Zebrafish animal cap aggregate assays. (A) The two co-cultured zebrafish animal caps were dissected from embryos injected with 100 pg Wnt5b-IRES-EGFP-CAAX (green) and 100 pg mCherry constructs + 1.0 pmol Wnt5b MO (red), respectively; a directional Wnt5b signal was created by juxtaposing the two caps. (B–G) Live images at the interface of co-cultured cap aggregates. (B) mCherry-CAAX cap (red) co-cultured with EGFP-CAAX cap (green) shows cell intermingling at the interface. (C) Fz2 (Fzd2)-mCherry + Wnt5b MO cap (red) co-cultured with Wnt5b-expressing cap (green) shows similar cell intermingling. (D) Wnt11-expressing cap (green) co-cultured with Ryk-mCherry–expressing cap (red) shows intermingled cells. (E) Ryk-mCherry + Wnt5b MO cap (red) co-cultured with Wnt5b-expressing cap (green) does not show intermingled cells. The region at the interface of the caps shows a distinct border with reduced Ryk-mCherry (asterisk). (F) High-magnification image of Ryk-mCherry–expressing cell projecting a protrusion away from the Wnt5b source. The arrowhead marks the leading edge of a Ryk-mCherry–expressing cell. (G) Ryk ICD-mCherry cap (red) co-cultured with Wnt5b-expressing cap (green) shows cell mixing. (H) Quantification of the number of cap aggregates that had cell intermingling at the interface. Bars, 20 µm.