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We discuss the upstream regulators of myogenesis that lead to the activation of myogenic determination genes and subsequent differentiation, focusing on the mouse model. Key upstream genes, such as Pax3 and Pax7, Six1 and Six4, or Pitx2, participate in gene regulatory networks at different sites of skeletal muscle formation. MicroRNAs also intervene, with emerging evidence for the role of other noncoding RNAs. Myogenic determination and subsequent differentiation depend on members of the MyoD family. We discuss new insights into mechanisms underlying the transcriptional activity of these factors.

We have analyzed the expression pattern of transcription factor AP-2 in mouse embryos to evaluate the potential of AP-2 as a regulator during vertebrate development. A partial cDNA encoding AP-2 was isolated from a mouse embryo cDNA library and used to prepare probes to measure AP-2 mRNA levels by RNase protection and RNA in situ hybridization. Between 10.5 and 15.5 days of embryogenesis, the relative abundance of AP-2 mRNA is greatest at 11.5 days and declines steadily thereafter. RNA in situ hybridization analysis of embryos between 8.5 and 12.5 days of gestation identified a novel expression pattern for AP-2. The principle part of this expression occurs in neural crest cells and their major derivatives, including cranial and spinal sensory ganglia and facial mesenchyme. AP-2 is also expressed in surface ectoderm and in a longitudinal column of the spinal cord and hindbrain that is contacted by neural crest-derived sensory ganglia. Additional expression of AP-2 occurs in limb bud mesenchyme and in meso-metanephric regions. This embryonic expression pattern is spatially and temporally consistent with a role for AP-2 in regulating transcription of genes involved in the morphogenesis of the peripheral nervous system, face, limbs, skin, and nephric tissues.

Our knowledge of the mechanisms that regulate transcription in higher eukaryotic cells has increased enormously during the past 2 years. Earlier studies, using a combination of in vitro mutagenesis and DNA-mediated gene transfer, identified two distinct types of cis-acting regulatory sequences: promoters, which are located close to the initiation site and act in a position-dependent fashion, and enhancers, which can be located far from the initiation site and act in a position-and orientation-independent fashion. Promoters can be subdivided into proximal elements, including the cap site itself and the TATA box, which is involved in fixing the site of initiation, and distal elements, which can be spread over several hundred base pairs. It is now clear that many promoters, particularly those of 'housekeeping' genes, lack TATA boxes and are instead composed of GC-rich elements that are often located within methylation-free islands (Bird 1986). Transcription controlled by this latter class of promoters often initiates at multiple sites. Many enhancer elements, for example, those of the immunoglobulin, insulin, and elastase genes, impose tissue-specific expression on adjacent promoters.These cis-acting elements operate by interacting with protein factors, many of which have now been identified by gel retardation and footprinting assays and some of which have been purified to homogeneity. In a few cases the corresponding genes have been cloned. In many of these studies well-characterized cis-acting elements of viruses, particularly of the DNA tumor viruses, have played a major role. At the recent ICRF-sponsored meeting on the Molecular Biology of SV40, Polyoma, and Adenoviruses (held in Cambridge, England, July 27-August 1, 1987) there was a major emphasis on such trans-acting factors. The results reported both identified new factors and considerably clarified the relationships amongst previously described factors.In this review we will discuss data reported at the meeting and, where appropriate, refer to recently published work. We will concentrate almost exclusively on work with DNA tumor viruses, referring to other systems only when there are direct connections to our major topic. Table 1 lists the factors of relevance, giving the DNA sequences they recognize, their synonyms, and the regulatory systems in which they are known to be involved.

skeletal muscle and skeletal muscle precursors, and on cell culture experiments, all of which pointed to func-and B. J. Wold § *Hamon Center for Basic Cancer Research tions in specification of myoblasts and execution of muscle differentiation. The major muscle phenotypes The University of Texas Southwestern Medical Center at Dallas described thus far are the failure of muscle differentiation in mice homozygous for myogenin null mutations Dallas, Texas 75235-9148 † Technische Universitat Braunschweig (Hasty et al., 1993; Nabeshima et al., 1993) and failure to produce the myogenic precursor cell population in Institut fur Biochemie und Biotechnologie 3300 Braunschweig mice deficient in both MyoD and Myf5 (Rudnicki et al., 1993). Unlike muscle deficits, the skeletal phenotypes Federal Republic of Germany ‡ Division of Eukaryotic Molecular Genetics were not expected, although they can account for the lethality of several MRF mutations. MRC National Institute for Medical Research London NW7 1AA MRF4 (Rhodes and Konieczny, 1989) is the final myogenic bHLH gene to be targeted in mice. It is located United Kingdom § Division of Biology 156-29 approximately 8 kb 5Ј of Myf5 on mouse chromosome 10. The proximity of MRF4 and Myf5 to each other raises

We have generalized the protocol of gene transfer, greatly increasing the variety of cells that can be used as recipients of foreign genes. Our approach has been to use a transient assay system that allows rapid screening of expression of foreign DNA. When the initial steps of gene transfer have been optimized with the transient system, these defined conditions are used to yield efficient stable transformation. We have seen that primate cells, including human cells, can be used in gene transfer experiments at levels sensitive enough to allow detection of single copy gene function. Recently we have also used this approach successfully with undifferentiated embryonic cells.

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