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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, 378 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. 380 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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