Otte AP et al. (1990), Characterization of a maternal type VI collagen...

XB-ART-25788

J Cell Biol 1990 Jul 01;1111:271-8.

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Characterization of a maternal type VI collagen in Xenopus embryos suggests a role for collagen in gastrulation.

Otte AP , Roy D , Siemerink M , Koster CH , Hochstenbach F , Timmermans A , Durston AJ .

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We characterized a novel extracellular matrix element that is present in the earliest developmental stages of Xenopus laevis, and is recognized by an mAb 3D7. Based on amino acid composition, breakdown patterns by bacterial collagenases, and the molecular weights of the components of the antigen (240, 200, and 140 kD), we found it very similar to mammalian collagen type VI. The antigen is evenly distributed in unfertilized eggs. Shortly after fertilization, it becomes localized intracellularly in the periphery of the cleaving embryo as well as in the extracellular spaces. During gastrulation, the antigen was localized in the cells lining the blastopore and in the extracellular space between the two cell layers, in the presumptive archenteron. When Fab elements of the 3D7 antibody were added to the culture medium, gastrulation was blocked, suggesting a role for the antigen in gastrulation movements.

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Species referenced: Xenopus laevis
Genes referenced: mrc1

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	Figure 1. Analysis of 3D7 antigen using SDS-PAGE and Western blotting or silver staining. (A) A blastula cell homogenate (10 #g) (lane 1) was subjected to 10 min at 10,000-g centrifugation to remove yolk platelets, and the supernatant was then resolved by SDSPAGE (5-15% polyacrylamide gradient gel), transferred to nitrocellulose, and immunostained using the 3D7 mAb (lanes 1-3). As negative control the second peroxidase-labeled antibody only was used (lane 4). Lane 2 shows immunoatfinity-purified antigen. Lane 3 shows concentrated culture medium containing secreted antigen. Binding of the antibodies was visualized using a peroxidase-conjugated second antibody. (B) SDS-PAGE of immunoaitinity-purified 3D7 antigen. Antigen was obtained from blastula cell homogenate as in A. Proteins were visualized by silver staining of the gel. The positions of the molecular weight markers are indicated to the left of the panels.
	Figure 2. Digestion of 3D7 antigen using collagenases. Blastula supernatant (lane 1) or purified antigen (lane 3) were incubated with 75 U/ml bacterial collagenase type I (Sigma Chemical Co.) (lanes 2 and 4) for 4 or 12 h (lane 5) at RT before SDS-PAGE and Western blotting. Blastula supernatant was also incubated with bacterial collagenase type I for 12 h at room temperature in the presence of 1 M DTT (lane 6). Further blastula supernatant was incubated with 0.5 mg/ml pepsin (Sigma Chemical Co.) at 4°C 12 h (lane 7). Binding of the antibodies was visualized using a peroxidase-conjugated second antibody. The results were obtained using different gels. The positions of molecular weight markers are indicated to the left of the panels.
	Figure 3. Distribution of the antigen recognized by 3D7 in sections of early Xenopus laevis embryos. 8-#m sections of glutaraldehyde/ paraformaldehyde-fixed embryos embedded in paraffin were stained by indirect immunofluorescence using the 3D7 antibody and FITCconjugated GAM IgG. (A) Unfertilized egg; (B) fertilized egg, 30 rain after fertilization; (C) stage 4 embryo; (D) stage 6 embryo; (E) stage 7 blastula; (F) stage 8 blastula; (G) stage 10 1/2-11 gastrula; (H) stage 16 neurula; (I) blastopore region of stage 10 1/4 gastrula; (J) detail of E, stage 7 blastula; (K) detail of D, stage 6 embryo; (L) detail of stage 8, mid-blastula embryo.
	Figure 4. Disappearance of immunocytological staining after the loss of cell-cell contacts in Ca 2+ and Mg 2÷ free medium. Stage 2 embryos were put in Ca 2+ and Mg2+-free MMR in which they disaggregate to loosely connected ceils. When embryos reached stage 6 they were either fixed immediately (A) or after incubation in Ca 2+ and Mg2÷-containing MMR for 45 min. (B). After fixation, the embryos were paraffin embedded and prepared for immunocytochemistry using the 3D7 antibody (C). Immunoblot of blastula supernatant of embryos in A and B. The 140-, 200-, and 240 kD components of the antigen from nontreated embryos are shown in lane 1. Disaggregated embryos are shown in lane 2. Embryos were reaggregated for 45 min (lane 3), or 2 h (lane 4) before analysis.
	Figure 5. 3D7 antibody blocks gastrulation when present in the incubation medium. The vitelline membranes were removed manually from stage 7 blastulas and the embryos were cultured in MMR containing monovalent or intact 3D7 antibodies (0.2 mg/ml) (B-D, Fand G) or nonsense hybridoma antibodies (0.2 mg/ml) (A and E). Photographs were taken during gastrula stages (A-C) or when the control (E) embryos reached stage 26. 3D7-treated embryos either exogastrulated (D) or were delayed in their development (F). Crosssections of nontreated embryos (G) and 3D7-treated embryos (H) were made when embryos had reached the stage shown in A and B. The involuting mesoderm is indicated (M), as well as the dorsal (DL) and ventral (VL) blastopore lip.
	Figure 6. A cell surface-binding antibody which does not block gastrulation. An antibody (2A9) that binds to gastrula cells (A and B) was added to the culture medium at the same concentration (0.2 mg/ml) as was used for 3D7 (Fig. 6). 2A9 did not interfere with gastrulation (C).

References [+] :

Boucaut, Biologically active synthetic peptides as probes of embryonic development: a competitive peptide inhibitor of fibronectin function inhibits gastrulation in amphibian embryos and neural crest cell migration in avian embryos. 1984, Pubmed