Abstract
During Xenopus laevis gastrulation, the basic body plan of the embryo is generated by movement of the marginal zone cells of the blastula into the blastocoel cavity. This morphogenetic process involves cell adhesion to the extracellular matrix protein fibronectin (FN). Regions of FN required for the attachment and migration of involuting marginal zone (IMZ) cells were analyzed in vitro using FN fusion protein substrates. IMZ cell attachment to FN is mediated by the Arg-Gly-Asp (RGD) sequence located in the type III-10 repeat and by the Pro-Pro-Arg- Arg-Ala-Arg (PPRRAR) sequence in the type III-13 repeat of the Hep II domain. IMZ cells spread and migrate persistently on fusion proteins containing both the RGD and synergy site sequence Pro-Pro-Ser-Arg-Asn (PPSRN) located in the type III-9 repeat. Cell recognition of the synergy site is positionally regulated in the early embryo. During gastrulation, IMZ cells will spread and migrate on FN whereas presumptive pre-involuting mesoderm, vegetal pole endoderm, and animal cap ectoderm will not. However, animal cap ectoderm cells acquire the ability to spread and migrate on the RGD/synergy region when treated with the mesoderm inducing factor activin-A. These data suggest that mesoderm induction activates the position-specific recognition of the synergy site of FN in vivo. Moreover, we demonstrate the functional importance of this site using a monoclonal antibody that blocks synergy region-dependent cell spreading and migration on FN. Normal IMZ movement is perturbed when this antibody is injected into the blastocoel cavity indicating that IMZ cell interaction with the synergy region is required for normal gastrulation.
Full Text
The Full Text of this article is available as a PDF (2.2 MB).
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Aota S., Nomizu M., Yamada K. M. The short amino acid sequence Pro-His-Ser-Arg-Asn in human fibronectin enhances cell-adhesive function. J Biol Chem. 1994 Oct 7;269(40):24756–24761. [PubMed] [Google Scholar]
- Barkalow F. J., Schwarzbauer J. E. Localization of the major heparin-binding site in fibronectin. J Biol Chem. 1991 Apr 25;266(12):7812–7818. [PubMed] [Google Scholar]
- Boucaut J. C., Darribère T., Boulekbache H., Thiery J. P. Prevention of gastrulation but not neurulation by antibodies to fibronectin in amphibian embryos. 1984 Jan 26-Feb 1Nature. 307(5949):364–367. doi: 10.1038/307364a0. [DOI] [PubMed] [Google Scholar]
- Boucaut J. C., Darribère T., Poole T. J., Aoyama H., Yamada K. M., Thiery J. P. 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. J Cell Biol. 1984 Nov;99(5):1822–1830. doi: 10.1083/jcb.99.5.1822. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Bowditch R. D., Hariharan M., Tominna E. F., Smith J. W., Yamada K. M., Getzoff E. D., Ginsberg M. H. Identification of a novel integrin binding site in fibronectin. Differential utilization by beta 3 integrins. J Biol Chem. 1994 Apr 8;269(14):10856–10863. [PubMed] [Google Scholar]
- Brickman M. C., Gerhart J. C. Heparitinase inhibition of mesoderm induction and gastrulation in Xenopus laevis embryos. Dev Biol. 1994 Aug;164(2):484–501. doi: 10.1006/dbio.1994.1218. [DOI] [PubMed] [Google Scholar]
- Busby T. F., Argraves W. S., Brew S. A., Pechik I., Gilliland G. L., Ingham K. C. Heparin binding by fibronectin module III-13 involves six discontinuous basic residues brought together to form a cationic cradle. J Biol Chem. 1995 Aug 4;270(31):18558–18562. doi: 10.1074/jbc.270.31.18558. [DOI] [PubMed] [Google Scholar]
- Chan B. M., Wong J. G., Rao A., Hemler M. E. T cell receptor-dependent, antigen-specific stimulation of a murine T cell clone induces a transient, VLA protein-mediated binding to extracellular matrix. J Immunol. 1991 Jul 15;147(2):398–404. [PubMed] [Google Scholar]
- Chapman M. D., Sutherland W. M., Platts-Mills T. A. Recognition of two Dermatophagoides pteronyssinus-specific epitopes on antigen P1 by using monoclonal antibodies: binding to each epitope can be inhibited by serum from dust mite-allergic patients. J Immunol. 1984 Nov;133(5):2488–2495. [PubMed] [Google Scholar]
- Danen E. H., Aota S., van Kraats A. A., Yamada K. M., Ruiter D. J., van Muijen G. N. Requirement for the synergy site for cell adhesion to fibronectin depends on the activation state of integrin alpha 5 beta 1. J Biol Chem. 1995 Sep 15;270(37):21612–21618. doi: 10.1074/jbc.270.37.21612. [DOI] [PubMed] [Google Scholar]
- Dawid I. B., Taira M. Axis determination in Xenopus: gradients and signals. Bioessays. 1994 Jun;16(6):385–386. doi: 10.1002/bies.950160603. [DOI] [PubMed] [Google Scholar]
- DeSimone D. W., Hynes R. O. Xenopus laevis integrins. Structural conservation and evolutionary divergence of integrin beta subunits. J Biol Chem. 1988 Apr 15;263(11):5333–5340. [PubMed] [Google Scholar]
- DeSimone D. W., Johnson K. E. The Xenopus embryo as a model system for the study of cell-extracellular matrix interactions. Methods Cell Biol. 1991;36:527–539. [PubMed] [Google Scholar]
- DeSimone D. W., Norton P. A., Hynes R. O. Identification and characterization of alternatively spliced fibronectin mRNAs expressed in early Xenopus embryos. Dev Biol. 1992 Feb;149(2):357–369. doi: 10.1016/0012-1606(92)90291-n. [DOI] [PubMed] [Google Scholar]
- Drake S. L., Klein D. J., Mickelson D. J., Oegema T. R., Furcht L. T., McCarthy J. B. Cell surface phosphatidylinositol-anchored heparan sulfate proteoglycan initiates mouse melanoma cell adhesion to a fibronectin-derived, heparin-binding synthetic peptide. J Cell Biol. 1992 Jun;117(6):1331–1341. doi: 10.1083/jcb.117.6.1331. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Garcia-Pardo A., Wayner E. A., Carter W. G., Ferreira O. C., Jr Human B lymphocytes define an alternative mechanism of adhesion to fibronectin. The interaction of the alpha 4 beta 1 integrin with the LHGPEILDVPST sequence of the type III connecting segment is sufficient to promote cell attachment. J Immunol. 1990 May 1;144(9):3361–3366. [PubMed] [Google Scholar]
- George E. L., Georges-Labouesse E. N., Patel-King R. S., Rayburn H., Hynes R. O. Defects in mesoderm, neural tube and vascular development in mouse embryos lacking fibronectin. Development. 1993 Dec;119(4):1079–1091. doi: 10.1242/dev.119.4.1079. [DOI] [PubMed] [Google Scholar]
- Guan J. L., Hynes R. O. Lymphoid cells recognize an alternatively spliced segment of fibronectin via the integrin receptor alpha 4 beta 1. Cell. 1990 Jan 12;60(1):53–61. doi: 10.1016/0092-8674(90)90715-q. [DOI] [PubMed] [Google Scholar]
- Guan K. L., Dixon J. E. Eukaryotic proteins expressed in Escherichia coli: an improved thrombin cleavage and purification procedure of fusion proteins with glutathione S-transferase. Anal Biochem. 1991 Feb 1;192(2):262–267. doi: 10.1016/0003-2697(91)90534-z. [DOI] [PubMed] [Google Scholar]
- Gurdon J. B. Methods for nuclear transplantation in amphibia. Methods Cell Biol. 1977;16:125–139. doi: 10.1016/s0091-679x(08)60096-5. [DOI] [PubMed] [Google Scholar]
- Harland R. M. In situ hybridization: an improved whole-mount method for Xenopus embryos. Methods Cell Biol. 1991;36:685–695. doi: 10.1016/s0091-679x(08)60307-6. [DOI] [PubMed] [Google Scholar]
- Haugen P. K., McCarthy J. B., Skubitz A. P., Furcht L. T., Letourneau P. C. Recognition of the A chain carboxy-terminal heparin binding region of fibronectin involves multiple sites: two contiguous sequences act independently to promote neural cell adhesion. J Cell Biol. 1990 Dec;111(6 Pt 1):2733–2745. doi: 10.1083/jcb.111.6.2733. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hemsley A., Arnheim N., Toney M. D., Cortopassi G., Galas D. J. A simple method for site-directed mutagenesis using the polymerase chain reaction. Nucleic Acids Res. 1989 Aug 25;17(16):6545–6551. doi: 10.1093/nar/17.16.6545. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hoogenraad N. J., Wraight C. J. The effect of pristane on ascites tumor formation and monoclonal antibody production. Methods Enzymol. 1986;121:375–381. doi: 10.1016/0076-6879(86)21036-8. [DOI] [PubMed] [Google Scholar]
- Howard J. E., Hirst E. M., Smith J. C. Are beta 1 integrins involved in Xenopus gastrulation? Mech Dev. 1992 Aug;38(2):109–119. doi: 10.1016/0925-4773(92)90003-3. [DOI] [PubMed] [Google Scholar]
- Howard J. E., Smith J. C. Analysis of gastrulation: different types of gastrulation movement are induced by different mesoderm-inducing factors in Xenopus laevis. Mech Dev. 1993 Sep;43(1):37–48. doi: 10.1016/0925-4773(93)90021-o. [DOI] [PubMed] [Google Scholar]
- Humphries M. J., Komoriya A., Akiyama S. K., Olden K., Yamada K. M. Identification of two distinct regions of the type III connecting segment of human plasma fibronectin that promote cell type-specific adhesion. J Biol Chem. 1987 May 15;262(14):6886–6892. [PubMed] [Google Scholar]
- Hynes R. O. Integrins: versatility, modulation, and signaling in cell adhesion. Cell. 1992 Apr 3;69(1):11–25. doi: 10.1016/0092-8674(92)90115-s. [DOI] [PubMed] [Google Scholar]
- Joos T. O., Whittaker C. A., Meng F., DeSimone D. W., Gnau V., Hausen P. Integrin alpha 5 during early development of Xenopus laevis. Mech Dev. 1995 Apr;50(2-3):187–199. doi: 10.1016/0925-4773(94)00335-k. [DOI] [PubMed] [Google Scholar]
- Keller R., Winklbauer R. Cellular basis of amphibian gastrulation. Curr Top Dev Biol. 1992;27:39–89. doi: 10.1016/s0070-2153(08)60532-3. [DOI] [PubMed] [Google Scholar]
- Komoriya A., Green L. J., Mervic M., Yamada S. S., Yamada K. M., Humphries M. J. The minimal essential sequence for a major cell type-specific adhesion site (CS1) within the alternatively spliced type III connecting segment domain of fibronectin is leucine-aspartic acid-valine. J Biol Chem. 1991 Aug 15;266(23):15075–15079. [PubMed] [Google Scholar]
- Lee G., Hynes R., Kirschner M. Temporal and spatial regulation of fibronectin in early Xenopus development. Cell. 1984 Mar;36(3):729–740. doi: 10.1016/0092-8674(84)90353-2. [DOI] [PubMed] [Google Scholar]
- McCarthy J. B., Chelberg M. K., Mickelson D. J., Furcht L. T. Localization and chemical synthesis of fibronectin peptides with melanoma adhesion and heparin binding activities. Biochemistry. 1988 Feb 23;27(4):1380–1388. doi: 10.1021/bi00404a044. [DOI] [PubMed] [Google Scholar]
- Mould A. P., Komoriya A., Yamada K. M., Humphries M. J. The CS5 peptide is a second site in the IIICS region of fibronectin recognized by the integrin alpha 4 beta 1. Inhibition of alpha 4 beta 1 function by RGD peptide homologues. J Biol Chem. 1991 Feb 25;266(6):3579–3585. [PubMed] [Google Scholar]
- Nakatsuji N., Johnson K. E. Conditioning of a culture substratum by the ectodermal layer promotes attachment and oriented locomotion by amphibian gastrula mesodermal cells. J Cell Sci. 1983 Jan;59:43–60. doi: 10.1242/jcs.59.1.43. [DOI] [PubMed] [Google Scholar]
- Newport J., Kirschner M. A major developmental transition in early Xenopus embryos: I. characterization and timing of cellular changes at the midblastula stage. Cell. 1982 Oct;30(3):675–686. doi: 10.1016/0092-8674(82)90272-0. [DOI] [PubMed] [Google Scholar]
- Niehrs C., Keller R., Cho K. W., De Robertis E. M. The homeobox gene goosecoid controls cell migration in Xenopus embryos. Cell. 1993 Feb 26;72(4):491–503. doi: 10.1016/0092-8674(93)90069-3. [DOI] [PubMed] [Google Scholar]
- Pierschbacher M. D., Ruoslahti E. Cell attachment activity of fibronectin can be duplicated by small synthetic fragments of the molecule. Nature. 1984 May 3;309(5963):30–33. doi: 10.1038/309030a0. [DOI] [PubMed] [Google Scholar]
- Pytela R., Pierschbacher M. D., Ruoslahti E. Identification and isolation of a 140 kd cell surface glycoprotein with properties expected of a fibronectin receptor. Cell. 1985 Jan;40(1):191–198. doi: 10.1016/0092-8674(85)90322-8. [DOI] [PubMed] [Google Scholar]
- Ransom D. G., Hens M. D., DeSimone D. W. Integrin expression in early amphibian embryos: cDNA cloning and characterization of Xenopus beta 1, beta 2, beta 3, and beta 6 subunits. Dev Biol. 1993 Nov;160(1):265–275. doi: 10.1006/dbio.1993.1304. [DOI] [PubMed] [Google Scholar]
- Ruoslahti E. Proteoglycans in cell regulation. J Biol Chem. 1989 Aug 15;264(23):13369–13372. [PubMed] [Google Scholar]
- Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Saunders S., Bernfield M. Cell surface proteoglycan binds mouse mammary epithelial cells to fibronectin and behaves as a receptor for interstitial matrix. J Cell Biol. 1988 Feb;106(2):423–430. doi: 10.1083/jcb.106.2.423. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Schwarzbauer J. E. Alternative splicing of fibronectin: three variants, three functions. Bioessays. 1991 Oct;13(10):527–533. doi: 10.1002/bies.950131006. [DOI] [PubMed] [Google Scholar]
- Sekiguchi K., Hakomori S. Functional domain structure of fibronectin. Proc Natl Acad Sci U S A. 1980 May;77(5):2661–2665. doi: 10.1073/pnas.77.5.2661. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Shih J., Keller R. The epithelium of the dorsal marginal zone of Xenopus has organizer properties. Development. 1992 Dec;116(4):887–899. doi: 10.1242/dev.116.4.887. [DOI] [PubMed] [Google Scholar]
- Shimizu Y., Van Seventer G. A., Horgan K. J., Shaw S. Regulated expression and binding of three VLA (beta 1) integrin receptors on T cells. Nature. 1990 May 17;345(6272):250–253. doi: 10.1038/345250a0. [DOI] [PubMed] [Google Scholar]
- Skorstengaard K., Jensen M. S., Petersen T. E., Magnusson S. Purification and complete primary structures of the heparin-, cell-, and DNA-binding domains of bovine plasma fibronectin. Eur J Biochem. 1986 Jan 2;154(1):15–29. doi: 10.1111/j.1432-1033.1986.tb09353.x. [DOI] [PubMed] [Google Scholar]
- Smith D. B., Johnson K. S. Single-step purification of polypeptides expressed in Escherichia coli as fusions with glutathione S-transferase. Gene. 1988 Jul 15;67(1):31–40. doi: 10.1016/0378-1119(88)90005-4. [DOI] [PubMed] [Google Scholar]
- Smith J. C., Price B. M., Green J. B., Weigel D., Herrmann B. G. Expression of a Xenopus homolog of Brachyury (T) is an immediate-early response to mesoderm induction. Cell. 1991 Oct 4;67(1):79–87. doi: 10.1016/0092-8674(91)90573-h. [DOI] [PubMed] [Google Scholar]
- Smith J. C., Symes K., Hynes R. O., DeSimone D. Mesoderm induction and the control of gastrulation in Xenopus laevis: the roles of fibronectin and integrins. Development. 1990 Feb;108(2):229–238. doi: 10.1242/dev.108.2.229. [DOI] [PubMed] [Google Scholar]
- Smith J. C., Yaqoob M., Symes K. Purification, partial characterization and biological effects of the XTC mesoderm-inducing factor. Development. 1988 Jul;103(3):591–600. doi: 10.1242/dev.103.3.591. [DOI] [PubMed] [Google Scholar]
- Sokol S. Y. The pregastrula establishment of gene expression pattern in Xenopus embryos: requirements for local cell interactions and for protein synthesis. Dev Biol. 1994 Dec;166(2):782–788. doi: 10.1006/dbio.1994.1356. [DOI] [PubMed] [Google Scholar]
- Wayner E. A., Kovach N. L. Activation-dependent recognition by hematopoietic cells of the LDV sequence in the V region of fibronectin. J Cell Biol. 1992 Jan;116(2):489–497. doi: 10.1083/jcb.116.2.489. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Whittaker C. A., DeSimone D. W. Integrin alpha subunit mRNAs are differentially expressed in early Xenopus embryos. Development. 1993 Apr;117(4):1239–1249. doi: 10.1242/dev.117.4.1239. [DOI] [PubMed] [Google Scholar]
- Wight T. N., Kinsella M. G., Qwarnström E. E. The role of proteoglycans in cell adhesion, migration and proliferation. Curr Opin Cell Biol. 1992 Oct;4(5):793–801. doi: 10.1016/0955-0674(92)90102-i. [DOI] [PubMed] [Google Scholar]
- Williams M. J., Hughes P. E., O'Toole T. E., Ginsberg M. H. The inner world of cell adhesion: integrin cytoplasmic domains. Trends Cell Biol. 1994 Apr;4(4):109–112. doi: 10.1016/0962-8924(94)90059-0. [DOI] [PubMed] [Google Scholar]
- Winklbauer R. Differential interaction of Xenopus embryonic cells with fibronectin in vitro. Dev Biol. 1988 Nov;130(1):175–183. doi: 10.1016/0012-1606(88)90424-1. [DOI] [PubMed] [Google Scholar]
- Winklbauer R. Mesodermal cell migration during Xenopus gastrulation. Dev Biol. 1990 Nov;142(1):155–168. doi: 10.1016/0012-1606(90)90159-g. [DOI] [PubMed] [Google Scholar]
- Winklbauer R., Selchow A. Motile behavior and protrusive activity of migratory mesoderm cells from the Xenopus gastrula. Dev Biol. 1992 Apr;150(2):335–351. doi: 10.1016/0012-1606(92)90246-d. [DOI] [PubMed] [Google Scholar]
- Winklbauer R., Selchow A., Nagel M., Angres B. Cell interaction and its role in mesoderm cell migration during Xenopus gastrulation. Dev Dyn. 1992 Dec;195(4):290–302. doi: 10.1002/aja.1001950407. [DOI] [PubMed] [Google Scholar]
- Woods A., Couchman J. R., Johansson S., Hök M. Adhesion and cytoskeletal organisation of fibroblasts in response to fibronectin fragments. EMBO J. 1986 Apr;5(4):665–670. doi: 10.1002/j.1460-2075.1986.tb04265.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Wu C., Keivens V. M., O'Toole T. E., McDonald J. A., Ginsberg M. H. Integrin activation and cytoskeletal interaction are essential for the assembly of a fibronectin matrix. Cell. 1995 Dec 1;83(5):715–724. doi: 10.1016/0092-8674(95)90184-1. [DOI] [PubMed] [Google Scholar]
- Yamada K. M. Fibronectins: structure, functions and receptors. Curr Opin Cell Biol. 1989 Oct;1(5):956–963. doi: 10.1016/0955-0674(89)90065-3. [DOI] [PubMed] [Google Scholar]