Alexa fluor 555 dextran

Embryo were obtained by natural mating and cultured in 0.1x MBS to desired stages as described previously^{30 (link)}. For in situ hybridization and immunohistochemistry, embryos were fixed at desired stages and processed as described⁶³ . MO 13-3, the antisense MO for ADAM13, was synthesized by Gene Tools, and the sequence was reported previously^{30 (link)}. For MO injections, 8-cell stage snai2:eGFP embryos were injected in a single dorsal-animal blastomere with 1.5 ng MO 13-3 using a PLI-100A microinjector (Harvard Apparatus), and Alexa Fluor 555 dextran (Invitrogen) was co-injected as a lineage tracer. For Wnt inhibitor treatment, embryos were cultured in XAV939 or IWR1-endo (both were from Selleckchem) from stage ~28 to ~35. Snai2-eGFP or Wnt reporter transgenic tadpoles were washed three times and subsequently cultured in 0.1x MBS until stage ~44 or ~47 (Fig. 6); wild-type tadpoles were immediately fixed and processed for in situ hybridization for snai2 or sox9 (Fig. 7A,B). For inhibition of Wnt target gene expression, 2-cell stage wild-type embryos were injected in one blastomere with 50 pg mRNA encoding EnR-LefΔN-GR^755A, and cultured to stage ~28, when 10 mM DEX (Sigma-Aldrich D4902) or DMSO was added. Embryos were further cultured to stage ~35, fixed, and processed for in situ hybridization for snai2 or sox9.