Animals and embryo manipulation

Wild-type X. tropicalis and X. laevis adults (male and female) were purchased from NASCO. The transgenic X. tropicalis Wnt reporter frogs were provided courtesy of Dr Kris Vleminckx (Flanders Institute for Biotechnology, Belgium), and the transgenic X. tropicalis snail2-eGFP line was generated as described elsewhere (J.L., M.P. and S.W., unpublished). Methods involving live animals were carried out in accordance with the guidelines and regulations approved and enforced by the Institutional Animal Care and Use Committees at West Virginia University, University of Delaware and University of Massachusetts at Amherst. Morpholino oligos were designed and synthesized by Gene Tools. Embryo preparation, injection and culturing were carried out as described previously (Wei et al., 2010b). Briefly, embryos were obtained by natural mating and injections were carried out to target specific blastomeres (Huang et al., 1998) using a PLI-100A microinjector (Harvard Apparatus). Alexa Fluor 555 or 488 dextran (Invitrogen) was co-injected as a lineage tracer. Embryos were cultured in 0.1× MBS to desired stages, and injected embryos were sorted according to the injected side using a Zeiss Axiozoom.v16 epifluorescence microscope. Embryo lysates were prepared as described below for cell lysates. For embryo dissociation, X. tropicalis embryos were injected in both blastomeres at the two-cell stage and cultured to stage ∼12.5. The embryos were placed in 1.5 ml centrifuge tubes with 0.1× MBS and 100 μM MG132 (or DMSO as control). The embryos were then dissociated using a 28 gauge 1 ml syringe (Exel) and incubated in 0.1× MBS and 100 μM MG132 or DMSO at 23°C for 5 h.