DEV029157 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. Model for FGF signaling in establishing polarity in notochord convergent extension. Ci-FGF3 emitted from the floor plate precursors (red circle) forms a medial-to-lateral gradient (red) and signals to the underlying notochord plate to reorganize the cytoskeleton and induces formation of lamellopodia (thick black lines) preferentially along the mediolateral (ML) axis, providing the driving force for ML intercalation. At the notochord cell membrane, activation of FGFR induces membrane localization of PKC-δ, which subsequently recruits Dishevelled to the membrane. This initiates the downstream cascades leading to local reorganization of the cytoskeleton and produces coordinated polarization along the ML axis.

• Supplemental Figure S2 -

  Fig. S2. FGF signaling is not required for notochord differentiation. A notochord-specific gene, Ci-Noto1, is expressed in both wild-type and dnFGFR notochord cells.

• Supplemental Figure S3 -

  Fig. S3. Real-time PCR quantification of the effect of Ci-FGF3 splice morpholinos.

• Supplemental Figure S4 -

  Fig. S4. FGF3 misexpression affects notochord intercalation not tissue fates. Embryos are co-electroporated with FGF3 misexpression constructs and Bra::GFP. Noto1 fluorescent in situ (A-C, green) and GFP antibody staining (A′-C′, red) were performed on late tailbud embryos (overlay, A′′-C′′). Those notochord cells that fail to intercalate (arrows) still express the notochord marker Noto1. They also express the Bra::GFP transgene, suggesting that they are derived from the early notochord lineage and differentiate like true notochord cells.

• Supplemental Figure S5 -

  Fig. S5. Average number of membrane protrusions in dissociated notochord cells (eight cells for each condition) under different conditions. As seen in representative Movies S3-S5, wild-type notochord cells form membrane protrusions at low frequency. Adding bFGF to the medium increases protrusion formation by ∼200%. This effect is inhibited by misexpression of a dnFGFR in these cells.

• Supplemental Figure S6 -

  Fig. S6. SU5402 inhibits FGF signaling during Xenopus mesoderm induction. The Xenopus mesoderm marker Brachyury is progressively reduced in embryos treated with higher doses of SU5402 from the 32-cell stage until stage 11. (A) Untreated. (B) DMSO control. (C) 40 µm SU5402. (D) 80 µm SU5402. (E) 120 µm SU5402. (F) 160 µm SU5402.

• Supplemental Figure S7 -

  Fig. S7. SU5402 treatment at 120 µM from stage 10.5 onwards does not affect mesoderm differentiation but inhibits convergent extension. (A-C) Xbra in situ on uninjected DMZ explants treated as indicated from stage 10.5-12, identical to those injected with Dsh-GFP or PKC-δ-GFP (five explants each). (D-F) Tor70 antibody (notochord) staining on whole embryos from stage 10.5 to tailbud under different conditions. (D′-F′) 12/101 antibody (muscle) staining on whole embryos from stage 10.5 to tailbud under different conditions. (A,D,D′) Untreated. (B,E,E′) DMSO. (C,F,F′) 120 µm SU5402.

• Movie 1 - Movie 1. Wild-type dorsal anterior quadrant embryo labeled with Bra::GFP showing that the notochord cells form protrusions and undergo intercalation along a particular axis. n=2.

• Movie 2 -

  Movie 2. Notochord cells in dnFGFR-Venus dorsal anterior quadrant fail to form protrusions or intercalate, resulting in a nascent notochord plate with no cell arrangement. n=2.

• Movie 3 -

  Movie 3. Wild-type notochord cells form membrane blebs in the medium. n=8.

• Movie 4 -

  Movie 4. bFGF induces massive bleb formation and at a much higher frequency. n=8.

• Movie 5 -

  Movie 5. dnFGFR-transformed notochord cell fails to respond to bFGF in the medium. n=8.