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First published online 5 May 2004
doi: 10.1242/dev.01141

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Guidance of mesoderm cell migration in the Xenopus gastrula requires PDGF signaling

¹ Department of Zoology, University of Toronto, 25 Harbord Street, Toronto, Ontario M5S 3G5, Canada
² Department of Biochemistry, Boston University School of Medicine, 715 Albany Street, Boston, MA 02118, USA

View larger version (23K): [in a new window]   Fig. 1. Experimental procedures. (A) Mesoderm migration on conditioned substratum. (Left) Mesoderm cells migrate on the extracellular matrix (ECM) of the BCR towards the animal pole (AP) (arrow). Blue, prospective ectoderm; pink, mesoderm (M); yellow, endoderm (E). (Right, top) A strip of BCR (bracket in left figure) is explanted at stage 10, and cultured for 1 hour with its matrix-forming surface down. (Right, bottom) The ECM of the BCR has become deposited at the bottom of the dish. A mesodermal explant (M) placed on the conditioned substratum migrates (arrow) to the animal pole position. (B) Construction of trPDGFA. The sequence corresponding to amino acid residues 198-227, that contain the matrix binding motif, was deleted from the lfPDGFA.

View larger version (38K): [in a new window]   Fig. 2. Directional migration and its dependence on PDGFR signaling. (A-C) Comparison of mesoderm migration on untreated tissue culture plastic (A), FN-coated plastic (B) and conditioned substratum (C). (D-I) Migration of PDGFR-inhibited mesoderm on normal conditioned substratum (see explanatory scheme above). Inhibition of PDGFR signaling by expression of dominant-negative PDGFR37 mRNA randomizes the direction of migration (D). Directed migration is restored by co-expression of wild-type PDGFR (E). Expression of missense PDGFR does not affect mesoderm migration (F). Mesoderm explants moving on conditioned substratum were treated during the 1 hour migration period with tyrphostin AG 1296 (G), control tyrphostin AG 43 (H) or Wortmannin (I). Both AG 1296 and wortmannin randomize the direction of migration. n, number of explants tested; results from at least three independent experiments. Explants in C,E,F,H prefer the animal pole side significantly (one-sided sign test, significance level =0.005 for (E) and 0.0005 for all others). In the same test, explants in A,B,D,G,I show no preference for the animal pole at any significance level.

View larger version (43K): [in a new window]   Fig. 3. Interference with PDGFA function in the BCR randomizes the migration of normal mesoderm. (A-F) Normal mesoderm on substratum conditioned with PDGFA-compromised BCR (see explanatory scheme above). Substratum was conditioned with BCR containing inhibitory dnPDGFA mRNA (A), or morpholino antisense oligonucleotides directed against PDGFA (B), 5-mismatch control morpholino (C), PDGFA morpholino together with lfPDGFA mRNA (D), lfPDGFA mRNA alone (E) or C-terminally truncated trPDGFA (F). (G,H) Mesoderm expressing trPDGFA mRNA (G) or normal mesoderm migrating in the presence of exogenous PDGFAA protein (H), on normal conditioned substratum. n, number of explants tested; results from at least three independent experiments. Explants in C,F prefer the animal pole direction significantly (significance level =0.0005). Explants in A,B,D,E,G,H show no preference for the animal pole [at =0.05 for (B,D) or any significance level for all others]. (I) Specificity of morpholino function. Animal caps were explanted at stage 9, and cultured for 3 days. (a) untreated animal caps; (b) caps from embryos co-injected with PDGFR and lfPDGFA mRNAs; (c) caps as in b, but with additional injection of PDGFA morpholino; (d) caps as in c, but incubated in 200 ng/ml of human recombinant PDGFA protein after explantation.

View larger version (118K): [in a new window]   Fig. 4. Effects of compromised PDGFA signaling on gastrula. Stage 10.5 gastrulae of uninjected controls (A,D,G) are compared with sibling embryos injected with inhibitory dnPDGFA mRNA (B,E,H) and wild-type lfPDGFA mRNA (C,F,I). (A-C) Gastrulae were fixed and fractured in the sagittal plane, dorsal is towards the right, animal towards the top; arrowheads, dorsal blastopore; small arrows, ventral blastopore; large arrows, pointed leading edges in controls, and corresponding positions in treated embryos. (D-F) In parallel, gastrulae were prepared for scanning electron microscopy. The BCR was removed to expose the surface of the leading edge mesoderm that had been in contact with the BCR substratum. Animal is towards the top. Arrow, cells with laterally or vegetally oriented protrusions. Scale bar: 50 µm. (G-I) BCRs of gastrulae were stained for the presence of a FN fibril matrix.

View larger version (47K): [in a new window]   Fig. 5. PDGFA signaling and protrusive activity. (A) Protrusions in the mesoderm of normal and of PDGFA signaling-compromised gastrulae, as determined from scanning electron microscope pictures. Percentage of cells with no underlapping protrusions, with protrusions pointing animally, or vegetally, were assessed in uninjected embryos (n=157 cells), and in embryos injected with dnPDGFA (n=155) or lfPDGFA mRNA (n=158), respectively. Cells were scored as animally oriented if the vector, cell center-protrusion, pointed upwards at any angle in carefully oriented SEM pictures, and vegetally oriented if otherwise. The lower percentage of protrusion-bearing cells in dnPDGFA and lfPDGFA embryos, when compared with controls, is highly significant (significance level =0.001, 2-test), the difference between dnPDGFA and lfPDGFA embryos is not significant. (B,C) Protrusions of single mesodermal cells on substratum conditioned with control BCR, with BCR overexpressing lfPDGFA or dnPDGFA. (B) The percentage of cells extending cytoplasmic protrusions (lamellipodia or pseudopodial blebs) was determined. The fraction of cells with no processes is significantly higher on dnPDGFA conditioned substratum (n=317 cells) when compared with control (n=323) or lfPDGFA (n=467) substratum (significance level =0.01, 2-test). (C) Examples of typical cell morphologies (rhodamine-phalloidin staining of the actin cytoskeleton) are shown.

View larger version (60K): [in a new window]   Fig. 6. Effect of compromised PDGFA signaling on mesoderm movement. (A-R) In situ hybridization. Expression of Xbra at stage 10 (A-C) and stage 11.5 (G-J), and of chordin at stage 10 (D-F) and stage 11.5 (K-N), in uninjected embryos (A,D,G,K), and in embryos injected with dnPDGFA (B,E,H,L) or lfPDGFA mRNA (C,F,I,M) or PDGF morpholino (J,N). (A-C) Vegetal view. (D-N) Dorsovegetal view with animal towards the top. Brackets indicate extending notochord region. (O-R) Expression of gsc in controls (O), and in dnPDGFA (P), lfPDGFA (Q) and PDGF morpholino (R) gastrulae fixed at stage 12 and fractured mid-sagittally. Small arrowhead, blastopore lip; large arrowhead, tip of archenteron; arrow, gsc expression. (S-V) Convergent extension in dorsal blastopore lips explanted at stage 10 from control (S), dnPDGFA expressing (T), lfPDGFA overexpressing (U) and PDGF morpholino injected (V) gastrulae, fixed at stage 14 (n=15, three independent experiments for each treatment).

View larger version (101K): [in a new window]   Fig. 7. Larval phenotypes after interference with PDGF signaling. Embryos injected at the four-cell stage with dominant negative PDGFR37 mRNA (A), PDGFA morpholino (B), dnPDGFA mRNA (C), wild-type lfPDGFA mRNA (D) and 5-mispaired control morpholino (E) are shown at the larval stage. Uninjected control larvae are at the left in each panel, injected ones are in the right column.