Ryk-expressing cells respond to Wnt5b stimulus by internalizing Ryk and protrusion formation. (A–F) Confocal images of hypoblast cells in 90% epiboly stage embryos, lateral view. (A and B) Donor embryo with Ryk-mCherry and Wnt5b MO (A); host embryo with Wnt5b-IRES-EGFP-CAAX (B). (C) Donor Ryk-mCherry + Wnt5b MO cells (red) transplanted into a Wnt5b-IRES-EGFP-CAAX host (green). (F) Red channel from C showing Ryk internalization and extensive protrusion formation in the transplanted cells. (D) Donor Ryk-mCherry + Wnt5b MO cells transplanted into an EGFP-CAAX + Wnt5b MO host. (E) Donor dnRyk-mCherry + Wnt5b MO cells transplanted into Wnt5b-IRES-EGFP-CAAX host (E). (G–I) Measurement of cell roundness frequency of transplanted cells. (G) Donor and host combination and the color code for each transplantation experiment (group 1–4) are listed. (H and I) A column chart shows the mean roundness and standard error (H), and a cumulative frequency chart shows the distribution of roundness for each group (I). 50 cells were measured for each group. One-way analysis of variance and Tukey’s honestly significant difference test were applied, showing that group 4 is significantly different than groups 1–3 (*, P < 0.01). Bar, 30 µm.