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First published online 18 May 2004
doi: 10.1242/jcs.01045

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Two classic cadherin-related molecules with no cadherin extracellular repeats in the cephalochordate amphioxus: distinct adhesive specificities and possible involvement in the development of multicell-layered structures

¹ JT Biohistory Research Hall, 1-1 Murasaki-cho, Takatsuki, Osaka 569-1125, Japan
² PRESTO, Japan Science and Technology Agency
³ College of Biological Science, Ocean University of Qingdao, 5 Yushan Road, Qingdao 266003, People's Republic of China

View larger version (32K): [in a new window]   Fig. 1. Comparison of the primary structures of amphioxus Bb1C and Bb2C, mouse E-cadherin, and sea urchin LvG-cadherin. The signal sequences and transmembrane segments are indicated by filled black boxes. The arrowhead shown for mouse E-cadherin indicates a proteolytic cleavage site that is utilized in the maturation of this protein (Shirayoshi et al., 1986). The domain organization of each classic cadherin is designated in the parentheses. Domain abbreviations: EC, cadherin extracellular repeat; NC, nonchordate classic cadherin-specific domain; CE, cysteine-rich EGF-like domain; LG, laminin G-like domain; CP, cytoplasmic domain; PCCD complex, primitive classic cadherin domain complex.

View larger version (81K): [in a new window]   Fig. 2. Amino acid sequences of Bb1C and Bb2C. (A) Alignment of the entire amino acid sequences of Bb1C (Bb1) and Bb2C (Bb2). The putative signal sequences and transmembrane segments are underlined. The boundaries of the domains are also indicated. Cysteine residues conserved between Bb1C and Bb2C are indicated by the character `+' while those present in either Bb1C or Bb2C are indicated by the character `0'. (B) Alignment of the amino acid sequences of the CPs of Bb1C, Bb2C, mouse E-cadherin (mE), ascidian Ci-cadherin, oyster Secadhrin, sea star Ap-cadherin, acorn worm Pf1-cadherin and Drosophila DN-cadherin. The abbreviations for these cadherins are shown in the legend of Fig. 3. Residues that are identical with those of Bb1C or Bb2C are highlighted. Numbers in parentheses represent the numbers of amino acid residues that were omitted at the indicated site. The p120- and ß-catenin-binding sites are indicated. The position of the primers used for PCR amplification, DQ1 and KL2, is also indicated.

View larger version (13K): [in a new window]   Fig. 3. Molecular phylogenetic trees of classic cadherin and ß-catenin/plakoglobin/Armadillo generated by the neighbor joining method. Numbers indicate bootstrap values. (A) A tree constructed using the CPs of selected classic cadherin family members. Bb1, Bb1C (AB075366); Bb2, Bb2C (AB120427); DE, Drosophila DE-cadherin (BAA05942); DN, Drosophila DN-cadherin (T00021); LvG, sea urchin LvG-cadherin (U34823); Ap, sea star Ap-cadherin (AB075365); Pf1, acorn worm Pf1-cadherin (AB075368); Pf2, acorn worm Pf2-cadherin (AB075369); Se, oyster Se-cadherin (AB075367); BS, ascidian BS-cadherin (U61755); Ci-I, ascidian Ciona intestinalis type I cadherin (AB031540); Cs-II, ascidian Ciona savjgnyi type II cadherin (AB057736); mE, mouse E-cadherin (X06115); mN, mouse N-cadherin (M31131); m6, mouse cadherin 6 (NM_007666); m11, mouse cadherin 11 (D31963). (B) A tree constructed using 541 amino acid sites of ß-catenin/plakoglobin/Armadillo. Bb.ßcat, Bb.ß-catenin (AB120428); Dm.Arm, Drosophila melanogaster Armadillo (P18824); At.Arm, spider Achaearanea tepidariorum Armadillo (AB120624); Hm.ßcat, Hydra magnipapillata ß-catenin (U36781); Uc.ßcat, spoon worm Urechis caupo ß-catenin (S33793); Tg.ßcat, sea urchin Tripneustes gratilla ß-catenin (P35223); Lv.ßcat, sea urchin Lytechinus variegates ß-catenin (AAC06340); Ci.ßcat, ascidian Ciona intestinalis ß-catenin (BAA92185); Drßcat, fish Danio rerio ß-catenin (NP_571134); Drplak, fish Danio rerio plakoglobin (NP_571252); Mm.ß-cat, mouse Mus musculus ß-catenin (NM_007614); Mm.plak, mouse Mus musculus plakoglobin (XP_126747).

View larger version (72K): [in a new window]   Fig. 4. The specificities of polyclonal antibodies to Bb1C, Bb2C and ß-catenin. (A) Western blot analysis of S2 cells transiently transfected with plasmids for Bb1C-GFP (lanes 1, 4 and 7), Bb2C-GFP (lanes 2, 5 and 8) or no insert (lanes 3, 6 and 9). The same blot was repeatedly used for detection with polyclonal antibodies to GFP (lanes 1-3), Bb1C (lanes 4-6) and Bb2C (lanes 7-9). (B-D) Endodermal epithelium of an amphioxus stage N3 embryo double-stained with the anti-Bb2C (B) and anti-ß-catenin (C) antibodies. (D) The two images were colored and merged (B in purple and C in green). The signals yielded by the two antibodies were coincidently detected at the apical portions of the lateral cell surfaces as seen in white (arrowheads). En, endoderm; Ec, ectoderm. (E) Western blot analysis of amphioxus stage L1 larvae (18 hour) to indicate the specificity of the anti-ß-catenin antibody. Two bands of about 100 and 106 kDa were detected.

View larger version (103K): [in a new window]   Fig. 5. Cell aggregation assays to test the adhesive specificities of Bb1C and Bb2C. (A-C) S2 cells transiently transfected with plasmids for Bb1C-GFP (A,B) and no insert (C) were tested for aggregation in the presence of 1 mM Ca2+ (A,C) or 1 mM EDTA (B). The cells expressing Bb2C-GFP were aggregated in a Ca2+-independent manner. (D) S2 cells separately transfected with plasmids for Bb1C and Bb2C-GFP were mixed and tested for aggregation. The resulting aggregates were fixed and stained for Bb1C (red). The cells expressing Bb1C (red) and those expressing Bb2C-GFP (green) aggregated separately.

View larger version (120K): [in a new window]   Fig. 6. Expression of Bb.ß-catenin and Bb2C in the early neurula. Embryos were double-stained for Bb.ß-catenin (A,C) and Bb2C (B,D). (A,B) Dorsal surface view of a stage N1 embryo at the same focal plane. Anterior is to the upper left. The epidermal ectoderm (Ep) has started to spread over the neural plate (Np). Staining for Bb2C yielded no specific signal in the epidermal ectoderm or the neural plate. (C,D) Internal view of a stage N1 embryo at the same focal plane. Anterior is to the lower left. The mesendodermal cell layer is undergoing somitogenesis (arrows). Since the observed embryo is compressed, the apical surfaces of some of the ventrally located, prospective endodermal cells are also in focus. Bb2C and Bb.ß-catenins were detected at the apical zones of the lateral surfaces of the mesendodermal cells (arrowheads). The strongest signals were detected at the blastopore region (Bp). Scale bar: 20 µm.

View larger version (177K): [in a new window]   Fig. 7. Expression of Bb.ß-catenin and Bb1C during neurulation. The embryos were double-stained for Bb.ß-catenin (A,C,E) and Bb1C (B,D,F). Anterior is to the left. (A,B) Dorsal surface view of a stage N1 embryo at the same focal plane. Signals for Bb.ß-catenin and Bb1C were detected at cell-cell contact sites in both the epidermal ectoderm (Ep) and the neural plate (Np). In cells at the edges of the epidermal ectoderm spreading over the neural plate (arrows), the characteristic concentrations of Bb1C and Bb.ß-catenin were poorly observed. Note that Bb2C was not detected during neurulation as shown in Fig. 6B. (C-F) Dorsal surface (C,D) and internal (E,F) views of a stage N2 embryo. The focal plane of E and F is separated from that of C and D by 9 µm. At the dorsalmost epidermal ectoderm, weaker levels of Bb1C were observed compared to more lateral areas, although the levels of Bb.ß-catenin showed no apparent differences (arrows in C and D). In the folded neural plate, Bb1C, together with Bb.ß-catenin, was highly concentrated at the apical sites of cell-cell contact (arrows in E and F). Nr, neuropore. Scale bar: 20 µm.

View larger version (97K): [in a new window]   Fig. 8. Expression of Bb1C and Bb2C in the late neurula. (A-C) A stage N3 embryo was triple-stained for Bb.ß-catenin (A), Bb1C (B) and Bb2C (C). Anterior is to the left. Dorsal is to the top. Bb1C was detected in the nerve cord (Nc) and differentiating notochord (No), but not or only faintly in the endoderm (En). Bb2C was detected in the notochord and endoderm, but not or little in the nerve cord. Note that in the notochord, the pattern of Bb1C expression differs from that of Bb.ß-catenin and of Bb2C (fat and thin arrows). (D) High magnification of the area boxed in B. The images for Bb.ß-catenin (green) and Bb1C (purple) were colored and merged. Bb1C colocalized with Bb.ß-catenin appears white (arrow). Arrowheads indicate the apical concentrations of Bb1C in the nerve cord (Nc) epithelial cells. (E,F) High magnification of the area boxed in A. In E, the images for Bb.ß-catenin (green) and Bb1C (purple) were colored and merged. In F, the image for Bb2C is shown. Arrows indicate interfaces between notochord dorsal (NoD) cells, at which Bb2C was detected, and arrowheads indicate interfaces between notochord ventral (NoV) cells and between notochord mid (NoM) cells. Some of the Bb1C and Bb2C signals were closely located. Note that NoM cells are interdigitated between the NoD and NoV cell layers. (G) Dorsolateral view of a stage N3 embryo stained for Bb.ß-catenin. Anterior is to the left. A single line of NoD cells is seen. The arrow points to the posterior region of the notochord, where NoM cells have not interdigitated. Scale bars: 20 µm.

View larger version (114K): [in a new window]   Fig. 9. Expression of Bb1C and Bb2C in the knife-shaped larva. (A-D) A stage L1 larva (24 hour) was simultaneously stained for Bb.ß-catenin (A), Bb1C (B,D in green), Bb2C (C,D in red) and DNA (D in blue). Arrowheads indicate the NoD, NoM and NoV cell layers of the notochord (No). Asterisks and thin white arrows indicate the lumen of the intestine (In) and the apical surfaces of the intestinal epithelial cells, respectively. Large white arrows point to the posterior end of the notochord. Green arrows in B point to lines of Bb1C concentration in the nerve cord (Nc). (E) Schematic representation of the area boxed in A. (F-H) High magnifications of B-D corresponding to the area boxed in A. In F, the arrows point to high concentrations of Bb1C at the interfaces between NoV cells. In G, the arrows point to high concentrations of Bb2C at the interfaces between NoD cells, while the arrowhead indicates the weaker concentrations of Bb2C between a NoD cell and NoM cells. Scale bars: 20 µm.

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