Diversification of epithelial adherens junctions with ... of epithelial adherens junctions with...

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

aJT Biohistory Research Hall, 1-1 Murasaki-cho, Takatsuki, Osaka 569-1125, JapanbTsukita Cell Axis Project, ERATO, Japan Science and Technology Corporation, Kyoto, JapancKewalo Marine Laboratory, Pacific Biomedical Research Center, University of Hawaii, 41 Ahui Street,

Honolulu, HI 96813-5511, USAdPRESTO, Japan Science and Technology Agency, Saitama, Japan�Author for correspondence (email: [email protected])

1Present address: Marine Biological Laboratory, Graduate School of Science, Hiroshima University, 2445 Mukaishima, Onomichi, Hiroshima 722-0073, Japan.

SUMMARY The adherens junction (AJ) is the mostuniversal junction found in bilaterian epithelia and mayrepresent one of the earliest types of cell–cell junctions. Theadhesion molecules responsible for forming AJs are theclassic cadherins (referred to simply as cadherins), whoseextracellular domain organization displays marked varietyamong species examined so far. In this study, we attempted toreconstruct the evolution of cadherin by analyzing new datafrom several arthropods (two insects, one noninsect hexapod,three crustaceans, and one chelicerate) and previouslypublished sequences for Drosophila melanogaster and otheranimals. The results of comparative analyses using theBLAST tool and immunohistochemical analyses revealedthat the extracellular domain organizations of a decapod, an

isopod, a spider, and a starfish cadherin, which are present atAJs in the embryonic epithelia are homologous. Independentreductive changes from the ancestral state were evidentin the epithelia of hexapods1branchiopod, vertebrates1

urochordates, and a cephalochordate. The form of cadherinsin hexapods is more closely related to that of a branchiopodthan to that of malacostracan crustaceans, and one of those ofvertebrates is more closely related to that of urochordatesthan to that of a cephalochordate. Although the sampling oftaxa is limited at this stage of research, we hypothesize thatthe reductive events in cadherin structure related to AJformation in the epithelia may possess information aboutbilaterian relationships as molecular synapomorphies.

INTRODUCTION

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 single-

pass transmembrane proteins with homotypic cell–cell adhe-

sion 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

indicate members of the classic cadherin family. The extra-

cellular structure of cadherin bridges the extracellular space

between neighboring cells via cis- and trans-interactions

(Gooding et al. 2004), whereas the intracellular structure pro-

vides an anchor for scaffolding the actin cytoskeleton via the

catenins (Gumbiner 2000). Cadherin functions are essential

for a variety of morphogenetic processes in bilaterian devel-

opment (Takeichi 1995; Tepass et al. 2000).

Despite the conservation of the CP domains, the extracel-

lular 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. How-

ever, it is not universal among bilaterians. In the extracellular

regions of known nonchordate cadherins, three different do-

main types in addition to the EC domain have been identified:

EVOLUTION & DEVELOPMENT 7:5, 376–389 (2005)

& BLACKWELL PUBLISHING, INC.376

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 nonchor-

date 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. elegansHMR-

1 cadherin, are localized to the epithelial AJs (Takeichi 1988;

Oda et al. 1994, 2002, 2004; Miller andMcClay 1997; Costa et

al. 1998). Different forms of cadherins are involved in AJ

formation in the epithelia of different animals. How this di-

versity 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 com-

parative 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 Educa-

tion, 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 Univer-

sity. Artemia franciscana (Tetra), Caridina japonica (Masuda,

Kyoto, Japan), and Folsomia candida (Spheroaqua, Hama-

matsu, 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 Quick-

Prep Micro mRNA purification kit (Amersham Biosciences,

Table 1. List of classic cadherins used in this study

Species

Name of

cadherin Form

Accession

no.

Arthropoda, Hexapoda, Diptera

Drosophila melanogaster DE-cadherin A1 BAA05942

DN-cadherin A2 T00021

Arthropoda, Hexapoda, Lepidoptera

Bombyx mori Bm1-cadherin1 ND AB1902943

Bm2-cadherin1 ND AB1902933

Arthropoda, Hexapoda, Orthoptera

Gryllus bimaculatus Gb1-cadherin A1 AB1902953

Gb2-cadherin1 ND2 AB1902963

Arthropoda, Hexapoda, Collembola

Folsomia candida Fc1-cadherin A1 AB1902973

Fc2-cadherin1 ND2 AB1902983

Arthropoda, Crustacea, Branchiopoda

Artemia franciscana Af1-cadherin A1 AB1902993

Af2-cadherin A2 AB1903003

Arthropoda, Crustacea, Decapoda

Caridina japonica Cj-cadherin A2 AB1903013

Arthropoda, Crustacea, Isopoda

Ligia exotica Le-cadherin A2 AB1903023

Arthropoda, Chelicerata, Arachnida

Achaearanea tepidariorum At-cadherin A2 AB1903033

Mollusca, Bivalvia

Saccostrea echinata Se-cadherin1 ND2 AB075367

Echinodermata, Echinoidea

Lytechinus variegatus LvG-cadherin E1 U34823

Echinodermata, Asteroidea

Asterina pectinifera Ap-cadherin E2 AB075365

Hemichordata, Enteropneusta

Ptychodera flava Pf1-cadherin H AB075368

Pf2-cadherin1 ND2 AB075369

Chordata, Cephalochordata

Branchiostoma belcheri Bb1-cadherin C AB075366

Bb2-cadherin C AB120427

Chordata, Urochordata, Ascidiacea

Ciona intestinalis Ci1-cadherin V1 AB031540

Ciona savignyi Cs2-cadherin V1 AB057736

Botryllus schlosseri BS-cadherin V1 U61755

Chordata, Vertebrata, Teleostei

Danio rerio Dr1-cadherin V1 NP571895

Danio rerio Dr2-cadherin V1 AAN61915

Chordata, Vertebrata, Mammalia

Mus musculus mE-cadherin V1 X06115

mN-cadherin V1 M31131

m6-cadherin V1 NP031692

m8-cadherin V1 NP031693

m10-cadherin V1 AAL67951

m11-cadherin V1 D31963

Chordata, Vertebrata, Aves

Gallus gallus cHz-cadherin V2 AY312363

1Only partial cDNA information is available.2The partial cDNA information indicates that an A2/E2-type PCCD

complex is present.3Identified in this study.

ND, not determined; PCCD, primitive classic cadherin domain.

Evolution of adherens junction cadherin 377Oda et al.

Piscataway, NJ, USA). cDNA was synthesized using a Time-

Saver 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 li-

braries. 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 fol-

lows: IN1, 50-ATHAAYTAYGAIGAIGARGGIGG-30; KL2,

50-CCRTACATRTCIGCIARYTT-30; HY2, 50-CCRTCICC-

YTCRTAIGCRTARTG-30; and NYA1, 50-AAYTAYGCN-

TAYGARGG-30(where H5A, C, or T; Y5C or T; I5

inosine; R5A or G; and N5A, 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 5min; 1

cycle of 941C for 40 sec, 551C for 2min 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 7min; 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 us-

ing the PCR DIG probe synthesis kit (Roche Diagnostics,

Mannheim, Germany), and the resulting signals were visual-

ized 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 two-

sequence 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 PHY-

LIP version 3.5 (Felsenstein 1993) and by the maximum par-

simony 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-

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

Fig. 1. The domain organizations of classic cadherins from ar-thropods (A1, A2), echinoderms (E1, E2), a hemichordate (H), acephalochordate (C), urochordates, and vertebrates (V1, V2) arediagrammed. Repeated domains are numbered from the N-termi-nus. The domain types are indicated at the bottom: NT, N-terminaldomain; EC, extracellular cadherin domain; NC, nonchordate-spe-cific domain; CE, cysteine-rich EGF-like domain; LG, lamininglobular-like domain; TM, transmembrane domain; CP, cytoplas-mic domain.

378 EVOLUTION & DEVELOPMENT Vol. 7, No. 5, September^October 2005

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-cad-

herin, aa171–aa751; Ap-cadherin, aa187–aa761; and Pf1-

cadherin, 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 immu-

nized 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 Biosci-

ences). The polypeptides contained in the fusion proteins were

as follows: Le-cadherin, aa193–aa792, aa957–aa1555; Cj-

cadherin, aa170–aa785; DN-cadherin, aa263–aa868; DE-

cadherin, 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.

Antibody staining

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% for-

maldehyde in PEM (100-mM PIPES, 1-mM EDTA, 2-mM

MgSO4, pH 6.9), followed by removal of the vitelline mem-

brane. 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 eth-

anol. They were then stored at � 201C until use. For anti-

body staining, samples were dehydrated, blocked with 5%

skim milk in PBS-T and then incubated with primary anti-

body 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 (1mg/ml) in PBS-T for 30min at

room temperature, washed with PBS three times, and incu-

bated with YOYO-1 iodide in PBST at a concentration of

0.1mM. To stain Ptychodera b-catenin, a commercially avail-

able 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).

RESULTS

Designations of the cadherin domainorganizations

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 indi-

cating the phylum or subphylum (e.g., A for Arthropoda) and

a number (Fig. 1). The form represented by mE- and mN-

cadherins 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-cad-

herin was H.

The states of the A1 and A2 forms wereevolutionarily stable in arthropods

To reconstruct the evolution of the cadherin form, our anal-

ysis initially focused on the phylum Arthropoda. We obtained

new data from three hexapod, three crustacean, and one che-

licerate species, and combined these data with publicly avail-

able 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 se-

quences of the CP domains were then analyzed using the

neighbor-joining method (Fig. 2A) and the maximum parsi-

mony 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 be-

tween the patterns of amino acid substitutions in the CP do-

mains 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 Af1-

cadherin, 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 informa-

tion 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 re-

sults suggest that the states of the A1 and A2 forms were

evolutionarily stable in arthropods.


The change from the A2/E2- to the A1-form can beexplained 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 E2-

form 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

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-formcadherins (C, D). The numbers at nodes indicate bootstrap values (%).


A 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 acidsequence of another cadherin (SEQ2). The E-values (Eo20) are plotted on the matrix. The lowest and second lowest E-values in each rowindicating the most significant matches between domains are highlighted with solid and dotted lines, respectively.


SEQ2 ApSEQ1 NT EC1 EC2 EC3 EC4 EC5 EC6 EC7 EC8 EC9 EC10 EC11 EC12 EC13 EC14 EC15 EC16 EC17 NC CE1 LG1 CE2 LG2 CE3

NT 0.20EC1 6.4 3.7 0.12 6.4 0.0523e-04 3.7EC2 8.3EC3 4.9EC4 2e-07 0.15EC5 6.4 3.7 3e-0619 0.0180.26 6.4 5e-062e-060.0180.00314EC6 19 8e-062.2 3e-04 0.34 0.0017e-04EC7 0.26 7e-070.003 11 2.2 0.12 0.0142.9

Cj EC8 0.014 0.0081.7 7e-15 1e-050.44 3e-083e-085e-065e-070.089EC9 1e-04 4.9 4.9 0.023 0.0050.04 0.089 3e-05EC10 3e-09 2.2 0.34 0.0083e-04EC11 0.98 0.068 2e-090.0010.0312e-05 0.26EC12 19 0.58 0.0687e-05 7e-050.58 1e-176e-042e-041e-082.9EC13 1.7 0.34 3e-07 0.15 2e-041e-130.34 0.15 0.15EC14 0.068 0.34 0.011 0.26 1e-040.0180.0313e-10 0.008EC15 0.014 0.005 0.98EC16 0.26 11 1e-05 0.15 0.0680.0050.15 9e-10EC17 2.9 8e-09NC 8e-27CE1 2e-13 8e-06LG1 7e-31CE2 0.34LG2 5e-10CE3 0.04 4.9 1e-13


NTEC1 2e-051.6EC2 18EC3EC4 5e-05 0.93EC5 8e-05 0.42 10 0.00213 2.1 0.003EC6 1e-04 0.084 18 10 10 1e-08EC7 13 2e-050.19 18 0.049

At EC8 3e-12 0.24 5e-050.0132e-05EC9 0.038 0.064 8e-05EC10 1e-04 0.14EC11 0.24 8e-100.71 0.084 18EC12 0.24 0.029 0.01 0.32 1e-15 0.0389e-07EC13 0.006 0.0490.008 2.1EC14 3e-04 4.6 0.01 2e-104.6 4.6EC15 3.5EC16 18 6e-05 10 0.71 3e-08EC17 2.7 3e-07

NC 4e-29CE1 3e-13 0.002LG1 3e-31CE2 0.005LG2 2e-16CE3 0.014 5e-15


NT 0.001EC1 0.005 1.7EC2 3e-13 6.3 2.8EC3 2e-16EC4 8e-14 14 0.004 14 2.8EC5 2e-04 2e-06 7e-058.3 0.0230.12 1e-060.002EC6 0.052 11 0.0025e-100.010 11 0.75 1.3 11EC7 0.014 0.0681.7 4e-151.3 2.2 0.0520.0239e-070.0010.0181e-09

LvG EC8 8.3EC9 0.20 7e-07 0.98 0.004 4e-04EC10 0.20 0.0031.3 4e-05 4e-050.34 1e-052e-05EC11 0.12 0.01011 1e-100.34 0.004EC12 2e-05 0.15 6e-06 0.34 2e-042.2 2e-143e-040.34 0.0892.2EC13 18 18 18 0.75 0.34 2.9 0.12 0.0180.15 0.010 0.26EC14 18 0.002 0.98 0.26 2e-141e-05EC15 11 0.003 6.4 11 1e-06EC16 3.7 3e-06NC 0.63 3e-52CE1 3e-20 0.002LG1 5e-46 2.4CE2 4e-04LG2 5e-46CE3 6e-04 0.20 2e-18

SEQ2 cHzSEQ1 EC1 EC2 EC3 EC4 EC5 EC6 EC7 EC8 EC9 EC10 EC11 EC12 EC13 EC14 EC15 NC CE1 LG1 CE2 LG2 CE3

NTEC1 0.18EC2 7.4 0.009 0.002 0.0123.3 9.7 6e-04 0.079EC3EC4 6e-07 17 0.004EC5 5e-04 0.10 0.0160.51 0.10 6e-04 1.5EC6 17 2e-06 0.18 2.6 0.18 0.88 0.021EC7 0.10 1e-05 0.67 0.67 0.016 0.0795.7 17

Ap EC8 0.0020.10 3e-11 0.0120.0477e-093e-043e-070.14 0.0213e-05EC9 0.14EC10 0.67 3e-080.14 0.88EC11 9e-065.7 3e-100.0022e-07 0.027EC12 0.0160.51 0.39 0.0070.14 0.0023e-120.88 0.036 3e-07EC13 7.4 1.5 5e-09EC14 17 0.51 2.0 1e-042.6 1.1 3e-051e-048e-051e-10 0.10 2e-06EC15 4.4 8e-05 1e-05 2.0 0.88 4e-06 0.036 4e-044e-05EC16 0.0017.4 7e-06 0.0098e-04 0.30 0.10 0.88 3e-120.18EC17 0.18 2.0 0.30 4e-05NC 7e-26CE1 8e-05 0.18LG1 2e-26CE2 0.88LG2 3e-14CE3 0.002 8e-12

A

B

C

D

Fig. 4. BLAST-based domain comparisons of the arthropod, echinoderm and vertebrate cadherins. Comparisons are shown between thefollowing 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.


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 com-

parisons 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 substi-

tutions. 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 be-

tween the A1- and the A2/E2-form cadherins (Fig. 5, A and

B). This revealed that the EC1–EC6 domains, the EC7 do-

main, 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, re-

spectively. 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-

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 theirexpression modes define the ‘‘ancestral’’ and‘‘derived’’ groups of arthropods

To investigate the polarity of cadherin evolution from a func-

tional 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 pat-

terns in the cricket and Artemia were similar to that of Dro-

sophila. 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 me-

senchymal tissues (Fig. 7, G and I). The expression patterns in

the malacostracan crustaceans and the chelicerate were dif-

ferent 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 anti-

serum 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 concen-

trated signals were detected in mesenchymal cells of the same

gastrula (Fig. 7, J and K; arrows). Whether these me-

senchymal cells expressed Ap-cadherin at weak levels was

A

B

Fig. 5. BLAST-based domaincomparisons of the arthropodA1- and A2-form cadherins. Com-parisons are shown between thefollowing pairs of cadherins: Gb1-and Cj-cadherin (A), and Af1- andAt-cadherin (B). Data are shownin the same manner as those inFig. 3.


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/E2-

form cadherin is derived. In agreement with this suggestion,

the A2/E2-form cadherins with the ancestral mode of expres-

sion and those with the derived mode of expression were

separated in the phylogenetic trees based on amino acid sub-

stitutions in the CP domains (Fig. 2, A and B) and the ex-

Fig. 6. Tests for antibody specificity. (A) Western blot analysis. The embryonic or larval lysates and the antibody used to probe the lysateare indicated at the top of each lane. The antibodies recognized proteins with sizes similar to DE- or DN-cadherin. The antibody raisedagainst Le-cadherin reacted with a protein in the lysate of Caridina, which was likely to be Cj-cadherin. (B) Schematic illustrations showingthe 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 ofthe anti-Le-cadherin antibody. (C) The six GST-fusion proteins (lanes 1–6) were separated by SDS-PAGE, blotted onto nitrocellulose, andprobed with an antibody against GST (top panel) or the anti-Le-cadherin antibody (bottom panel). The anti-Le-cadherin antibody cross-reacted 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 DN-cadherin in situ. The data indicate that the anti-Le-cadherin antibody recognizes a wide range of crustacean and hexapod A2-formcadherins with no reaction with the A1-form cadherins.

Fig. 7. Expression of various cadherins in the epithelial and/or mesenchymal tissues of developing animals. (A, B) Embryos of Gryllusbimaculatus were stained with anti-Gb1-cadherin (A) or anti-Le-cadherin antibody (B). The anti-Le-cadherin antibody reacted with Cj- andDN-cadherins (see Fig. 6) and possibly with Gb2-cadherin. The staining patterns are consistent with expression patterns determined bywhole mount in situ hybridization with probes prepared for Gb1- and Gb2-cadherin transcripts (data not shown). (C, D) Larvae of Artemiafranciscana were stained with anti-Af1-cadherin (C) or anti-Af2-cadherin (D) antibody. (E–G) An embryo of Ligia exotica (E) and embryosof Caridina japonica (F, G) were stained with anti-Le-cadherin antibody. (H, I) An embryo of Achaearanea tepidariorum stained with anti-At-cadherin antibody is shown with the focal planes adjusted to the ectodermal layer (H) and to the mesodermal layer (I). (J, K) A gastrulaof Asterina pectinifera was double-stained with anti-Ap-cadherin antibody (J) and YOYO-1 iodide (K). Small arrowheads indicate me-senchymal cells located between the ectoderm and endoderm layers. (L–O) A gastrula of Ptychodera flava was double-stained with anti-Pf1-cadherin (L, N) and b-catenin (M, O) antibodies. Surface (L, M) and sagittal (N, O) views of the gastrula are shown. Arrowheads indicatethe 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 20mm.

"


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

subset of Arthropoda and the structural relationship between

the A2/E2 and the A1 form, define the ‘‘ancestral’’ and ‘‘de-

rived’’ 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.


Independent reductive changes from the A2/E2form generated the A1, V1, and C forms

The conservation of cadherin forms between Arthropoda and

Echinodermata, which are generally considered to be phylo-

genetically 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 an-

cestor, as exemplified by the A1 form in the arthropod lin-

eage. To evaluate this possibility, we examined the V1-form

cadherins, which are found only in vertebrates and urochor-

dates. The topology of the trees based on amino acid sub-

stitutions 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 gen-

erate 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 transi-

tions 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), al-

though the sampling of taxa is limited at this stage of research.

Diversification of the epithelial AJs with changesin 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 immunohisto-

chemical 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 cad-

herin 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 b-catenin (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 concentra-

tions 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 an-

cestral form in epithelial tissues. It is suggested that the cad-

herin 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 ho-

mologous parts of different cadherins. In our analyses of ar-

thropod 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 ex-

plained 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 relationshipsamong bilaterians

Our immunohistochemical data indicated that the epithelia of

different taxa have the AJs constituted by different forms of


A B C

D E

F

G

H

I

J

Fig. 8. BLAST-based domain comparisons of the vertebrate, urochordate, hemichordate, and other cadherins. Comparisons are shownbetween 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.


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 epi-

thelial cell–cell adhesion might have contributed to the sep-

aration 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 ep-

ithelial cells at their surface were connected by the mutated (or

derived) cadherin. We also speculate that the adhesion alter-

ations might have affected the cell functions and cell be-

haviors 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 an-

cestral, 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 ver-

tebrates 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 valid-

ity of these phylogenetic hypotheses depends on the com-

pleteness of information on the expression and subcellular

localization of cadherins in the phyla. In this respect, expres-

sion 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 re-

lationships. Taking these into account, our phylogenetic hy-

potheses 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 sister-

group 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 gener-

ally considered to be one of the most important synapomorp-

hies 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 structur-

ally reduced cadherins. Finally, a continued effort to integrate

data from cell biology, developmental biology, genomics and

paleontology is essential to validate our hypotheses.

AcknowledgmentsWe would like to thank N. Sumida, K. Kimura, E. Shoguchi and H.Wada for animals; H. Uemiya for identification of the collembolanspecies; M. Iwami for cDNA libraries; and two anonymous reviewersfor helpful comments on the manuscript. We are also grateful to S.Tsukita for supervision; K. Nakamura for encouragement; Z. H. Sufor 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 technicalassistance.

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