Development 108, 229-238 (1990) 229 Printed in Great Britain ©The Company of Biologists Limited 1990 Mesoderm induction and the control of gastrulation in Xenopus laevis: the roles of fibronectin and integrins J. C. SMITH1, K. SYMESH, R. O. HYNES2'3 and D. DeSIMONE3* ' Laboratory of Embryogenesis, National Institute for Medical Research, The Ridgeway, Mill Hill, London NW71AA, UK ^Howard Hughes Medical Institute and ^Center for Cancer Research, Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA * Present address: University of Virginia, Health Sciences Center, Department of Anatomy and Cell Biology, Box 439, School of Medicine, Charlottesville, VA 22908, USA t Present address: Department of Cell and Molecular Biology, 385 LSA, University of California, Berkeley, CA 94720, USA Summary Exposure of isolated Xenopus animal pole ectoderm to diated cell migration is not required for convergent the XTC mesoderm-inducing factor (XTC-MIF) causes extension. the tissue to undergo gastrulation-like movements. In We have investigated the molecular basis of XTC- this paper, we take advantage of this observation to MIF-induced gastrulation-like movements by measuring investigate the control of various aspects of gastrulation rates of synthesis of fibronectin and of the integrin f}y in Xenopus. chain in induced and control explants. No significant Blastomcres derived from induced animal pole regions differences were observed, and this suggests that gastru- are able, like marginal zone cells, but unlike control lation is not initiated simply by control of synthesis of animal pole blastomeres, to spread and migrate on a these molecules. In future work, we intend to investigate fibronectin-coated surface. Dispersed animal pole cells synthesis of other integrin subunits and to examine are also able to respond to XTC-MIF in this way; this is possible post-translational modifications to fibronectin one of the few mesoderm-specific responses to induction and the integrins. that has been observed in single cells. The ability of induced animal pole cells to spread on fibronectin is abolished by the peptide GRGDSP. How- Key words: gastrulation, fibronectin, integrins, mesoderm ever, the elongation of intact explants is unaffected by induction, mesoderm-inducing factors, XTC-MIF, this peptide. This may indicate that fibronectin-me- amphibian embryo, Xenopus laevis. Introduction these molecules, and this idea is supported by studies of the temporal expression of fibronectin and integrins. Two lines of evidence indicate that fibronectin plays an The rate of synthesis of fibronectin increases dramati- important role in amphibian gastrulation. First, fibro- cally at the mid-blastula transition (MBT; see Newport nectin is localised to the roof of the blastocoel, the and Kirschner, 1982), and the protein is first detectable surface on which presumptive mesodermal cells migrate immunocytochemically about three hours later, at the (Boucaut and Darribere, 1983; Lee et al. 1984; Nakat- beginning of gastrulation (Boucaut and Darribere, suji et al. 1985a). Second, if antibodies to fibronectin or 1983; Lee et al. 1984). This increase in fibronectin its receptors, integrins, are microinjected into the synthesis does not require RNA synthesis, and must blastocoels of amphibian embryos, gastrulation is in- involve activation of maternal message. The integTins hibited (Boucaut et al. 1984a; Darribere et al. 1988). consist of a-and /S subunits (see Hynes, 1987). Analysis Similar results are obtained by injecting synthetic pep- of Xenopus Pi subunit mRNA shows that expression tides corresponding to a major cell-binding site of the begins around the gastrula stage (DeSimone and fibronectin molecule (Boucaut et al. 19846). Hynes, 1988); information is not yet available about the Although this work demonstrates that fibronectin a subunits. and its receptors are required for gastrulation to pro- These observations do not, .of course, prove that ceed, it does not address the question of how gastru- gastrulation is controlled by synthesis of fibronectin and lation is initiated and controlled. The simplest sugges- its receptor. The similarity in timing may be coinciden- tion is that gastrulation is triggered by the synthesis of tal, and indeed transcription of many genes first occurs 230 J. C. Smith and others at the MBT or soon afterwards (see, for example, multiwell or microtitre plates (Nunc), were treated at room 1 Sargent and Dawid, 1983; Kreig and Melton, 1985). In temperature for 4-18 h with 20-100/tg ml" bovine or rat this paper, we test the functions of fibronectin and plasma fibronectin, kindly supplied by Dr Heather Streeter integrins more directly by taking advantage of the (NIMR) or Mr Terry Butters and Dr Colin Hughes (NIMR). recent observation that the XTC-mesoderm-inducing The surfaces were then rinsed and 'blocked' with NAM containing 0.5 % bovine serum albumin (BSA) for 20min. factor (XTC-MIF; see Smith, 1987; Rosa et al. 1988; Smith et al. 1988; Dawid et al. 1989; Smith, 1989) Embryo dissections induces gastrulation-like movements in isolated animal Mid-blastula Xenopus embryos (stages 8-9) were dissected pole regions (Symes and Smith, 1987; Cooke and into animal, marginal zone, and vegetal pole regions using Smith, 1989). This observation makes it possible to electrolytically sharpened tungsten needles. manipulate gastrulation and thus ask how its various aspects are controlled. Cell spreading assays Our results indicate that animal pole blastomeres Animal pole or dorsal marginal zone regions were transferred exposed to XTC-MIF, whether as intact explants or as to calcium- and magnesium-free medium (CMFM: 100 ITIM- NaCl, 5mM-KCl, lmM-NaHCO , 2.5mM-sodium phosphate, isolated cells, are able to spread and migrate on a 3 pH7.5 and 0.25 % (w/v) gentamycin sulphate; Sargent et al. fibronectin-coated substrate. In this respect, they re- 1986). The outer layer of cells, which is difficult to dissociate, semble prospective mesodermal cells, but differ from was discarded and the inner layer was disaggregated into a uninduced animal pole cells (Nakatsuji, 1986). How- single-cell suspension. These cells were seeded onto fibronec- ever, acquisition of the ability to spread on fibronectin tin-coated surfaces in NAM containing 0.5% BSA and, is not accompanied by an increase in synthesis of where appropriate, XTC-MIF, and they were scored 1-2 h fibronectin or of the integTin ft chain. We conclude later. from this that initiation of gastrulation is not controlled Cells could be divided into three classes (see Fig. 1 for through synthesis of these proteins, although it is examples). Non-adherent cells are spherical and move about possible that existing molecules are redistributed within the dish when it is disturbed. Adherent cells cannot easily be cells or undergo post-translational modification. dislodged by disturbing the dish, but like non-adherent cells they are roughly spherical. They do not flatten but frequently The ability of both XTC-MIF-induced animal pole undergo 'circus movements', during which cytoplasmic pro- blastomeres and of presumptive mesodermal cells to trusions move slowly around the circumference of the cell. spread on fibronectin is abolished by the peptide Spread cells adhere to the substrate, flatten, and send out GRGDSP, which contains the fibronectin cell attach- processes. They can sometimes be seen to migrate over the ment site (Pierschbacher and Ruoslahti, 1984; Yamada fibronectin-coated substrate (see Fig. 4). Spread cells are and Kennedy, 1984). However, the elongation of clearly distinct from adherent cells. induced animal pole explants is unaffected by this Cultures were scored independently by two observers peptide. This suggests that the 'convergent extension' (J.C.S. and K.S.), one of whom was ignorant of the coding of movements of gastrulation (see Keller, 1986; Keller and the experiment. The two assessments did not differ signifi- Danilchik, 1988; Keller and Tibbetts, 1989) do not cantly. depend on interactions involving RGD sites in fibronec- Antibodies and inhibitory peptides tin or other matrix molecules. These experiments used a rabbit antiserum raised against Xenopus plasma fibronectin (Heasman et al. 1981) and a rabbit antiserum raised against a 39-amino acid peptide Materials and methods corresponding to a COOH-terminal portion of vertebrate integrin /Si subunits (Marcantonio and Hynes, 1988). The Embryos synthetic peptides GRGDSP and GRGESP were synthesized Embryos of Xenopus laevis were obtained by artificial fertiliz- on an Applied Biosystems Peptide Synthesizer and purified by ation as described by Smith and Slack (1983). They were HPLC by Gene Yee, to whom we are very grateful. chemically dejellied using 2% cysteine hydrochloride (pH7.8-8.1), washed and transferred to Petri dishes coated Metabolic labelling of embryonic tissue and with 1% Noble agar and containing 10% normal amphibian immunoprecipitation medium (NAM: Slack, 1984). The embryos were staged Dissected pieces of mid-blastula Xenopus embryos were according to Nieuwkoop and Faber (1967). incubated in NAM, with or without XTC-MIF as appropriate, in the presence of 0.3-0.9 mCimP1 [35S]methionine at room XTC-mesoderm-inducing factor temperature (19-22°C). At the end of the incubation period, XTC-MIF was partially purified from heated XTC-cell-con- the tissues were rinsed three or four times in NAM and frozen ditioned medium by DEAE-Sepharose chromatography fol- on dry ice in a minimum volume of fluid. lowed by phenyl Sepharose chromatography, as described by Samples for immunoprecipitation using the anti-fibronectin Cooke et al. (1987) and Smith et al. (1988). One unit of antibody were homogenized in 2 M-urea as described by Lee et mesoderm-inducing activity is defined as the minimum quan- al. (1984). Samples for analysis using the anti-integrin anti- tity that must be present in lml medium for induction to body were homogenized in O.lM-NaCl, 1% Triton X-100, occur. The sample of partially purified XTC-MIF used in lmM-PMSF, 0.22 trypsin inhibitor unit (TIU) ml"' aprotinin, these experiments contained approximately 7.7X103 units 1 and 20mM-Tris, pH7.6.