PDGF Signalling Is Required for Gastrulation of Xenopus Laevis

Development 121, 3099-3110 (1995) 3099 Printed in Great Britain © The Company of Biologists Limited 1995 PDGF signalling is required for gastrulation of Xenopus laevis Paris Ataliotis1, Karen Symes1, Margaret M. Chou1, Lap Ho2 and Mark Mercola1,* 1Department of Cell Biology, Harvard Medical School, 25 Shattuck Street, Boston MA 02115, USA 2Department of Pharmacology, Cornell University Medical College, 1300 York Avenue, New York NY 10021, USA *Author for correspondence SUMMARY During Xenopus gastrulation, platelet-derived growth closure of the blastopore, leads to abnormal gastrulation factor (PDGF) receptor-α is expressed in involuting and a loss of anterior structures. Convergent extension is marginal zone cells which migrate over ectodermal cells not inhibited in these embryos, but rather, involuting meso- expressing PDGF-A. To investigate the role of PDGF sig- dermal cells fail to adhere to the overlying ectoderm. PDGF nalling during this process, we have generated a novel point may therefore be required for mesodermal cell-substratum mutant of PDGF receptor-α analogous to the W37 mutation interaction. of c-kit. This molecule is a specific, potent, dominant inhibitor of PDGF signalling in vivo. Injection of RNA encoding this protein into Xenopus embryos prevents Key words: Xenopus, PDGF, gastrulation, dominant negative INTRODUCTION to block colony stimulating factor-1-dependent transformation of Rat-2 cells (Reith et al., 1993). Gastrulation involves a complex, co-ordinated series of cell Here we demonstrate that a point mutant of Xenopus movements that give rise to the body plan in all vertebrates. In PDGFR-α analogous to the W37 mutation of c-kit lacks the frog, Xenopus laevis, this process begins with the involu- detectable kinase activity and acts in a dominant negative tion of cells on the dorsal side of the embryo. Apical constric- manner to inhibit signalling by the wild-type receptor in vivo. tion of the outermost epithelial cells gives rise to the blasto- We have used this mutant receptor to disrupt PDGF-A sig- pore lip and precedes the involution of mesodermal cells from nalling in the Xenopus embryo and show that PDGF is the marginal zone (for review, see Keller, 1991). As these necessary for normal gastrulation but is not required for mesodermal cells involute, they migrate over the inner surface mesoderm induction. We further demonstrate that defects arise of the blasotcoel roof toward the future anterior end of the due to the inappropriate behaviour of mesodermal cells and embryo. The migration and fate of these involuting cells are discuss possible mechanisms that may lead to these abnormal- presumed to be controlled by extracellular signals, although ities. these are as yet poorly understood. A candidate signalling molecule that may be involved in gastrulation is platelet-derived growth factor A (PDGF-A, MATERIALS AND METHODS Jones et al., 1993). We now show by in situ hybridisation that PDGF-A mRNA is present in the blastocoel roof of the early Embryos gastrula stage Xenopus embryo. This tissue forms the substra- Xenopus embryos were fertilized in vitro and chemically dejellied tum for the migration of marginal zone cells which express the using 2% cysteine-HCl, pH 7.8, then maintained in 10% Marc’s receptor for PDGF-A, PDGFR-α. modified Ringer’s (0.1× MMR, Peng, 1991) at temperatures between In order to assess the role of PDGF-A during Xenopus gas- 13 and 20°C until they had reached the appropriate stages (according trulation, we have generated a novel, dominant negative mutant to Nieuwkoop and Faber, 1967) for injection or analysis. Microinjec- of its receptor, PDGFR-α. This mutant is based on the W37 tion was performed in a solution of 3% Ficoll in 1× MMR using mutation of the homologous receptor tyrosine kinase c-kit bevelled, glass capillary needles. Xenopus animal poles were (Nocka et al., 1990; Reith et al., 1990). The amino acid residue explanted at stage 8 and maintained in 75% MMR/0.1% bovine serum affected in W37 is conserved throughout the PDGF receptor albumin until control embryos had reached the required stage. Growth factors (50 ng/ml PDGF-AA, Gibco-BRL, 500 ng/ml bFGF, Amgen family and is therefore thought to be essential for function. and 2 U/ml βA activin, from transfected COS cell supernatant) were 37 Indeed, a mutation of c-fms corresponding to W c-kit added directly to this medium immediately after explants were abolishes the kinase activity of this receptor. More importantly, excised. Exogastrulae were induced by manually removing the the W-like fms mutants can act in a trans-dominant manner to vitelline membrane of embryos at stage 8 and transferring to 1× MMR prevent tyrosine phosphorylation of the wild-type receptor and in dishes coated with 1% agarose in 1× MMR. β-galactosidase activity 3100 P. Ataliotis and others was detected by standard procedures using the chromogenic substrate slides and hybridised to 35S-labeled RNA probes as described X-gal. (O’Keefe et al., 1991). Serial sections were hybridised to sense and antisense cRNA probes for Xenopus PDGF-A (Mercola et al., 1988) In situ hybridisation and PDGFR-α (Jones et al., 1993). In no case was a hybridisation For radioactive in situ hybridisation experiments, albino embryos signal detected with sense probes. After washing, slides were coated were fixed in 4% paraformaldehyde in 70% PBS for 1 hour at 4°C with Kodak NTB-2 emulsion and exposed for 2 weeks at 4°C. For prior to embedding in paraffin. 8 µm sections were mounted on glass whole-mount in situ hybridisation, pigmented embryos were fixed in Fig. 1. In situ hybridisation of PDGF A and PDGFRα mRNA during the gastrula stages of development. Serial sections of gastrula staged embryos were probed with 35S-labelled antisense PDGF-A and PDGFR-α cRNA probes. PDGF A is expressed in the presumptive ectoderm (A,C,E) and PDGFR-α is expressed in the presumptive mesoderm (B,D,F). (A,B) Sagittal sections through the dorsal midline of a stage 10.25 embryo. (C,D) Parasagittal and (E,F) horizontal sections through a stage 12 embryo. An, animal pole; Vg vegetal pole; D, dorsal; V ventral; A, anterior; P, posterior; bl. blastocoel; a, archenteron. Scale bar, 100 µm. PDGF in Xenopus gastrulation 3101 4% formaldehyde, dehydrated in methanol and then processed as RT-PCR described (Ho et al., 1994). Digoxigenin-labelled cRNA probes for RNA prepared from animal pole explants was reverse transcribed goosecoid and brachyury (Amaya et al., 1993) were the kind gift of using the Stratascript RT-PCR kit (Stratagene) according to manu- Pascal Stein. Embryos were dehydrated in methanol and in some cases facturer’s instructions. cDNA was amplified by PCR in the presence bleached in 10% hydrogen peroxide and cleared in benzyl of [32P]dATP using primers specific for EF1-α and brachyury as alcohol:benzyl benzoate (BA:BB, 1:2) prior to photography. described (Wilson and Melton, 1994). Amplified cDNA products were detected by autoradiography following electrophoresis on 5% Site-directed mutagenesis non-denaturing polyacrylamide gels. A cDNA clone encoding the full-length Xenopus PDGFR-α (Jones et al., 1993) was subcloned as a SacI/BglII fragment into pALTER-1 (Promega) and mutagenised by single-stranded, site-directed mutage- RESULTS nesis according to manufacturer’s instructions. A control frameshift mutation was generated in the full-length receptor by digestion with Distribution of PDGF-A and PDGFR-α in Xenopus KpnI, blunting and re-ligation, causing premature termination in the embryos extracellular domain of the protein at amino acid 159. Using in situ hybridisation we have determined that PDGF-A DNA cloning/RNA production mRNA is distributed uniformly throughout the inner layers of ectodermal cells of the blastocoel roof, but not in the superfi- The wild-type, frameshift and mutagenised Xenopus PDGFR-α were ′ cial layer of early gastrula stage Xenopus embryos (Fig. 1A). subcloned into pGHE2, a vector containing a poly(A) tail and 5 and α 3′ untranslated sequences from Xenopus β-globin (a gift from the late PDGFR- mRNA is localised to the equatorial region of the Peter Hess, Harvard Medical School) such that sense RNA transcripts embryo in cells of the marginal zone (Fig. 1B). During gas- were produced by using T7 RNA polymerase. Capped mRNA was trulation, cells of the marginal zone begin to involute at the made using the mMessage Machine system (Ambion) according to dorsal side of the embryo and move over the inner surface of manufacturer’s instructions. β-galactosidase mRNA containing a the blastocoel roof towards the future anterior end of the nuclear localisation signal was produced from the SP64nuc-βgal embryo (reviewed in Keller, 1991). These cells express plasmid (Vize and Melton, 1991). PDGFR-α and first come into contact with cells expressing PDGF-A shortly after the onset of gastrulation. This comple- Phosphotyrosine detection mentary pattern of ligand and receptor expression persists COS cells were passaged at 1×106 cells per 100 mm dish, 24 hours throughout gastrulation (Fig. 1C-F) and suggests that sig- before DEAE-Dextran transfection. Cells were transfected with either nalling between these cell populations may play an important 10 µg pMT2, 2 µg or 5 µg Xenopus PDGFR-α in pMT2, 10 µg µ α µ role in controlling cellular movement or differentiation during PDGFR-37 in pMT2, 5 g Xenopus PDGFR- plus 5 g PDGFR-37 this process. or 2 µg Xenopus PDGFR-α plus 8 µg PDGFR-37. The total amount µ of DNA was adjusted to 10 g with pMT2 where necessary. After 24 Construction of a dominant negative mutant of hours, cells were transferred to DMEM containing 0.5% calf serum α for a further 24 hours. Cells were treated with 20 ng/ml PDGF-BB PDGFR- (Gibco-BRL) for 10 minutes, washed in ice-cold PBS, then lysed as To examine the function of PDGF-A during Xenopus devel- described previously (Chen and Blenis, 1990).

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