XB-ART-2418Development March 1, 2005; 132 (5): 987-97.
Myocardin is sufficient and necessary for cardiac gene expression in Xenopus.
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.
PubMed ID: 15673566
Article link: Development
Grant support: HL63926 NHLBI NIH HHS
Genes referenced: actc1 actl6a cnn2 gata4 mef2a mrtfa mrtfb myf5 myf6 myh6 myl2 mylpf myocd myod1 myog nkx2-5 srf tagln tbx5 tbxt tnni3
Morpholinos referenced: myocd MO1 myocd MO2
Article Images: [+] show captions
|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 embryo myocardin 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, anterior left.|
|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.|