XB-ART-25018Development March 1, 1991; 111 (3): 829-44.
Differential expression of two cadherins in Xenopus laevis.
Using a cadherin fraction from Xenopus tissue culture cells as an immunogen, two monoclonal antibodies were obtained that allowed the characterization of two distinct cadherins in the Xenopus embryo. The two cadherins differ in molecular weight, in their time of appearance during development and in their spatial pattern of expression. One of the antigens was identified as E-cadherin. It appears in the embryonic ectoderm during gastrulation when epidermal differentiation commences and it disappears from the neural plate area upon neural induction. The second antigen could not be allocated to any of the known cadherin subtypes and was termed U-cadherin. It is present in the egg and becomes deposited in newly formed inner cell membranes during cleavage, the outer apical membranes of the embryo remaining devoid of the cadherin throughout development. U-cadherin is found on membranes of all cells up to the late neurula stages. A conspicuous polarized expression of the antigen on the membranes of individual inner cells suggests its participation in the segregation of cell layers and organ anlagen. These findings are discussed in the context of current hypotheses on the role of cadherins in establishing the spatial structure of the embryo.
PubMed ID: 1879345
Article link: Development
Species referenced: Xenopus laevis
Genes referenced: cad cald1 cdh1 cdh3 mcf2 prss1 tbx2
Antibodies: Cdh1 Ab8 Cdh3 Ab3 Epidermis Ab1
Article Images: [+] show captions
|Fig. 1. Characterization of the antigens from A6 cells recognized by mAbs 6D5 and 10H3. (a-d) Isolation of a 90xl(fi MT protein fraction from supernatants of A6 cells after trypsin treatment. Supernatants from trypsin-digested A6 cells were used for lentil lectin affinity chromatography as described under Materials and methods. Lane a: supernatant of A6 cells treated with trypsin in the absence of Ca (500^1 per lane). Lane b: supernatant from A6 cells treated with trypsin in the presence of Ca (500^1 per lane). Lane c: wash through of the lentil lectin chromatography (500^1 per lane). Lane d: eluate from the lentil lectin column (300 ^il per lane which corresponds 10% of the eluate). The arrowhead indicates the 90X103 Mr protein fraction, (e-h) Identification of the 90X103 MT protein fraction by mAbs 6D5 and 10H3 on immunoblots. Supernatants from A6 cells treated with trypsin in the presence of Ca2+ (lanes e and g) or in the absence of Ca2+ (lanes f and h) were immunoblotted with mAb 6D5 (lanes e and f) or mAb 10H3 (lanes g and h). (i-m) Determination of a protein moiety as epitopes for mAbs 6D5 and 10H3. Cell lysates of untreated A6 cells (lanes k and m) or tunicamycin-pretreated A6 cells (10/igmP1) were immunoblotted with mAb 6D5 (lanes i and k) or mAb 10H3 (lanes 1 and m). Approximately 100 ng of protein was loaded on each lane. Arrowheads indicate the two main protein bands recognized by the two antibodies. Bars indicate relative molecular masses from top to bottom as follows: 200, 116, 97, 68, 43 (x10^3).|
|Fig. 2. Comparison of the 21 N-terminal amino acids from a microsequence analysis of the 90X103 MT protein fraction and the corresponding amino acid sequences of cadherins in different species. Residues identical with those of the 90x10* MT protein fraction are enclosed in boxes. Numbers on the right indicate % identity of the obtained 90X103 Mt protein fraction with the listed cadherins. The N terminus is marked by an arrowhead. X=not identified amino acid residue; M, mouse; H, human; C, chicken; X, Xenopus; cad, cadherin; p.f., protein fraction. Sequences from top to bottom are reported by Nagafuchi et al. 1987 and Ringwald et al. 1987; Wheelock et al. 1987; Gallin et al. 1987; Miyatami et al. 1989; Nose et al. 1987; Hatta et al. 1988; Shimoyama et al. 1989; Detrick et al. 1990.|
|Fig. 3. Antibody inhibition of the reaggregation of dissociated A6 cells. Cells of an A6 monolayer culture were dissociated in Ca2+-free buffer (A) and incubated for 2h in culture medium supplemented with either control IgG P3 (B), purified mAb 10H3 (C) or mAb 6D5 (D) in a concentration of 10/xgml"1 each. Bar, 50um.|
|Fig. 4. Immunostainings of Xenopus tissues. Cryostat sections of the head region of a stage 47 tadpole (A-C), liver (D-F) and heart (G-I) were immunostained with mAb 10H3 (A,D,G), mAb 6D5 (B,E,H) or inert control IgG (C,F,I). Arrows and arrowheads inert control IgG (C,F,I). Arrows and arrowheads in H indicate the intercalated discs and the costameres in heart tissue, respectively. Bars, 30um.|
|Fig. 5. Identification of two different cadherins in embryos by mAbs 10H3 and 6D5. Protein extracts of embryos of different stages were prepared as described in Materials and methods. The equivalent of 5 embryos per lane was used for electrophoresis and immunoblotting. (A) Comparison of molecular masses of cadherins recognized by mAbs 10H3 and 6D5 in protein extracts of stage 20 embryos. A6 cell lysates (100/ig/lane, lanes a and d) and the protein extract of stage 20 embryos (lanes b and c) were immunoblotted with mAb 10H3 (lanes a and b) or mAb 6D5 (lanes c and d). (B) and (C) Western blot analysis of the antigens recognized by mAbs 10H3 (B) and 6D5 (C) during development. Stages from a-g: 1, 3, 8 10, 12, 14, 20. Numbers indicate relative molecular masses xlO3.|
|Fig. 6.' Aggregation assays of blastomeres of the early embryo and inner cells of the animal cap of late blastulae. (A-C) Fertilized eggs were demembranated and incubated in Ca2+-free buffer until the 8-cell stage was reached. The incubation was continued in Ca2+-free buffer (A) or in Ca2+-containing buffer supplemented with 10/igmF1 of purified mAb 10H3 (B) or mAb 6D5 (C). Photographs were taken 4h post-fertilization. (D-F) Inner cells of the animal half of a stage 9 blastula were isolated and dissociated in Ca2+-free buffer containing lmin EDTA. Cells were further incubated either in Ca2+-free buffer (D) or in Ca2+-containing buffer supplemented with purified mAb 10H3 (E) or mAb 6D5 (F) in a concentration of lO^gml"1 each. Photographs were taken after 5h of incubation. By this time control embryos had reached stage 13. Bars, 500um.|
|Fig. 7. Distribution of E-cadherin in the embryo during early development. Embryos were immunostained with mAb 10H3 embedded in glycolmethacrylate and sectioned. A schematic drawing (A) indicates the cell layers seen in the sagittal section of an embryo at stage 12.5 (B). The arrow in A indicates the original animal pole region. C represents a magnification of the original animal pole region in B. (D) A schematic drawing indicates the cell layers seen in the transverse section of an embryo stage 14 (E). (F) Transverse section of an embryo stage 20. np, neural plate; pe, presumptive epidermis; bl, blastocoel; ae, archenteron, dbl, dorsal blastopore lip; vbl, ventral blastopore lip; be, bottle cells; no, notochord; pm, paraxial mesoderm; ar, archenteron roof; nt, neural tube;. Bars represent in B and E 200/an, in C and F 50um.|
|Fig. 8. Localization of U-cadherin to the newly inserted cell membranes in the first cleavage stage. Embryos in the first cleavage stage were immunostained with mAb 6D5, embedded in glycolmethacrylate and sectioned. (A) Immunostaining. (B) Same section in phase contrast. Arrows indicate the staining of newly formed membranes in the first cleavage (A). Bar, 50um.|
|Fig. 9. Distribution of U-cadherin in the dorsal blastopore lip region. Pieces of embryos stage 10.5 containing the dorsal blastopore lip region were stained with mAb 6D5 (A) or control IgG P3 (B), embedded in glycolmethacrylate and sectioned. Arrowhead indicates apically constricted bottle cells. Arrows point in the direction of the animal pole, en, endoderm; me, mesoderm; ec, ectoderm. Bar, 100um.|
|Fig. 10. Distribution of U-cadherin in neurula stages. Anterior halves of embryos stage 13 (A) and dorsal halves of embryos stage 17 (B) and stage 21 (C and D) were stained with mAb 6D5 (A, B and C) or control IgG P3 (D). The tissues were embedded in glycolmethacrylate and transverse sections were cut through the middle of the dorsal regions. Arrows in B and C indicate cell membranes devoid of staining, np, neural plate; no, notochord; pm, paraxial mesoderm; ar, archenteron roof; af, archenteron floor; so, somite; nt, neural tube. Bar, 30um.|