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First published online 2 October 2008
doi: 10.1242/dev.023416

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PDGF signalling controls the migration of mesoderm cells during chick gastrulation by regulating N-cadherin expression

Xuesong Yang^*, Holly Chrisman and Cornelis J. Weijer

Division of Cell and Developmental Biology, Wellcome Trust Biocentre, College of Life Sciences, University of Dundee, Dundee DD1 5EH, UK.

View larger version (116K): [in this window] [in a new window]   Fig. 1. Expression Pdgfa, Pdgfr, Pdgfb and Pdgfc during early chick development. (A-F) Pdgfa expression in HH3-8 chick embryos. (A'-F') Sections through the embryos shown in A-F at the level of the arrows. (G-L) Pdgfr expression in HH3-8 embryos. (G'-L') Sections through the embryos shown in G-L at the level of the arrows. (M,N) Expression of Pdgfb is almost undetectable in HH3-8 embryos. (O-Q) Expression of Pdgfc. (Q'-Q''') In later embryos (HH6-8), Pdgfc is expressed in the brain and forming neural tube and in the forming somites. Probes used in these and all further figures are indicated in the bottom left-hand corner of relevant panels.

View larger version (122K): [in this window] [in a new window]   Fig. 2. Inhibition of PDGF signalling inhibits the migration of mesoderm cells away from the streak. (A) Chick embryo transfected on one side of the streak with a GFP expression construct (pCAβ/GFP). (A') Fluorescent image of section through the embryo at the level of the arrow in A. (B) Embryo transfected with a dominant-negative (dn) Pdgfr construct. (B') Section through the embryo at the level of the arrow in B. (C-C'') GFP-expressing streak cells grafted in a control embryo of similar age migrate away from the site of transplantation in the anterior primitive streak to form somites and lateral plate mesoderm. (C') Fluorescent image of embryo shown in C. (C'') Section taken at the arrow in C'. (D-D'') Streak cells expressing a dn-PDGFR do not migrate away from the site of transplantation in the streak. (D') Section of embryo taken at the level of the arrow in D. (D'') β-catenin staining of the section shown in D'. (E-E'') Embryo 20 hours after injection with 0.5 µl 100 µg/ml mouse dn-PDGFR-Fc and transplantation of GFP-expressing middle streak cells. Depletion of PDGFR ligands results in failure of most GFP-expressing cells to migrate away from the site of implantation. (E') Fluorescent image of embryo shown in E. (E'') Section taken at the arrow in E'.

View larger version (118K): [in this window] [in a new window]   Fig. 3. Downregulation of endogenous Pdgfa expression inhibits movement of cells out of the streak. (A) Chick embryo 22 hours after transfection with a mixture of GFP and Smartpool Pdgfa siRNA. Labelled cells migrate to the streak, but do not move out of the streak; development is arrested at the extended streak stage. (A') Pdgfa in situ hybridisation of the embryo shown in A. (B) Embryo transfected with GFP and a control siRNA (cyclophylin). GFP-expressing cells migrate away to form paraxial mesodermal structures. (B') Pdgfa in situ hybridisation of embryo shown in B. (C) Pdgfa in situ hybridisation of an embryo 22 hours after half-sided Pdgfa siRNA knockdown. (C') Section taken at the position of the arrow in C. (C'') High-magnification image of section shown in C' after staining of nuclei with propidum iodide. (D) Pdgfa in situ hybridisation of an embryo 22 hours after half-sided transfection with siRNA-dilution buffer. (D') Section taken at the position of the arrow in D. (D'') High-magnification image of section in D' after staining of nuclei with propidum iodide.

View larger version (59K): [in this window] [in a new window]   Fig. 4. Half-sided knockdown of Pdgfa results in reduced migration in the side of reduced Pdgfa expression. (A) Merged brightfield and fluorescence image of a chick embryo 20 hours after transfection with Pdgfa siRNA on one side of the streak. (E) GFP-expressing middle streak cells from a control embryo were transplanted in a homotypic position in the streak of the siRNA-transfected embryo. (B) Fluorescent image of the embryo shown in A. (C) In situ hybridisation of the embryo shown in A. (D) Tracks of the fluorescently labelled cells followed over the 20 hours of the experiment. (F) The accumulated results from 23 embryos show that in 13 out of the 23 experiments, the cells move preferentially into areas of normal Pdgfa expression, as shown in B,D; in nine experiments, there was no clear effect and in one experiment the cells migrated towards low PDGF (see Movie 1 in the supplementary material). To score the migration, we binned cases in which 80-100% of the tracks went to one side of the streak as strongly positive, cases in which 60-80% of the cells went to one side as positive, and cases where 40-60% went to one side as normal.

View larger version (71K): [in this window] [in a new window]   Fig. 5. Signalling though the PDGFA receptor enhances, and is required for, cell migration. (A) GFP-expressing streak cells are grafted in the area opaca between a bead soaked in PBS and a bead soaked in rat PDGFAA short form (20 µg/ml). (B) Merged fluorescence and brightfield image at the start of the experiment. Arrows indicate the position of the PBS- and PDGFA-soaked beads as shown in A. (C) Image of the same region after 20 hours incubation. (D) Fluorescence image after 20 hours development. (E) Cell tracks followed over the duration of the experiment. For further details, see Movie 2 in the supplementary material. A positive response was scored when many cells were seen to migrate towards the beads and to accumulate on their surface. Owing to movements of the embryos during many of the experiments and despite computational fixation of the position of the beads, the resulting tracks are often distorted. (F) Middle streak cells expressing either GFP alone or together with dn-Pdgfr are grafted into the area opaca on the right and left sides, respectively, of a bead soaked in FGF4 (100 µg/ml). (G) Merged fluorescence-brightfield image taken at the start of the experiment. Arrow indicates the position of the FGF4-soaked bead as shown in F. (H) Image of the same area after 20 hours of the experiment. (I) Fluorescence image after 20 hours. (J) Cell tracks over the course of the experiment. For further details, see Movie 3 in the supplementary material.

View larger version (114K): [in this window] [in a new window]   Fig. 6. Effect of PDGF signalling on N-cadherin expression. (A) Reduced N-cadherin expression as detected by immunofluorescence in a chick embryo in which Pdgfa has been knocked down in one half of the embryo. The embryo was transfected at stage HH3+ and allowed to develop for 20 hours in early chick (EC) culture. Brightfield image (A1) and N-cadherin immunofluorescence (A1') of section of the embryo in A at level 1. Brightfield image (A2) and N-cadherin immunofluorescence (A2') of section of the embryo in A at level 2. (B) Implantation of a heparin bead soaked in PDGFA results in increased N-cadherin expression at the side of implantation of the bead, which is confirmed in N-cadherin immunofluorescence (B1') and merged brightfield-immunofluorescence (B1) images of section taken at position 1 in B. (C,D) Expression of a dn-N-cadherin construct results in cells migrating to the streak but failure to migrate out, in contrast to control cells expressing just GFP. (E,F) Expression of dn-N-cadherin inhibits chemotaxis of middle streak cells to a bead soaked in FGF4. (E) Merged brightfield and fluorescence images taken at the start of the experiment. (F) The same embryo after 20 hours of development. (G) Local application of an FGF4 bead in a developing embryo results in the attraction of many N-cadherin-expressing cells to the FGF4 bead, which is not the case with a PBS-soaked control bead. Sections through the PBS control and FGF4 beads show that the FGF4 bead has attracted many N-cadherin-expressing cells (G2), whereas the PBS bead has not (G1).

View larger version (110K): [in this window] [in a new window]   Fig. 7. Expression of N-cadherin rescues mesodermal cell migration after inhibition of PDGF signalling. (A) Cells co-transfected with a dn-PDGFR expression vector (dn-NPDGFR-pCAβ/GFP) and full-length N-cadherin expression vector (N-Cad-pCAβ/GFP) 20 hours after transfection. Arrow, level of section in A1,A2. (A1) GFP-expressing cells migrate into the mesoderm on both sides of the streak. (A2) N-cadherin expression as detected by 6B3 antibody. (B) Cells co-transfected with dn-NPDGFR-pCAβ/GFP and pCAβ/GFP 20 hours after transfection. Arrow, level of section in B1,B2. (B1) GFP-expressing cells fail to migrate into the mesoderm on either side of the streak. (B2) N-cadherin expression as detected by 6B3 antibody. B2 was taken at the same exposure as A2, showing that N-cadherin overexpression levels are high compared with endogenous expression levels, which are almost undetectable at these settings.

View larger version (103K): [in this window] [in a new window]   Fig. 8. Coincidence of PDGF and PI3 kinase signalling domains. (A,B) Expression of Pdgfr (A) and PDGFA (B). (C) AKT (PKB) activation, measured as phospho-AKT (S473), shows a remarkably similar pattern to that of Pdgfr expression (A). (C') Section through the embryo at the arrow in C, showing that the mesoderm cells have activated the PI3 kinase pathway. (D) Application of an ectopic PDGFA bead results in localised activation of the PI3 kinase pathway, measured as AKT phosphorylation (S473) around the bead. (D') Section through the embryo in D at the site of the bead (arrow) shows that the cells that activate the PI3 kinase pathway are mostly mesodermal. (E) AKT staining of an embryo incubated for 10 hours on a substrate containing the PDGFR inhibitor AG1296 (50 µM).

View larger version (25K): [in this window] [in a new window]   Fig. 9. PDGF and FGF signalling control mesodermal cell migration. PDGFA (green) is expressed in the epiblast on both sides of the streak in a posterior-to-anterior-directed gradient that controls the migration of PDGFR-expressing mesoderm cells. PDGF signalling is most likely to have a dual role in mesodermal cell migration. Through a PI3 kinase-dependent signalling pathway, it controls the expression of N-cadherin on mesoderm cells, which allows them to gain the traction necessary to migrate. Secondly, in cooperation with FGF signalling, it provides instructive information for the anterolateral migration of the mesoderm cells towards their final destination (Yang et al., 2002). The black arrows indicate the patterns of movement of the mesoderm cells after their ingression through the streak. The yellow arrow indicates the direction of regression of Hensen's node.

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