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Fig. 3. Myocardin activates ectopic
expression of myocardial markers
in the Xenopus embryo. (A-H) 125
pg of myocardin mRNA was
injected into one cell of an eightcell
embryo, which was then
assayed for cardiac markers by
whole-mount in situ hybridization.
No expression of the MHCα gene
is observed in uninjected stage 14
embryos (A), however widespread
transcription of MHCα is observed
in myocardin-injected embryos
(B). Similarly, cardiac α-actin is
observed specifically in the presomitic
mesoderm at stage 14
control embryos (C), while
myocardin injected embryos
display widespread expression of
cardiac α-actin on the side of
injection (D). (E) Section through
the embryo in D shows ectopic
cardiac α-actin expression
(arrows) in the ectodermal and
mesodermal tissue layers. Ectopic
cardiac marker expression is not
observed in endodermal tissues.
(F) MHCα expression is heartspecific
at stage 28 in un-injected control embryos, but myocardin overexpression, (G), causes MHCα transcription in ectopic locations. Arrows
indicate normal cardiac expression. (H) Section through the embryo in G shows patches of ectopic MHCα expression in the neural tube (nt) and
eye. (I) Fluorescence microscopy of a stage 29 Xenopus embryo co-transgenic for NβT-GFP and NβT-myocardin showing GFP expression in
neural tissues. (J) In situ hybridization analysis of NβT-GFP/NβT-myocardin co-transgenic embryos using a MHCα probe shows ectopic
expression of MHCα in neural tissues. |
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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
MHCα 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 onesided
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 MHCα
or Nkx2-5 transcripts to mark the location of
myocardial cells and to confirm a reduction in MHCα expression on the injected side (right side) of the MO-injected embryo. Uninjected
controls showing normal heart tube morphogenesis are included for comparison. |
