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BACKGROUND: Cranial neural-crest (CNC) cells originate from the lateral edge of the anterior neuroepithelium and migrate to form parts of the peripheral nervous system, muscles, cartilage, and bones of the face. Neural crest-cell migration involves the loss of adhesion from the surrounding neuroepithelium and a corresponding increase in cell adhesion to the extracellular matrix (ECM) present in migratory pathways. While proteolytic activity is likely to contribute to the regulation of neural crest-cell adhesion and migration, the role of a neural crest-specific protease in these processes has yet to be demonstrated. We previously showed that CNC cells express ADAM 13, a cell surface metalloprotease/disintegrin. Proteins of this family are known to act in cell-cell adhesion and as sheddases. ADAMs have also been proposed to degrade the ECM, but this has not yet been shown in a physiological context.
RESULTS: Using a tissue transplantation technique, we show that Xenopus CNC cells overexpressing wild-type ADAM 13 migrate along the same hyoid, branchial, and mandibular pathways used by normal CNC cells. In contrast, CNC cell grafts that express protease-defective ADAM 13 fail to migrate along the hyoid and branchial pathways. In addition, ectopic expression of wild-type ADAM 13 results in a gain-of-function phenotype in embryos, namely the abnormal positioning of trunk neural-crest cells. We further show that explanted embryonic tissues expressing wild-type, but not protease-defective, ADAM 13 display decreased cell-matrix adhesion. Purified ADAM 13 can cleave fibronectin, and tissue culture cells that express wild-type, but not protease-defective, ADAM 13 can remodel a fibronectin substrate.
CONCLUSIONS: Our findings support the hypothesis that the protease activity of ADAM 13 plays a critical role in neural crest-cell migration along defined pathways. We propose that the ADAM 13-dependent modification of ECM and/or other guidance molecules is a key step in the directed migration of the CNC.
Figure 2. Overexpression of ADAM 13 alters neural crest-cell positioning in vivo. Embryos were injected into the animal pole of one blastomere at the two-cell stage with (b,d,j,k) 1 ng wild-type ADAM 13, (c,g,h,l,m) the E/A point mutant, or (i) the E/AδCyto transcripts. Injected embryos were cultured to (a) early-neurula stage 13 or (d) tailbud stages and processed for (a) Xslug mRNA expression (blue), (j) Xtwist, and the Myc-tag epitope (light brown). The green dot indicates the injected side of each embryo; red and blue arrowheads indicate the cranial and trunk crests, respectively. Dorsal views of (a) noninjected control embryos, (b) wild-type embryos, or (c) E/A mutants at stage 13 show no difference in the Xslug expression pattern. (d) Dorsal views of wild-type transcript-injected stage 25 (left) and 22 (right) embryos. (e) A higher-magnification view of the stage 22 embryo in (d) shows that the three cranial-crest segments visible on the control side (mandibular, m; hyoid, h; and branchial, b) appear as a single mass on the injected side (red arrowhead). (d,e) Islands of ectopic Xslug- positive trunk crest cells (blue arrowheads) are also noted in the flank on the injected side. (f) A horizontal section of a stage 24/25 embryo confirms the location of the cranial crest expressing Xslug on the injected side. Xslug expression in the head is weak at this stage, as seen in the noninjected control side (e.g., stage 25 embryo on the left in [d]). (g) Dorsal views of tailbud embryos (stage 24, left; stage 22, right) expressing the E/A point mutant. The pattern and position of the trunk crest is normal, although subtle differences in the cranial crest are observed; (h) the extent of migration of cranial crest segments on the injected side is decreased relative to that of the control side (red arrows). (i) Stronger defects are observed in embryos overexpressing the E/AδCyto protein. (j) Lateral views of tailbud stage embryos injected with wild-type ADAM 13 (j,k) or the E/A mutant (l,m). Injected embryos were stained with the cranial neural crest-cell marker Xtwist. As seen with Xslug, the cranial-crest segments of the embryo injected with wild-type ADAM 13 are not as clearly defined as those in the contra-lateral noninjected side (compare [j] and [k]). Interestingly, overexpression of the E/A ADAM 13 (compare [l] and [m]) has dramatically reduced the migration of cells from the hyoid and branchial crest segments (red arrowhead)
Figure 3. Overexpression of ADAM 13 does not affect central nervous-system formation. Whole-mount in situ hybridization of embryos expressing (a,c,d) wild-type ADAM 13 or (b,e) the E/A mutant constructs were processed with (a) the pan-neural marker Xsox-2 and (d,e) the neuronal marker N-tubulin. Black dots indicate the transcript-injected half of each embryo. Embryos expressing wild-type ADAM 13 lack Xsox-2-positive cells in the vicinity of the otic vesicle; (a) such cells are present in the E/A and noninjected control halves of each embryo (arrowheads). (c) Transverse section at the level of the putative otic vesicle of an embryo in a stage similar to that in (a). Dorsal views of both (d) wild-type and (e) E/A-expressing embryos at the early-neurula stage (stage 15). (d,e) Lateral expression of N-tubulin within the putative trigeminal ganglion (black arrowheads) is absent from (d) the wild-type-injected side. (f) Dorsal views of neurula-stage (stage 19) embryos overexpressing (f,g) wild-type or (h,i) E/A ADAM 13 and hybridized with the Xkrox-20 probe. The localization of Xkrox-20 in rhombomeres 3 and 5 is identical in the uninjected control side compared to the injected side of each embryo
Figure 4. CNC grafts. Transcripts encoding GFP (0.3 ng) alone or in combination with wild-type (0.7 ng) or E/A (0.7 ng) ADAM 13 were injected in one blastomere of two-cell-stage embryos. Labeled cranial neural-crest explants were taken from stage 17/18 Xenopus embryos and grafted into unlabeled sibling embryos at the same position (CNC was removed from the host at this position). Lateral views of tailbud stage (stage 24) grafted embryos, with (a) bright field images on the left and (a′′) fluorescent images on the right. (a,a′) Embryos that were grafted with cranial-neural crest that expressed both GFP and wild-type ADAM 13 were indistinguishable from (not shown) embryos grafted with CNC expressing GFP alone. (a′) Fluorescent neural-crest cells migrate along pathways normally used by CNC streams (m, mandibular; h, hyoid; ba, branchial anterior; bp, branchial posterior). (b) In contrast, cranial neural-crest cells expressing both the E/A ADAM 13 mutant and GFP (b′) do not migrate or (c′) only migrate in the mandibular segment. The cement gland (dotted line) and (e) the future eye are used in each embryo as reference points for the evaluation of neural crest-cell position and the extent of migration
Figure 5. Ectopic expression of ADAM 13 causes perturbation of cell spreading on fibronectin. Animal cap explants expressing (b) wild-type ADAM 13 or (e,f) the protease-defective E/A point mutant were cultured on fibronectin substrates in the presence of 20 U/ml activin-A. (a) Under these conditions, noninjected explants undergo convergence extension movements (arrow), while cells in contact with the substrate spread and migrate away from the explant as a cohesive sheet. (b) Explants expressing wild-type ADAM 13 also undergo convergence extension movements (arrow), but (b,c) the cells in contact with the substrate are round and dispersed. These cells remain viable during the course of the assay as determined by trypan blue exclusion (data not shown). (d) Extensive cell spreading occurs in cap cell explants expressing wild-type ADAM 13 in the presence of 0.25 mM BB-3103 protease inhibitor. (e,f) Cell spreading is also observed in animal cap explants expressing the protease-defective E/A mutant construct. (c,f) Higher-magnification views of explants in (b) and (e), respectively. (a,b,d,e, arrows) Converging and extending explant tissue above the plane of focus. The scale bars in (e) and (f) represent 1 mm
Figure 6. Xenopus XTC cells transfected with wild-type ADAM 13 remodel a fibronectin substrate. Xenopus XTC cells were plated on fluorescent fibronectin for 14 hr prior to fixation. Cells were permeabilized and processed for immunofluorescence with a mouse polyclonal antibody directed against (a) endogenous Xenopus ADAM 13 or (b) mAb 9E10 directed against the Myc-tag epitope. Fluorescence was observed with a confocal microscope. (a′′) Fluorescent fibronectin substrates correspond to the same fields as (a) immunostained cells. (a) Endogenous ADAM 13 is preferentially localized to the perinuclear endoplasmic reticulum and to cellular extensions at the cell periphery in nontransfected XTC cells (arrowheads). (a′) These cellular extensions colocalize with minor alterations in the fluorescent fibronectin substrate (arrowheads). (b) Cells transfected with wild-type ADAM 13 dramatically modified (b′) the substrate. In contrast, (c′) the fluorescent fibronectin substrate in contact with (c) E/A-transfected cells is comparable to the substrate of (a′) nontransfected control cells
Figure 7. The E/A ADAM 13 inhibits endogenous ADAM 13 remodeling of fibronectin. Xenopus XTC cells were plated on fluorescent fibronectin for 24 hr prior to fixation. (a) Cells expressing the E/A ADAM 13 were detected with the 9E10 mAb. Fields containing both transfected (arrowhead 3) and nontransfected (arrowheads 1 and 2) cells were selected. The fluorescent fibronectin substrate within the same field is presented in (a′). (a′) During this longer period of incubation (24 hr), nontransfected cells remodeled the substrate (arrowheads 1 and 2), while the cell expressing the E/A ADAM 13 did not (arrowhead 3). (b) Surface-biotinylated XTC cells transfected with either wild-type or E/A ADAM 13 were extracted and immunoprecipitated with mAb 9E10. Immunoprecipitated proteins were separated by SDS-PAGE and transferred to nitrocellulose. Blots were either incubated in mAb 9E10 for the detection of total ADAM 13 protein (left panel) or in streptavidin-HRP for the identification of ADAM 13 at the cell surface (right panel). In each case only the mature form (M) of wild-type ADAM 13 or the E/A mutant is present at the cell surface. (c) Embryos overexpressing wild-type ADAM 13 were extracted in the presence of BB-3103. Immunoprecipitated wild-type ADAM 13 was incubated with immunoprecipitated fibronectin in the absence (lane 1) or the presence (lane 2) of 0.5 mM BB-3103 for 1 hr at 20. The immunoprecipitated fibronectin was analyzed by a Western blot with the 4H2 mAb (left panel). The same blot was then probed with mAb 9E10 for the detection of wild-type ADAM 13 (right panel). The mature (M) form of ADAM 13 is only seen in lane 2′, and this observation confirms the efficiency of the BB-3103 inhibitor in these experiments. The arrowheads in the left panel of (c) point to intact fibronectin (arrowhead a) or the major specific degradation product (arrowhead b, 90 kDa). The asterisk points to a 120 kDa fibronectin fragment, the formation of which is independent of ADAM 13
