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???displayArticle.abstract??? Nck is a widely expressed SH2/SH3 adaptor protein containing one SH2 and three SH3 domains. Although Nck is assumed to mediate the formation of protein-protein complexes during signaling, little is currently known about its specific function. We have constructed a series of Nck SH3 and SH2 domain mutants, expressed them in Xenopus laevis embryos, and monitored injected embryos for developmental abnormalities. This approach allows correlation of developmental phenotypes with the presence or absence of specific Nck protein-binding domains. We show that microinjection of RNA-encoding Nck with an inactivating mutation in the third SH3 domain (NckK229) into dorsal blastomeres of early embryos caused anterior truncation with high frequency, and membrane localization of both the first and second SH3 domains together was sufficient to induce this anterior-truncation phenotype. Molecular marker analysis of explants revealed that the expression of NckK229 ventralized dorsal mesoderm. Lineage tracing experiments demonstrated that the expression of Nck K229 in dorsal blastomeres affected the migratory properties of mesoderm cells in gastrulation and led to the adoption of a more posterior fate. These data suggest that protein(s) that bind the first and second SH3 domains of Nck can affect the response to signals that establish dorso-ventral patterning, and that protein(s) that bind the third SH3 domain antagonize the ventralizing effect of the first two SH3 domains.
Figure 1 Xenopus Nck mRNA expression during embryonic development. Equal amounts of poly(A)+ RNA extracted from embryos at stage indicated above were loaded in each lane and blot was hybridized to full-length Xenopus Nck cDNA probe. Hybridization with ornithine decarboxylase (ODC) riboprobe (Top) is a control for RNA loading.
Figure 2 Ventralization induced by expression of Nck constructs. The structure of proteins encoded by microinjected RNAs is represented. Synthetic mRNA was injected into marginal zone region of the two dorsal blastomeres at four-cell stage. Phenotypes were scored morphologically at stage 36 according to the dorso-anterior index described previously (26). SH2 and SH3 domains are represented by shaded boxes; mutant domains in which ligand binding activity was ablated are indicated by large “X†on diagram. Amino acid number of residues substituted by lysine (K) is indicated on left. For truncation mutants (Bottom), number in shaded box indicates first, second, or third SH3 domain. Wavy line, myristoylation signal. ∗∗, 10 embryos (10 of 19) also had “split tail†resulting from incomplete closure of the blastopore. ∗, All embryos had “split tail.â€
Figure 3 Nck K229 interferes with head and anterior notochord development. (A) Phenotype of embryos microinjected with KALL control mRNA (top embryo) or K229 mRNA (bottom four embryos) as in Fig. 2 legend. (B–E) Histological analysis of KALL-injected control embryos (B and D) and K229-injected embryos (C and E). Embryos were fixed at stage 36, sectioned, and stained with hematoxylin/eosin. (B and C) Transverse sections at the level of the eye in the control embryo. Note absence of notochord (no), headmesenchyme and brainventricle (bv) in C. (D and E) Sagittal (midline) sections. Note absence of headmesenchyme (hm) and anterior notochord in E. (Bar = 100 μm.)
Figure 5 Nck K229 disrupts migration of mesoderm cells. mRNA (0.1 ng) encoding nuclear-localized β-galactosidase plus 0.2 ng of either KALL control (A and C) or K229 (B and D) mRNA was injected into C1 blastomeres of 32-cell embryos. Embryos were fixed and stained with X-Gal at stage 12 (A and B) or stage 30 (C and D). (A and B) Embryos are positioned with animal pole at top and dorsal side to the right. (Bar = 100 μm.)
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