DIFFERENTIAL EXPRESSION OF THE MYOCYTE ENHANCER FACTOR-2 FAMILY OF TRANSCRIPTION FACTORS IN DEVELOPMENT - THE CARDIAC FACTOR BBF-1 IS AN EARLY MARKER FOR CARDIOGENESIS

In the present study, we have used single chicken blastoderms of defin ed early developmental stages, beginning with the prestreak stage, sta ge 1 (V. Hamburger and H. L. Hamilton, J. Morphol. 88:49-92, 1951), to analyze the onset of cardiac myogenesis by monitoring the appearance of selected cardiac muscle tissue-specific gene transcripts and the fu nctional expression of the myocyte enhancer factor 2 (MEF-2) proteins. Using gene-specific oligonucleotide primers in reverse transcriptase PCR assay, we have demonstrated that the cardiac myosin light-chain 2 (MLC2) and alpha-actin gene transcripts appear as early as stage 5, i. e., immediately after the cardiogenic fate assignment at stage 4. Cons istent with this observation is the developmental expression pattern o f DNA-binding activity of BBF-1, a cardiac muscle-specific member of t he MEF-2 protein family, which also begins at stage 5 prior to MEF-2. Differential expression of DNA-binding complexes is also observed,vith another AT-rich DNA sequence (CArG box) as probe, but the binding pat tern with the ubiquitous TATA-binding proteins remains unchanged durin g the same developmental period. Thus, the cardiogenic commitment and differentiation of the precardiac mesoderm, as exemplified by the appe arance of cardiac MEF-2, MLC2, and alpha-actin gene products, occur ea rlier than previously thought and appear to be closely linked. The ons et of skeletal myogenic program follows that of the cardiogenic progra m with the appearance of skeletal MLC2 at stage 8. We also observed th at mRNA for the MEF-2 family of proteins appears as early as stage 2 a nd that for CMD-1, the chicken counterpart of MyoD, appears at stage 5 . The temporal separation of activation of cardiac and skeletal MLC2 g enes, which appears immediately after the respective fate assignments, and those of cardiac MEF-2 and CMD-1, which occur before, are consist ent with the established appearance of the myogenic programs and with the acquisition pattern of the two tissue-specific morphological chara cteristics in the early embryo. The preferential appearance of BBF-1 a ctivity in precardiac mesoderm, relative to that of MEF-2, indicates t hat these two protein factors are distinct members of the MEF-2 family and provides a compelling argument in support of the potential role o f BBF-1 as a regulator of the cardiogenic cell lineage determination, while cardiac MEF-2 might be involved in maintenance of the cardiac di fferentiative state.