Ataliotis P et al. (1995), PDGF signalling is required for gastrulation of...

XB-ART-19327

Development 1995 Sep 01;1219:3099-110.

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PDGF signalling is required for gastrulation of Xenopus laevis.

Ataliotis P , Symes K , Chou MM , Ho L , Mercola M .

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During Xenopus gastrulation, platelet-derived growth factor (PDGF) receptor-alpha is expressed in involuting marginal zone cells which migrate over ectodermal cells expressing PDGF-A. To investigate the role of PDGF signalling during this process, we have generated a novel point mutant of PDGF receptor-alpha analogous to the W37 mutation 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 closure of the blastopore, leads to abnormal gastrulation and a loss of anterior structures. Convergent extension is not inhibited in these embryos, but rather, involuting mesodermal cells fail to adhere to the overlying ectoderm. PDGF may therefore be required for mesodermal cell-substratum interaction.

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Species referenced: Xenopus laevis
Genes referenced: fgf2 gsc kit mrc1 pdgfa pdgfb pdgfra tbxt

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	Fig. 1. In situ hybridisation of PDGF A and PDGFRa mRNA during the gastrula stages of development. Serial sections of gastrula staged embryos were probed with 35S-labelled antisense PDGF-A and PDGFR-a cRNA probes. PDGF A is expressed in the presumptive ectoderm (A,C,E) and PDGFR-a 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 mm.
	Fig. 2. Schematic representation of PDGFR-37 mutation. PDGFR-a consists of five immunoglobulin-like repeats in the extracellular domain, a single transmembrane domain (dark stippling) and a split catalytic domain (hatching) with intervening kinase insert. The nucleotide and predicted amino acid sequence of Xenopus PDGFR-a in the region adjacent to the first catalytic domain are shown at top, and the corresponding region in PDGFR-37 below. The sequence of the mutagenesis oligonucleotide is shown in bold type.
	Fig. 3. PDGFR-37 lacks tyrosine kinase activity and acts as a dominant inhibitor. (A) Quiescent, transiently transfected COS cells were treated with PDGF (+) or control medium (-). Tyrosine phosphorylated proteins were detected following SDS-PAGE. Endogenous PDGF receptor migrates at ~170´103 Mr (left pointing arrowhead). Transfected Xenopus PDGF receptor migrates at ~180´103 Mr (right pointing arrowhead). Phosphorylated endogenous receptor was detected in PDGF-treated COS cells transfected with 10 mg of an empty expression vector (pMT2) and in non-transfected NIH-3T3 cells (NIH-3T3). COS cells transfected with 2 mg or 5 mg of Xenopus wild-type PDGFR-a (PDGFR2, 5) contained a small amount of phosphorylated receptor in untreated cells (PDGFR2-, PDGFR5-) which increased on addition of PDGF (PDGFR2+, PDGFR5+). Endogenous phosphorylated PDGF receptor was also detected in PDGF-treated COS cells transfected with 10 mg of PDGFR-37, but no phosphorylation of the transfected mutant receptor was detected (PDGFR-37). Cotransfection of an equal amount of PDGFR-37 and PDGFR-a (5 mg of each) resulted in a reduction of Xenopus receptor phosphorylation in response to PDGF (compare 1:1+ to PDGFR5+). A four-fold excess of PDGFR-37 to PDGFR-a reduced this phosphorylation below the level of detection (compare 4:1+ to PDGFR2+). NIH-3T3 cells act as a control for PDGF stimulation and detection of phosphotyrosine. (B) NIH-3T3 cells were transfected with either 20 mg of pMT2 (pMT2), 2 mg of v-sis plus 18 mg of pMT2 (v-sis) or 2 mg of v-sis plus 18 mg of PDGFR-37 (v-sis PDGFR-37). Cells were passaged into medium containing 0.5% calf serum and colony formation assessed 14 days after transfection. Note that v-sis-dependent colony formation was inhibited by co-transfection of PDGFR-37. (C) Xenopus embryos were injected in the animal pole region of both blastomeres at the 2-cell stage with RNA encoding either wild-type PDGFR-a (PDGFR-wt), PDGFR-37 or the PDGFR-a frameshift. Animal pole explants were made when embryos reached stage 8 and placed into either 75% MMR alone (untreated), or 75% MMR containing either 50 ng/ml PDGF-AA (PDGF), 500 ng/ml bFGF or 2 U/ml activin. Explants were photographed when sibling embryos had reached stage 16-17. Explants injected with the PDGFR-a frameshift control RNA did not elongate in the presence or absence of PDGF, but did elongate in the presence of activin or FGF. Explants from PDGFR-a-injected embryos elongated in the presence of activin, FGF or PDGF, but not in the absence of PDGF. Explants from PDGFR-37-injected embryos did not elongate in the absence or presence of PDGF, but elongated in the presence of activin or FGF, demonstrating that PDGFR-37 does not inhibit FGF or activinmediated signalling.
	Fig. 4. PDGFR-37 does not block the induction of brachyury by FGF or activin. Xenopus embryos were injected in the animal pole region of both blastomeres at the 2-cell stage with RNA encoding either PDGFR-37 or the PDGFR-a frameshift. Animal pole explants were made when embryos reached stage 8 and placed into either 75% MMR alone (untreated), or 75% MMR containing either 50 ng/ml PDGF-AA (PDGF), 500 ng/ml bFGF or 2 U/ml activin. Explants were frozen when sibling embryos had reached stage 13-14, then processed to extract RNA followed by RT-PCR with primers specific for EF1-a and brachyury (Xbra). Brachyury RNA was not detected in untreated animal pole explants from PDGFR-a frameshift-injected embryos, was strongly induced by FGF or activin, but not by PDGF. Explants form PDGFR-37-injected embryos displayed an identical response to all these growth factor additions.
	Fig. 5. PDGFR-37 causes defects in Xenopus gastrulation. Xenopus embryos were injected with 50-200 pg of mRNA encoding PDGFR-37 or a PDGFR-a frameshift mutation in the lateral region of both blastomeres at the 2-cell stage. Defects became apparent between stages 11 and 12. Vegetal view of stage 12 embryos injected with PDGFR-a frameshift (A) or PDGFR-37 RNA (B). Note failure of blastopore closure in B. Appearance of embryos at stage 27 (C). The PDGFR-a frameshiftinjected embryo (upper) looks normal (lateral view, anterior to left) while the PDGFR-37-injected embryo has reduced head structures and the neural folds have not closed (dorsal view, anterior to left). (D) PDGFR-a frameshift-injected embryo at stage 40. (E) PDGFR-37- injected embryos at stage 40 have poorly developed heads (anterior at left), open neural folds and shortened anterior-posterior axis. The defects caused by PDGFR-37 are rescued by wild-type PDGFR- a. Embryos were injected with 140 pg of PDGFR-a frameshift RNA which did not cause any defects (F). Embryos were also unaffected by 70 pg of PDGFR-a (G). Injection of 70 pg of PDGFR-37 caused defects in 44% of embryos (H) which were reversed by co-injection of 70 pg of PDGFR-37 plus 70 pg of PDGFR-a (I).
	Fig. 6. Histological analysis of PDGFR-37-injected embryos at stage 33-34. Transverse sections of PDGFR-a frameshift (A,C) and PDGFR- 37-injected (B,D) embryos through the level of the eye (A, B) and otic vesicle (C,D). Note absence of notochord (no) in B, and misshapen neural tube (nt). The notochord and neural tube are abnormal in D. Sagittal sections of PDGFR-a frameshift (E,F) and PDGFR-37 (G,H)- injected embryos. The notochord forms a straight, well organised structure in control embryos but is misshapen or missing from certain regions of PDGFR-37-injected embryos. Muscle is largely absent from the dorsal and anterior regions of PDGFR-37-injected embryos and is often poorly organised when present. Large masses of endodermal tissue (endo) are exposed on the dorsal side of PDGFR-37-injected embryos (G). All embryos are oriented with dorsal at the top and anterior to the left (E-H). In this experiment, 40 of 64 embryos appeared abnormal, 13 appeared normal and 11 were dead. Seven abnormal embryos with a recognisable anterior-posterior axis were entirely sectioned and all displayed similar phenotypes.
	Fig. 7. PDGFR-37 does not affect expression of goosecoid or brachyury in whole embryos. Embryos injected with PDGFR-a frameshift (A,C) or PDGFR-37 (B,D) RNA were processed for whole-mount in situ hybridisation with cRNA probes for goosecoid at stage 10-10.25 (A,B) or brachyury at stage 11 (C,D). goosecoid expression was seen in the organiser region of control embryos, directly above the dorsal lip. The embryos in A and B are viewed from the vegetal side with the dorsal side of the embryo toward the top. Embryos in C and D have been cleared and are viewed form the vegetal side to show the circumferential expression pattern of brachyury.
	Fig. 8. PDGFR-37 disrupts the migration of involuting mesoderm. RNA encoding b-galactosidase (containing a nuclear localisation signal) was co-injected into the equatorial region of the presumptive dorsal side of both blastomeres of the 2-cell embryo with either PDGFR-a frameshift (A) or PDGFR-37 RNA (B). Embryos are positioned with the animal pole at the top and the dorsal side to the right. Extensive involution and migration of X-gal-stained dorsal mesoderm cells occurred in PDGFR-a frameshift-injected embryos (A). PDGFR-37-injected embryos had X-gal-stained cells in the blastocoel and in the marginal zone prior to involution (B). Few X-galstained cells were present in the anterior mesoderm. Note also, the poor demarcation of involuting mesoderm in PDGFR-37-injected embryos and the failure of the mesoderm to extend across the blastocoel roof on the dorsal, but not the ventral, side of these embryos.
	Fig. 9. PDGFR-37-expressing mesoderm is not affected in exogastrulae. Embryos were co-injected with b-galactosidase and either PDGFR-a frameshift (A) or PDGFR-37 (B) RNA on the presumptive dorsal side of both blastomeres at the 2-cell stage and were induced to exogastrulate. Exogastrulae were stained as whole mounts with X-gal. As before, convergent extension was unaffected in PDGFR-37-injected embryos. The ectoderm (pigmented tissue at right of figure) is separated from the endoderm (large, yolky cells at left of figure) by the convergently extended mesoderm. X-gal-stained cells extend throughout this mesoderm, with the X-gal staining concentrated in the nuclei of these cells (in contrast to X-gal-stained cells in the blastocoel in Fig. 8B).