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Abstract
Embryonic development involves a series of cell adhesive interactions that provide mechanical and instructive information required for morphogenesis. The ADAMs family of membrane-anchored proteins, containing a disintegrin and metalloprotease domain, is well suited for participating in such developmental events. They encode not only a potential adhesive function, through an integrin-binding disintegrin domain, but also a potential antiadhesive function, through a zinc-dependent metalloprotease domain. In order to investigate the role of ADAMs in early development we cloned a cDNA encoding a novel member of the ADAM family from a Xenopus laevis neurula stage library. We call this cDNA, and the 915-amino-acid protein it encodes, ADAM 13, X-ADAM 13 RNA is expressed during embryogenesis from the midblastula stage through tadpole stage 45. X-ADAM 13 is localized to somitic mesoderm and cranial neural crest cells during gastrulation, neurulation, and in tail bud stages. Sequence analyses of the X-ADAM 13 metalloprotease and disintegrin domains indicate that the protein is likely to be involved in both proteolytic and cell-adhesive functions. The X-ADAM 13 sequence is most closely related to that of mouse meltrin alpha, which is implicated in myoblast fusion. Our data suggest that X-ADAM 13 may be involved in neural crest cell adhesion and migration as well as myoblast differentiation.
FIG. 6. Localization of X-ADAM 13 mRNA in early embryos. (A) Lateral view of a blastula (stage 8) and a neurula (stage 13) stage embryo. X-ADAM 13 mRNA is not detected in the blastula. During neurulation the mRNA is localized to the anterior placodal ectoderm (arrows and arrowhead). (B) Dorsal view of a stage 13 neurula. The neural plate (np) and notochord (c) are indicated. (C) Anterodorsal view of stage 20 embryo. Three distinct groups of cells are stained in the anterior region. These populations of cells arise from the border of the neural tube and expand on either side ventrally (arrows and arrowhead). The most anterior of these groups of cells surrounds the optic vesicle (arrowhead). (D) Dorsal view of stage 20 embryo. X-ADAM 13 mRNA is also localized to the forming somites (blue arrows). At this angle, the anterior neural crest staining is observed around both the optic vesicle and the brain (arrowhead). Black arrows point to the same cells as in C. (E) During tailbud formation, the mRNA is present in the head, somites, and proctodeum (pd). The staining present in the anterior part of the embryo progresses ventrally as development proceeds (arrows). Note the extensive staining of facial structures of the head (arrowhead) with the exception of the cement gland (cg). (F) Magnification of a dorsal view of an early neurula (stage 13). When the color reaction is allowed to proceed overnight, secondary sites of specific expression become apparent. Note the paraxial mesodermotochord boundary where presumptive somite cells are detected with the antisense probe (white arrowhead). No staining is detected in the notochord (c). (G) Magnification of the dorsoanterior view of an early tailbud (stage 20). In the somites, the staining is restricted to the region containing the nuclei (white arrows) and absent from the intersomitic border (red arrowheads).
FIG. 9. Localization of X-ADAM 13 protein during development. Whole-mount embryos were stained with the affinity-purified cytoplasmic tail antibody and alkaline phosphatase-conjugated secondary antibody. (A) Lateral view of an early tailbud (stage 22, top) and late neurula (stage 19, bottom) stage embryo. The staining is consistent with mRNA localization in the somites (red arrowhead) and in the cranial neural crest (black arrows and arrowhead. (B) Magnification of the anterior portion of a tailbud (stage 22) embryo. The somitic staining is strongest at the intersomitic border (red arrowheads). In the most anteriorsomites, the entire cell surface is stained. In the head area, staining is localized to the four cranial neural crest-derived segments (black arrows and arrowheads): posterior branchial crest (p), anterior branchial crest (a), hyoid crest (h), and mandibular crest segment (m). Staining of this last segment (m) completely surrounds the optic vesicle at this stage (black arrowhead). (C) Section of tailbud stage embryo triple-labeled with DATF-conjugated anti-rabbit secondary antibody, Texas-red-conjugated anti-mouse secondary antibody and DAPI. The X-ADAM 13 protein appears in green, fibronectin appears in red, and DAPI-labeled nuclei appear in blue. In this frontal section that passes through the somites and notochord, X-ADAM 13 protein is localized to individual myocyte end junctions. The fibronectin staining is present in the extracellular space between somites (appears yellow due to overlap with green X- ADAM 13 staining) as well as the periphery of the notochord (stained red). Nuclear staining indicates the organization of somitic cells. Primary antibodies are 6615 (anti-X-ADAM 13) and 4H2 (anti-fibronectin: Ramos and DeSimone, 1996).
Fig 10. Immunolocalization of X-ADAM 13 protein by immuno- fluorescence. Cryosectioned embryos were labeled with the 6615 affinity-purified antibody and DATF-conjugated secondary antibody. (A) Horizontal section through the head of a tailbud (stage 24) Xenopus embryo. The section passes throught the neural tube (nt) and the optic vesicles (ov). Note staining of cells along the outer layer of the brain and the optic vesicle. No staining is apparent in the epidermis. (B) Higher magnification view of a horizontal section through the head. This section passes through the neural tube (nt), the notochord (c) and the optic vesicle and is, therefore, more dorsal than in A. From left to right, the fluorescence is concentrated at the border between the sensory layer of the epidermis and the wall of the optic vesicle. The protein is also present in cells along the lateral region of the neural tube
