Fig. 4. Myocardin induces transcription of endogenous cardiac and smooth muscle marker genes in animal cap explants. Myocardin-expressing 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 -actin, MHC, 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.