ formation in the epithelial tissues. These lines of evidence,
combined with the localized distribution of the A1 form in the
Evolution of adherens junction cadherin
385
subset of Arthropoda and the structural relationship between
the A2/E2 and the A1 form, define the ‘‘ancestral’’ and ‘‘derived’’ groups of arthropods. Thus, it appears that the derived
arthropods arose through an event in which the A1 form
replaced the A2/E2 form in the epithelial tissues.
386
EVOLUTION & DEVELOPMENT
Vol. 7, No. 5, September^October 2005
Independent reductive changes from the A2/E2
form generated the A1, V1, and C forms
The conservation of cadherin forms between Arthropoda and
Echinodermata, which are generally considered to be phylogenetically distant from each other (Erwin and Davidson
2002; Brusca and Brusca 2003), suggests that the AJs in the
epithelia of the last common arthropod-echinoderm ancestor
used the A2/E2 form of cadherin. We considered a possibility
that the variations in cadherin form might have arisen in
descendants of the last common arthropod–echinoderm ancestor, as exemplified by the A1 form in the arthropod lineage. To evaluate this possibility, we examined the V1-form
cadherins, which are found only in vertebrates and urochordates. The topology of the trees based on amino acid substitutions in the CP domains (Fig. 2, A and B), as well as the
detected collinear similarities between the five EC domains of
the V1-form cadherins (Fig. 8, A–E), favor a single origin for
the V1 form. BLAST-based domain comparisons revealed the
five EC domains of some V1-form cadherins to be collinearly
similar to the C-terminal five EC domains of the A2/E2- and
V2-form cadherins (Fig. 8, F–H). These observations imply
that the V1 form may have evolved from the A2/E2 form
through a reductive change similar to, but distinct from, the
change that gave rise to the A1 form (Fig. 9). Interestingly,
this explanation is also applicable to the C form, identified
only in the cephalochordate (Oda et al. 2002, 2004), the H
form, identified only in the hemichordate (Oda et al. 2002),
and the C. elegans HMR-1 cadherin (Costa et al. 1998). As
shown in Fig. 9 and our previous study (Oda et al. 2002), all
the EC domains and the NC domain may have been lost to
result in the C form and nine of the 17 EC domains and a part
of the CE3 domain to result in the H form. The reductive
changes in cadherin form could have occurred in descendants
of the last common arthropod–echinoderm ancestor to generate the observed diversity. Judging from the relationships
between the inferred reductive changes, it is likely that the A1,
V1, and C forms, and possibly other reduced forms arose
independently from the ancestral state. However, the transitions from H to V1 and from E1 to V1, C and A1 cannot be
ruled out. The independent origins of the reduced forms are
also supported by the mutually exclusive distributions of the
reduced forms among bilaterians and the patterns of amino
acid substitutions in the CP domains (Fig. 2, A and B), although the sampling of taxa is limited at this stage of research.
Diversification of the epithelial AJs with changes
in cadherin form
It has been reported that the V1 and C forms, like the A1
form, are localized to AJs in the epithelia (Takeichi 1988; Oda
et al. 2002, 2004). Although there has been no immunohistochemical analysis performed on the urochordate V1-form
cadherins, the sequenced Ciona intestinalis genome revealed
that this urochordate species possesses no cadherins except
two V1-form cadherins which correspond to the vertebrate
types I and II cadherins (Takeichi 1995; Sasakura et al. 2003).
The chick V2-form cHz-cadherin is expressed in only limited
cell populations (Tanabe et al. 2004). Although V2-like cadherin genes are also present in fish genomes (Tanabe et al.
2004), no expression data have been reported. In this study, to
examine the tissue distribution and subcellular localization of
the H-form Pf1-cadherin in Ptychodera embryos, we raised an
antiserum against Pf1-cadherin. This antiserum stained the
apical cell–cell contact sites of epithelial cells located at the
surface and the inside of the Ptychodera gastrula (Fig. 7, L
and N), where the cadherin appeared to be colocalized with bcatenin (Fig. 7, M and O). However, no concentrated signals
for Pf1-cadherin were detected in the epithelial cells enclosing
the protocoel despite the observation that strong concentrations of b-catenin were detected in the cells (Fig. 7, N and O;
arrowheads). These observations indicate that the H-form
Pf1-cadherin, like the A1 form in the insects, may be involved
in AJ formation in the ectodermal and endodermal epithelia
of the Ptychodera embryo. Together, these imply that each of
the reduced forms of cadherin was formed to replace the ancestral form in epithelial tissues. It is suggested that the cadherin form was altered at multiple separate points during
early bilaterian evolution to diversify the epithelial AJs.
DISCUSSION
In this study, we attempted to reconstruct the evolution of
classic-type cadherin. The Hennigian method was applied to
the extracellular domain organizations of cadherin molecules.
The two-sequence BLAST tool was used to detect the homologous parts of different cadherins. In our analyses of arthropod and deuterostome cadherins, shifts in the structural
state of cadherin were detected as rare changes in ancient
genomes (Rokas and Holland 2000), allowing us to deduce
that the A2/E2 form is ancestral. In potential out-groups of
bilaterians, no classic-type cadherin genes have been reported.
Whether any other form that is more primitive than the A2/
E2 form exists in extant metazoans remains unknown. A
theoretically important point of this study is that the events
leading to the likely derived forms of cadherin can be explained by independent reductive changes from the ancestral
state. The validity of this generalization should be tested
through further accumulation of information about cadherin
genes in the genomes of bilaterians and other metazoans.
Phylogenetic hypotheses for deep relationships
among bilaterians
Our immunohistochemical data indicated that the epithelia of
different taxa have the AJs constituted by different forms of
Oda et al.
Evolution of adherens junction cadherin
A
B
D
E
387
C
F
G
H
I
J
Fig. 8. BLAST-based domain comparisons of the vertebrate, urochordate, hemichordate, and other cadherins. Comparisons are shown
between these pairs of cadherins: mE- and mN-cadherin (A), m8- and mN-cadherin (B), Ci1- and mN-cadherin (C), Cs2- and m8-cadherin
(D), Ci1- and Cs2-cadherin (E), mN- and Ap-cadherin (F), mN- and At-cadherin (G), Cs2- and cHz-cadherin (H), Pf1- and Ap-cadherin (I),
and Pf1- and cHz-cadherin (J). Data are shown in the same manner as those in Fig. 3.
388
EVOLUTION & DEVELOPMENT
Vol. 7, No. 5, September^October 2005
Fig. 9. A schematic diagram showing inferred reductive changes
from the ancestral A2/E2 form of cadherin that gave rise to the
various derived forms of cadherin. This study suggests that the
regions indicated by black lines were lost to generate each derived
form. The broken line indicates an ambiguous lost region.
cadherin. Interestingly, the derived forms of cadherin appear
preferentially in the epithelia outlining the developing embryo.
We consider a possibility that changes in the mode of epithelial cell–cell adhesion might have contributed to the separation of animal lineages particularly during early stages of
bilaterian evolution. Drastic deletion mutations in a cadherin
gene that not only retained homotypic cell–cell recognition
and adhesion function of the cadherin but also altered its
adhesion specificity might have led to the appearance of a new
independent population of multicellular organisms whose epithelial cells at their surface were connected by the mutated (or
derived) cadherin. We also speculate that the adhesion alterations might have affected the cell functions and cell behaviors involved in morphogenesis of early embryos, and
offered rare opportunities to drastically change body plans.
Although the sampling of taxa is limited at this stage of
research, we hypothesize that the reductive events in cadherin
structure related to AJ formation in the epithelia may possess
information about bilaterian relationships as molecular
synapomorphies. Assuming that the state in which the A2/
E2 form of cadherin functions at AJs in the epithelia is ancestral, the presence of each reduced form of cadherin at the
epithelial AJs may be a molecular synapomorphy. The A1
form may indicate a clade including extant hexapods and
branchiopods, and the V1 form a clade including extant vertebrates and urochordates. These potential molecular
synapomorphies favor the phylogenetic hypotheses in which
hexapods are more closely related to branchiopods than to
malacostracan crustaceans, and vertebrates are more closely
related to urochordates than to cephalochordates. The validity of these phylogenetic hypotheses depends on the completeness of information on the expression and subcellular
localization of cadherins in the phyla. In this respect, expression data for the fish cadherin genes with A2/E2-like forms
(Tanabe et al. 2004) are important, but these have not been
reported.
The relationships we propose conflict with those that have
been proposed by others based on nucleotide substitutions and
morphology (Turbeville et al. 1994; Wada and Satoh 1994;
Cameron et al. 2000; Wilson et al. 2000; Giribet et al. 2001;
Nardi et al. 2003). However, it should be noted that in such
conventional phylogenetic analyses, different substitution rates
among sites and lineages (Aguinaldo et al. 1997; Abouheif et
al. 1998), saturation of mutations at variable sites (Philippe
and Laurent 1998) and convergent evolution and secondary
simplifications in morphology (Jenner 2004) might lead to
misleading results particularly in attempts to resolve deep relationships. Taking these into account, our phylogenetic hypotheses based on the cadherin forms are worth testing.
The arthropod relationships we propose are consistent
with the idea that hexapods are regarded as part of Crustacea
(Boore et al. 1998; Garcia-Machado et al. 1999; Giribet et al.
2001). A recent finding that neural crest-like cells are present
in a urochordate species (Jeffery et al. 2004) has made the
chordate relationships we propose attractive. Also, the sistergroup relationship between vertebrates and urochordates may
be favored with the shared possession of tight junctions in the
epithelia (Lane et al. 1994; Kollmar et al. 2001; Tsukita et al.
2001; Sasakura et al. 2003). Although the somites are generally considered to be one of the most important synapomorphies linking vertebrates and cephalochordates, if one assumes
that the segmentation of vertebrates is homologous to that of
arthropods (De Robertis 1997; Tautz 2004), secondary loss of
the metamerism may account for the urochordate condition.
Our hypotheses need to be further tested. Identification
and characterization of a more extensive array of cadherin
genes present in bilaterians and potentially nonbilaterian
metazoans are required to prove the rarity of cadherin form
changes and the mutually exclusive distributions of structurally reduced cadherins. Finally, a continued effort to integrate
data from cell biology, developmental biology, genomics and
paleontology is essential to validate our hypotheses.
Acknowledgments
We would like to thank N. Sumida, K. Kimura, E. Shoguchi and H.
Wada for animals; H. Uemiya for identification of the collembolan
species; M. Iwami for cDNA libraries; and two anonymous reviewers
for helpful comments on the manuscript. We are also grateful to S.
Tsukita for supervision; K. Nakamura for encouragement; Z. H. Su
for technical advice with molecular phylogenetic analysis; K. Tanabe,
S. Nakagawa, and M. Takeichi for sharing data before publication;
and M. Irie, M. Okubo, S. Okajima, and A. Noda for technical
assistance.
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