Direction of Wnt5b–Ryk signaling is correlated with cell mobility and polarized cell protrusions. (A) A wnt5b RNA–injected cap (red) and a Wnt5b MO–injected cap were juxtaposed and co-cultured to generate a directional Wnt source. Cells with ryk RNA + EGFP-CAAX or Ryk MO + EGFP-CAAX (green) were transplanted to the center of the aggregate interface. (B) An example of cell tracking of one ryk RNA–injected cell showing active extension and retraction of lamellipodia-like protrusions over time (arrowheads). (C) Overlay traces of a ryk RNA–injected cell (Video 5) and a Ryk MO–injected cell (Video 6); the arrow marks the direction of Wnt5b gradient (high to low) that was used to calculate cell roundness and was significantly different between ryk RNA– and Ryk MO–injected cells. Error bars represent standard error (n = 24; Student’s t test: *, P < 0.01). (D) Representative migration path of individual cells recorded by time-lapse imaging over 20 min at 1-min intervals; start and end points are indicated by yellow and red triangles, respectively; Wnt5b source to the left. Total speed in all directions (black curved arrow) and net speed away from the Wnt5b source (red dashed arrow) are charted comparing Ryk MO–injected cells with Ryk RNA–injected cells (n = 10). Error bars represent standard error. (E–G) Polarized lamellipodia-like protrusions in 80–90% epiboly stage embryos. (E) Schematic of the mosaic injection experimental approach. (F) Ryk-mCherry cells (red) adjacent to Wnt5b-IRES-nls-EGFP cells (green) show directional protrusions (arrowhead). (G) mCherry-CAAX and Ryk MO cell (red) adjacent to Wnt5b-IRES-nls-EGFP cells (green) display random projections (arrowhead). (F and G) White lines outline a Wnt5b-expressing cell (green) and an adjacent cell injected with mCherry constructs (red). Bars: (B) 10 µm; (D) 5 µm; (F and G) 30 µm.