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Figure 3. Localization of ADAM13, ADAM10, and XCG mRNA in early Xenopus embryos. (A) In an anterior view at stage 16, ADAM13 (purple, black arrow) mRNA is found in the neural crest and just dorsal to the cement gland anlagen (as marked by XCG mRNA expression in red, arrowhead), but the two transcripts do not overlap. (B) ADAM10 (light purple, open arrow) mRNA is localized to the entire neural plate and placodes, and encompasses the expression field of XCG. No distinct red color revealing XCG mRNA expression is seen because it is overlaid by the light purple ADAM10 costaining. (C) In a schematic of the Xenopus embryo at stage 16, the pink represents the entire neural plate and surrounding placodal and cement gland anlagen that express ADAM10 mRNA (open arrow). The ADAM10-expressing area encompasses both the regions of ADAM13 mRNA expression (blue, black arrow) and XCG (red, arrowhead) (Nieuwkoop and Faber, 1994). |
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Figure 4. Overexpression of wild-type ADAM13, but not ADAM10, results in hyperplasia of the cement gland. (A) Transcripts encoding ADAM constructs are microinjected into the animal pole of one blastomere at the two-cell stage. The embryos are allowed to recover until stage 22+, and then fixed for whole mount in situ hybridization with the cement gland marker XCG. (B) The increase in XCG mRNA levels caused by ADAM13 overexpression was quantified by RNA spot blot. Data are represented as relative values of expression based on average pixel densities obtained using the PhosphorImager. All levels of expression were within the linear range of this detection method. Data are shown as means ± SD of five independent experiments, each with pooled RNAs from 10 embryos. By stage 17, a twofold increase in XCG mRNA levels (P < 0.05) was found in embryos expressing ADAM13, but not E/A ADAM13 or ADAM10. The differences in XCG mRNA levels quantified by RNA spot blot were also observed by whole mount in situ hybridization. (C) GFP-injected embryos had no alterations in the cement gland marker XCG (black arrow), whereas large, ectopic islands of XCG-positive cells (D, arrow) were found when overexpressing ADAM13. Expression of the E/A ADAM13 mutant (E) or ADAM10 (F) had no affect on cement gland formation. NI, noninjected control; E/A13, E/A ADAM13. |
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Figure 7. Summary of ADAM10/13 construct expression in embryos. The molecular weights in kD were calculated by MacVector software and include the addition of the six myc tags and predicted N-linked glycosylations. After in situ hybridization with XCG, embryos were scored for mild or severe cement gland perturbations. The embryos with mild phenotypes were subdivided into two classes, in some cases (shown with a back slash), defined as one to three small spots of ectopic XCG expression or three to four small spots accompanied by <10% expansion of the area of the main cement gland. Severe phenotypes had numerous large islands of ectopic cement gland or substantial expansions (>10%) of the existing cement gland. Up arrows indicate the ability of a construct to induce severe cement gland hyperplasia (corresponding to arrows in Fig. 5). Constructs with high levels of cell surface expression in the met form (as compared with ADAM13) have two pluses (++) and embryos with lower levels of met forms are given one plus (+). N, the number of embryos assayed. aLow cell surface expression, but robust activity. *Previously we reported separate experiments involving the coexpression of equal amounts of transcript (0.5 ng each) encoding wild-type and E/A ADAM13, which led to a marked reduction (62%) in cement gland hyperplasia. We conclude that E/A ADAM13 can function as a dominant-negative inhibitor of wild-type ADAM13 in the cement gland assay; the details are reported in Alfandari et al. (2001). |
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Figure 8. Anterior views of representative stage-22 Xenopus embryos showing XCG mRNA expression. (A) Embryos injected with GFP alone have no alterations in XCG expression. (B) Like ADAM13, the 10PM/13 chimeric construct causes a large expansion in XCG. The 10/13DCE (C) and 10/13DC (D) chimeras also perturb the cement gland. (E) An E/A point mutant in the catalytically active site of the 10/13DC chimera ablates its ability to cause hyperplasia of the cement gland. (F) Replacing ADAM13's disintegrin and cysteine-rich domains (chimera 13/10DC) did not fully ablate the XCG expansion, but it did lessen its severity. Replacing the disintegrin domain of ADAM13 with that of ADAM10 (chimera 13/10D) did not alter the function (G), whereas replacing only ADAM10's cysteine-rich domain with that of ADAM13 (10/13C) was sufficient to give that chimera the ability to cause alterations in XCG (H). Swapping ADAM13's cysteine-rich domain caused both a quantitative (6% severe phenotype vs. 33% for wild-type ADAM13; Fig. 7) and qualitative (I) decrease in activity. (J) Three point mutations in the disintegrin loop of ADAM13 diminish its ability to cause the enlargement of the cement gland, but do not affect its ability to cause its own degradation in vitro (unpublished data). |
