DEV023416 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. siRNA-mediated knockdown of Pdgfa results in reduced N-cadherin expression. (A) Section through the streak of a stage HH8 embryo 20 hours after transfection with Pdgfa siRNA in the right-hand side of a HH3+ embryo. Immunostained for N-cadherin (6B3), showing reduced N-cadherin expression on the siRNA-transfected side. (B) High-magnification confocal image of a field of view taken at the position of the left arrow in A. Cells show high levels of N-cadherin expression on their surface, with highest levels in cellular protrusions at sites of cell-cell contact. (C) High-magnification confocal image of a field of view taken at the position of the right-hand arrow in A. Cells show significantly reduced levels of N-cadherin expression on their surface; some points of higher expression at points of cell-cell contact remain.

• Supplemental Figure S2 -

  Fig. S2. N-cadherin RNA expression is not affected by siRNA-mediated Pdgfa downregulation. (A) In situ hybridisation for N-cadherin in a stage HH7 embryo 20 hours after transfection with pdgfa siRNA on the left-hand side of the streak of an HH3+ embryo. (B-G) Sections through the embryo taken at positions B-G of the embryo shown in A. Note the strong expression of N-cadherin confined to the mesoderm and the absence of any downregulation on the siRNA-transfected side of the embryo.

• Supplemental Figure S3 -

  Fig. S3. Expression of dn-PDGFRα results in downregulation of N-cadherin expression. (A) High-magnification image of a pCAβ/GFP-transfected cell that has migrated into the mesoderm. (B) N-cadherin staining of the transfected and surrounding cells showing N-cadherin expression on the cell surface of the transfected cell. (C) Merged image of GFP fluorescence (A, green) and N-cadherin expression (B, red). (D) High-magnification image of a group dn-PDGFRα-pCAβ/GFP-transfected cells that have just migrated into the mesoderm. Note the lack of clear polarity of the transfected cells. (E) N-cadherin staining of the transfected and surrounding cells showing the absence of detectable N-cadherin expression on the cell surfaces of the transfected cells. (F) Merged image of GFP fluorescence (D, green) and N-cadherin expression (E, red).

• Supplemental Figure S4 -

  Fig. S4. Downregulation of PDGFA expression does not affect integrin α6, integrin β1, laminin or fibronectin expression. HH3+ stage embryos were transfected with Pdgfa siRNA. Embryos were fixed and stained with antibodies against integrin α6 (A,B), integrin β1 (C,D), laminin (E,F) and fibronectin (G,H) followed by sectioning. (A,C,E,G) Sections through the siRNA-transfected sides of the embryos; (B,D,F,H) sections through the non-transfected sides of the embryos at the same position.

• Supplemental Figure S5 - >Fig. S5. Inhibition of PDGF and PI kinase signalling results in reduced N-cadherin expression. (A) Control embryo 6 hours after incubation on a control plate containing 0.5% DMSO. (A1) N-cadherin staining for the embryo shown in A. (A2,A3) Brightfield and fluorescence images of section through the embryo shown in A,A1 at the level of the arrow. Note the strong N-cadherin staining in the mesoderm in A3. (B) embryo 6 hours after incubation on a plate containing the PI3 kinase inhibitor LY249002 (50 µM). (B1) N-cadherin staining for the embryo shown in B. (B2,B3) Brightfield and fluorescence images of section through the embryo shown in B,B1 at the level of the arrow. Note the absence of detectable N-cadherin staining in the mesoderm in B3. (C) Embryo 6 hours after incubation on a plate containing the PDGFR inhibitor AG1296 (20 µM). (C1) N-cadherin staining for the embryo shown in C. (C2,C3) Brightfield and fluorescence images of section through the embryo shown in C,C1 at the level of the arrow. Note the absence of detectable N-cadherin staining in the mesoderm in C3.

• Movie 1 -

  Movie 1. Time-lapse sequence showing that half-sided knockdown of Pdgfa results in reduced migration on the side of reduced Pdgfa expression. See legend to Fig. 6. The fluorescence image was taken every 3 minutes and recorded for 9 hours from the time of transplantation. Left panel shows the fluorescence images, the right-hand panel the cell migration trajectories.

• Movie 2 -

  Movie 2. Time-lapse sequence showing the mesodermal cell chemotaxis toward PDGFAA beads in an area opaca graft experiment. See Fig. 7A-E. The fluorescence image was taken every 3 minutes and recorded for 9 hours from the time of transplantation. Left panel shows the fluorescence images, the right-hand panel the cell migration trajectories.

• Movie 3 -

  Movie 3. Time-lapse sequence showing that the expression of dn-Pdgfrα blocks chemotaxis towards FGF4. See legend to Fig. 7E-I. The fluorescence image was taken every 3 minutes and recorded for 9 hours from the time of transplantation. Left panel shows the fluorescence images, the right-hand panel the cell migration trajectories.