DMZ explants were dissected at stage 10.25 and cultivated in petri dishes coated with 1% BSA in 1x MBSH until their siblings reached stage 12.
Animal caps of stage 9 embryos were cut manually, placed on BSA coated glass cover slides and fixed with cover glass. To generate a cultivation chamber, the cover glass was separated from the cover slides by a thin layer of silicone. Recombinant Wnt5a protein (R&D Systems) was added by carefully changing MBSH in the cultivation chamber with MBSH containing 0,1 µg/µl Wnt5a and 0,1% BSA.
Time-lapse movies and z-stacks were captured using the Z1 Cell Observer Spinning Disc inverse microscope (Zeiss, Jena, Gemany). For the z-distribution of fluorophore-tagged proteins in animal cap explants ten optical sections with 1 µm distance were captured, starting from the basal side at position 0 µm, where cell protrutions were visible.
Animal caps of stage 9 embryos were cut manually, placed on BSA coated glass cover slides and fixed with cover glass. To generate a cultivation chamber, the cover glass was separated from the cover slides by a thin layer of silicone. Recombinant Wnt5a protein (R&D Systems) was added by carefully changing MBSH in the cultivation chamber with MBSH containing 0,1 µg/µl Wnt5a and 0,1% BSA.
Time-lapse movies and z-stacks were captured using the Z1 Cell Observer Spinning Disc inverse microscope (Zeiss, Jena, Gemany). For the z-distribution of fluorophore-tagged proteins in animal cap explants ten optical sections with 1 µm distance were captured, starting from the basal side at position 0 µm, where cell protrutions were visible.