Fig. 6. Inhibition of myocardin activity using antisense morpholino (MO) oligos. (A,B) Control experiment where myocardin MO1 inhibits translation of a transcript containing the myocardin 5'UTR fused to the EGFP coding region. mRNA (400 pg) was injected into one-cell Xenopus embryos with or without 10 ng of myocardin MO1 and the embryos were then assayed for the presence of GFP transcript and protein at stage 17. The presence of MO1 did not affect the levels of EGFP transcript as detected by RT-PCR (A) but did significantly reduce the amount of translated GFP protein as detected by western blotting (B). (C) Xenopus embryos were injected with 10 ng of myocardin MO1 into one blastomere at the two-cell stage and cultured until stage 29, when cardiac differentiation markers are normally expressed in the symmetric heart patches. Uninjected control embryos (labeled C) or myocardin MO1-injected embryos (labeled MO) were assayed by in situ hybridization. Myocardin MO1 inhibited expression of MHC and MLC2 on the side of injection (right side of figure) but did not affect the expression of Nkx2-5. (D) Sections through the heart of uninjected (labeled C) and one-sided MO1-injected (labeled MO) Xenopus embryos at the linear heart tube stage (stage 34). Embryos were assayed by in situ hybridization for expression of either MHC or Nkx2-5 transcripts to mark the location of myocardial cells and to confirm a reduction in MHC expression on the injected side (right side) of the MO-injected embryo. Uninjected controls showing normal heart tube morphogenesis are included for comparison.