Pli 100a microinjector

Wild-type X. tropicalis adults (male and female) were purchased from the NASCO. 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 the University of Delaware. Morpholinos (MOs) were designed using Gene Tools, LLC, OR. The Hspb1 MO is a 25-mer with the sequence 5′ GTA TTC TGC GTT CTG ACA TTT TC 3′. The control MO is the standard control MO obtained from Gene Tools with the sequence 5′ CCT CTT ACC TCA GTT ACA ATT TAT A 3′. Embryos were collected and injected with a PLI-100A microinjector (Harvard Apparatus) as described previously (106 (link)). Control or HSPB1 morpholino (1.5 ng per blastomere) was injected into a single dorsal-animal blastomere at the eight-cell stage; Alexa Fluor 568 dextran (Invitrogen #D22912) was co-injected as a lineage tracer. For the rescue experiments, mouse Hspb1 mRNA was generated by in vitro transcription as described (107 (link)) and co-injected with the HSPB1 morpholino (50 pg mRNA per blastomere). Injected embryos were cultured in 0.1X MBS to desired stages, and eye phenotypes were observed using a Zeiss Axiozoom.v16 epifluorescence microscope.

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.