We have examined the role of two RSRF/MEF2 proteins in the onset of skeletal and cardiac muscle differentiation in early Xenopus embryos. In normal development, zygotic expression of SL1 (MEF2D) precedes that of SL2 (MEF2A) by several hours, but neither gene is expressed prior to the accumulation of MyoD and Myf5 transcripts in the somitic mesoderm. Ectopic expression of the myogenic factors in explants of presumptive ectoderm induces expression of both SLI and SL2, whereas in reciprocal experiments, neither RSRF protein activates the endogenous myoD or Myf5 genes. We conclude that SL1 and SL2 lie downstream of these myogenic factors in the skeletal myogenic pathway. SL1 is distinguished from SL2 in being expressed in the presumptive heart region of the early tailbud embryo, prior to detection of any markers for cardiac muscle differentiation. Furthermore, ectopic SL1 induces the expression of an endogenous cardiac muscle-specific myosin light-chain (XMLC2) gene in cultured blastula animal pole explants, whereas SL2 has no comparable effect. These results demonstrate that in addition to a possible role in skeletal myogenesis, SL1 also acts in vivo as a regulator of cardiac muscle-specific transcription.[Key Words: RSRF/MEF-2; myosin light chain; cardiac-specific transcription; Xenopus] Received February 3, 1994; revised version accepted April 20, 1994. The RSRF (or MEF2) genes encode a family of sequencespecific DNA-binding proteins that are thought to play an important role in the differentiation of several cell types, including muscle and neuronal tissue. Four different RSRF genes have been identified so far, and their transcripts have been detected in a wide variety of vertebrate tissues and cultured cell lines (Pollock and Treisman 1991;Chambers et al. 1992;Yu et al. 1992;Breitbart et al. 1993;Leifer et al. 1993;McDermott et al. 1993). Each gene gives rise to multiple transcripts through alternate splicing, and the RSRF protein family potentially contains many distinct polypeptides. These proteins possess an amino-terminal portion that is virtually invariant throughout the family and encompasses the DNA-binding domain. The remainder of their sequence shows little conservation between family members.RSRF proteins recognize an A/T-rich DNA sequence motif (Pollock and Treisman 1990; Chambers et al. 1992) and can bind to the target sequence either as homodimers or as heterodimers with other members of the ~Corresponding author.RSRF family. A similar sequence specificity has been found for a number of previously characterized DNAbinding activities, and it is now clear that these contain RSRF proteins. One such binding activity, termed MEF2, is prevalent in muscle cells (Gossett et al. 1989). MEF2 activity increases during normal differentiation of skeletal myoblasts and is rapidly induced in fibroblasts in response to myogenic conversion by members of the MyoD family (Cserjesi and Olson 1991;Lassar et al. 1991). Sequences matching the RSRF/MEF2-binding site consensus have been found in the regulatory regions ...
We have isolated a cDNA fragment encoding a portion of the myosin heavy chain alpha-isoform (XMHC alpha) in the amphibian, Xenopus laevis. The XMHC alpha transcript is highly enriched in adult heart RNA and is expressed exclusively in embryonic heart tissue. It therefore provides a tissue-specific marker for cardiac muscle differentiation during early embryogenesis. Using an RNAase protection assay, we can detect the onset of cardiac muscle differentiation in an anterior, ventral region of tailbud embryos, many hours before the appearance of a beating heart. Whole-mount in situ RNA hybridisation indicates that expression of the XMHC alpha gene is restricted to the developing heart primordium. XMHC alpha gene expression can also be induced in isolated animal pole explants of blastulae by treatment with the growth factor, activin A. Induction is dose-dependent, requiring high doses of the growth factor compared with that required for myotomal (skeletal) muscle differentiation. In contrast, no XMHC alpha transcripts are detected in explants incubated with basic FGF, despite the induction of myotomal muscle differentiation. Activin-induced explants show a similar temporal pattern of XMHC alpha gene expression to that found in normal embryogenesis. Furthermore, cells expressing this gene appear clustered in one or two foci within fused explant aggregates, which often show regular, spontaneous contractions after several days in culture. These results show that terminal differentiation of cardiac muscle can occur in growth factor-induced explants and may be distinguished from skeletal muscle differentiation by the dose and nature of the inducing factor.
the Fgf8b mouse isoform. The cell line HEK-293 was transfected with this construct to obtain a permanent clone cell line in which the Fgf8b-EGFP could be secreted on the extracellular matrix. The inductive activity of Fgf8b-EGFP protein was proved either in vitro (cell cultures) as in ex-vivo (organotypic explant cultures of E9.5 mouse neural tube). Ectopically, Fgf8b-EGFP induces the phosphorilation of the final effector of the FGF signalling cascade (ERK1/2) and also the expression of downstream isthmic genes (Dusp6, Spry, SEF, En1/2). Using passive carriers (loaded heparin beads) or active productive sources (HEK-293-Fgf8b-EGFP cells) we have analysed the gradiental component of FGF8b over the neuroepithelium in time and space.
Despite recent progress in understanding of how left-right (L-R) asymmetry is generated during vertebrate development, many important questions still unanswered. One such question concerns the mechanism by which the signal responsible for the generation of L-R asymmetry is transferred from the node to the lateral plate. This signal, whose identity remains unknown, is
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