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???displayArticle.abstract??? Myocardin is a cardiac- and smooth muscle-specific cofactor for the ubiquitous transcription factor serum response factor (SRF). Using gain-of-function approaches in the Xenopus embryo, we show that myocardin is sufficient to activate transcription of a wide range of cardiac and smooth muscle differentiation markers in non-muscle cell types. We also demonstrate that, for the myosin light chain 2 gene (MLC2), myocardin cooperates with the zinc-finger transcription factor Gata4 to activate expression. Inhibition of myocardin activity in Xenopus embryos using morpholino knockdown methods results in inhibition of cardiac development and the absence of expression of cardiac differentiation markers and severe disruption of cardiac morphological processes. We conclude that myocardin is an essential component of the regulatory pathway for myocardial differentiation.
Fig. 2. Developmental expression of Xenopus myocardin and MRTF genes. The expression of Xenopus myocardin (A-A'”) was analyzed by whole-mount in situ hybridization and compared to the expression patterns of the cardiac differentiation marker, MHCα (B-B'”), and the pre-cardiac marker, Nkx2-5 (C-C'”) at the stages indicated. Myocardin expression in the stage 24 embryo is localized to the pre-differentiation cardiac mesoderm in a more restricted domain than Nkx2-5, which is also expressed in the pharyngeal arch region (compare A' with C'). MHCα expression is located in an identical domain to myocardin at stage 27 (compare A” with B”). A'”, B'” and C'” are ventral views of the stage 27 embryos illustrated. (D) In the heart of a stage 45 embryomyocardin expression is located throughout the myocardial layer of the atrium (a), ventricle (v), and outflow tract (ot). (E) Myocardin is expressed in the visceral smooth muscle in stage 42 embryos. (F) Higher magnification reveals myocardin expression in individual smooth muscle cells adjacent the dorsal aortae and in the smooth muscle layer of the gut. DA, dorsal aorta; SM, smooth muscle. (G, H) In situ hybridization analysis of stage 27 embryos shows that the myocardin-related transcription factors, MRTF-A and MRTF-B, are not expressed in the pre-cardiac mesoderm (ventral views). (I) RT-PCR analysis of myocardin, MRTF-A and MRTF-B expression in early Xenopus embryos and isolated heart patches from stage 28 embryos confirms a lack of MRTF-A and B expression in the pre-cardiac mesoderm.
myocd (myocardin) gene expression in Xenopus laevis, NF stage 30 embryo, in situ hybridization, lateral view, anteriorleft.
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.
Fig. 4. Myocardin induces transcription of endogenous cardiac and
smooth muscle marker genes in animal cap explants. Myocardinexpressing
animal pole explants were cultured until stage 12.5 and
assayed for cardiac and smooth muscle gene expression by RT-PCR.
(A) Uninjected animal caps differentiate into epidermal tissue and
never express mesodermal derivatives, including cardiac or smooth
muscle markers (lane labeled uninjected). Myocardin-injected caps
however, express a wide range of cardiac and smooth muscle
differentiation markers (lane labeled myocardin), including cardiac
α-actin, MHCα, cardiac TnI, SM22, calponin H1 and smooth muscle
actin. The myocardin cofactor SRF and the MADS box transcription
factor Mef2a, are upregulated in myocardin expressing caps. The
cardiogenic genes, Nkx2-5 and Gata4 are not expressed in
myocardin-injected animal caps. The lane labeled 12.5 WE,
represents the normal expression of the assayed genes in the whole
embryo at the time that the animal cap explants were assayed. (B)
Myocardin does not activate genes of the skeletal muscle or
mesodermal pathways. Myocardin-injected animal caps were
assayed by RT-PCR for the activation of mesodermal and skeletal
muscle markers. The general mesoderm marker brachyury (Xbra) is
not expressed in myocardin-injected caps. Furthermore, myocardin
does not activate expression of the skeletal muscle transcription
regulators, MyoD, Myf5, MRF4 and myogenin, or the skeletal
muscle-specific differentiation marker skMLC.
Fig. 5. Myocardin acts in combination with other cardiac
transcription factors to activate endogenous MLC2 expression in
animal cap explants. (A) Expression of myocardin alone (lane
labeled myocardin) activates SM22 and MHCα expression, but is not
sufficient to activate expression of the MLC2 gene. Expression of
Nkx2-5, Gata4, or Tbx5 alone, or the combination of these three
factors (lane labeled N+G+T) is not sufficient to activate expression
of MLC2 or SM22 or MHCa. However, when myocardin is
coexpressed with Nkx2-5, Gata4 and Tbx5, MLC2 gene expression is
activated (lane labeled M+N+G+T). M, myocardin; N, Nkx2-5; G,
Gata4; T, Tbx5. (B) Co-expression of combinations of transcription
factors in animal cap explants shows that any combination of
myocardin and Gata4 is sufficient to activate MLC2 expression.
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.
