and differentiation of specialized cell types in vertebrates. The isolation of a family of muscle regulatory genes which includes MyoD, myogenin, Myf-5 and MRF4 ...
Journal of Cell Science 104, 957-960 (1993) Printed in Great Britain © The Company of Biologists Limited 1993
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COMMENTARY The role of Myf-5 in somitogenesis and the development of skeletal muscles in vertebrates Hans-Henning Arnold and Thomas Braun Department of Cell and Molecular Biology, University of Braunschweig, Konstantin-Uhde-Straße 5, 3300 Braunschweig, FRG
INTRODUCTION Muscle research has become a paradigm for the investigation of developmental processes that trigger determination and differentiation of specialized cell types in vertebrates. The isolation of a family of muscle regulatory genes which includes MyoD, myogenin, Myf-5 and MRF4 (also called herculin and Myf-6) represented a breakthrough in our attempt to identify important genetic components required for myogenesis (for recent reviews see: Emerson, 1990; Olson, 1990; Weintraub et al., 1991). About twenty years ago, the first experimental evidence was provided in muscle cell culture systems that suggested the existence of a “master gene” that controls the myogenic fate of cells (O’Neill and Stockdale, 1974; Holtzer et al., 1975). This hypothesis has been revived recently by the identification of the myogenic regulatory genes. While MyoD was the first gene to be cloned that had the ability to induce the muscle phenotype in cultured fibroblasts (Davis et al., 1987), it is now clear that the other members of this family have similar properties and may serve overlapping biological functions. In this Commentary, we will focus on the myogenic protein Myf-5, its importance as muscle-specific transcription factor and its possible role in somitogenesis during mouse development.
Myf-5 IS A CELL TYPE-SPECIFIC TRANSCRIPTION FACTOR Myf-5 (Braun et al., 1989) belongs to a large group of related regulatory proteins that have been identified in numerous organisms ranging from Drosophila to man. These proteins share homology within a segment of approximately 70 amino acids, designated as basic-helix-loophelix (bHLH) motif, because it contains a region rich in basic amino acids adjacent to a sequence postulated to form two amphipathic α-helices, which are connected by an intervening loop structure of variable length (Murre et al., 1989). Myf-5 as well as the other members of the family of myogenic regulatory proteins accumulates in nuclei of established and primary muscle cells in culture, and in the skeletal musculature and its somitic precursors in vertebrate
embryos. No muscle-specific HLH proteins have yet been detected in cardiac and smooth muscle cells. While muscle cell lines of embryological origin tend to contain more Myf5 transcripts, cell lines derived from satellite cells of adult muscle express more MyoD, an observation that may reflect their expression patterns in vivo (see discussion below). In tissue culture cells there is also evidence that myogenic bHLH proteins may autoregulate their own gene expression as well as that of the other family members, complicating the assessment of individual functions of Myf proteins. The activity of the Myf-5 gene appears to be negatively controlled by MyoD during mouse myogenesis as targeted inactivation of the MyoD gene results in elevated levels of Myf-5 transcripts (Rudnicki et al., 1992). The important role that Myf-5 may play in the determination and differentiation of myoblasts has most intriguingly been demonstrated by its remarkable ability to induce the muscle phenotpye in a variety of non-muscle cells, most effectively in 10T1/2 fibroblasts, which appear to be particularly permissive for this myogenic conversion (Myf-5 shares this activity with the other three myogenic factors). The induction of myogenesis in 10T1/2 cells by forced expression of Myf-5 (for instance, from a viral LTR promoter) is accompanied by transcriptional activation of an array of numerous, genetically unlinked muscle-specific genes including the genes for MyoD, myogenin and MRF4, which are subject to the above-mentioned cross-activation by Myf-5. Interestingly, the Myf-5 gene itself can not be auto- or cross-activated efficiently in 10T1/2 cells, which may suggest that it acts upstream from the other genes in a functional hierarchy or cascade of gene activations. This was supported by experiments with rat L6 myoblasts, in which Myf-5 activity has been inhibited by the adenovirus protein E1A resulting in a complete block of myogenin transcription (Braun et al., 1992a). Mutational analysis of the Myf-5 protein has revealed that transcriptional activation of muscle-specific target genes by Myf-5 requires transactivator domains located in the amino- and carboxyl-terminal regions of the protein, outside of the bHLH domain (Braun et al., 1990). However, the basic region that mediates sequence-specific DNAbinding also cooperates with these transactivator domains, Key words: Myf-5, somitogenesis, skeletal muscle
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whereby it specifies transcription of muscle-specific genes through a mechanism that is yet unknown. The ability of Myf-5 to activate specifically the transcription of muscle but no other genes to which it also can bind is dependent on few amino acids within the basic region, referred to as muscle recognition motif (Winter et al., 1992). Similar results were also described for MyoD and myogenin (for review see: Olson, 1990; Weintraub et al., 1991). Future investigations will be needed to clarify whether the specification of transactivation that is clearly distinguishable from DNA binding is caused by a binding-site-induced conformational change of the myogenic factors or by distinct coactivator molecules that selectively interact with them. Myf-5, as all bHLH proteins, binds as heterodimer to the DNA consensus motif CANNTG, known as E-box, which is present in one or several copies in control regions of many muscle-specific genes. Dimerization which is mediated through the HLH domain increases the affinity to the E-box and makes DNA-binding more efficient. While Myf5 as well as MyoD, myogenin and MRF4 readily form complexes with the universal bHLH proteins E12 and E47, they do not interact with each other. The structural requirements within the amphipathic helices that determine the specificity of heterodimerization are unknown. Overexpression of Myf-5 in cell culture systems has revealed no specific target genes that would not also be activated by MyoD or myogenin. However, the apparent lack of specificity among the different myogenic transcription factors may be the result of the experimental conditions under which large quantities of the proteins are produced, which probably obscure more subtle differences. In summary, there is abundant evidence in tissue culture systems that Myf-5, similar to MyoD, myogenin and presumably MRF4, acts as specific transcription factor that activates the expression of muscle-specific genes either directly or indirectly by activating other transcription factors that also participate in muscle gene expression. Although these observations in in vitro systems seem to support the concept of the “master switch” gene in myogenesis, the precise role of myogenic factors in vivo, in particular a function in determining myogenic cell fate during embryonic development, cannot be deduced from these results.
matome providing fibroblasts of the dorsal dermis as well as proliferating myoblasts and the myotome, which develops into axial skeletal muscles. The dermomyotome also provides the source of migrating myoblasts that form premuscle masses in the limbs and elsewhere. Thus, somites yield muscle precursor cells that become committed to two different myogenic lineages, the precursors for dorsal and lateral body muscles and the progenitors for the limb musculature (Christ et al., 1990; Ordahl and Le Douarin, 1992). The Myf-5 gene is first activated in cells of the dorsomedial edge in the epithelial and ball-shaped somites beginning in the cranial region of the mouse embryo at day 8 p.c. (Ott et al., 1991). It continues to be expressed in maturing somites throughout the dermomyotome, particularly in cells of the dorsal region adjacent to the neural tube. Myf5 transcripts are still present in differentiated myotomal cells and start to appear in the premuscle masses of the limb buds at around 10.5 days p.c. The Myf-5 gene is then downregulated and by day 12 p.c. transcripts are no longer detectable by in situ hybridization. The actual appearance of Myf-5 protein in the embryo has not yet been determined. The onset of Myf-5 expression precedes that of myogenin, herculin (MRF4/Myf-6) and MyoD, the other members of the myogenic gene family which become activated in myotomal cells sucessively in this order (Bober et al., 1991). Myf-5 expression also precedes α-actin transcripts, encoding the first contractile protein found in myotomal muscle. Thus, the only myogenic transcript detectable in the somite prior to myotome formation is Myf5, which suggests that it may be involved in the determination process that directs precursor cells to the myogenic lineage where it may serve as molecular marker for future myotomal cells. It also suggests that, if cross-activation of the myogenic bHLH genes that is observed in cultured cells plays any role in vivo, Myf-5 should be the first gene in this activation cascade. The exploration of functional aspects with respect to early Myf-5 expression during development of the mouse embryo became recently amenable in a mouse mutant which carries an inactive Myf5 gene. TARGETED INACTIVATION OF THE Myf-5 GENE AFFECTS EARLY SOMITE DEVELOPMENT
Myf-5 IS EXPRESSED IN SOMITES IN THE MOUSE EMBRYO PRIOR TO MUSCLE FORMATION The first indication that the myogenic regulatory genes may indeed play a role during myogenesis in vivo came from in situ hybridizations, which elucidated their spatio-temporal expression patterns during mouse development (Sassoon et al., 1990). Skeletal muscle in the vertebrate embryo derives from somites which form in a cranio-caudal sequence from the segmental plate mesoderm located on both sides of the neural tube and notochord along the longitudinal axis. Initially, somites appear as spherical structures, which consist primarily of epithelial-like cells that later differentiate into several cell types. The ventral half of the somite gives rise to the mesechymal cells of the sclerotome and the dorsal half forms the dermomyotome. Further subdivision of the dermomyotome results in the der-
To study the consequences of a “loss of function” mutation in its natural biological context, a Myf-5 gene copy inactivated by insertion of the neomycin resistance gene into the first coding exon which disrupts the open reading frame has been introduced into the mouse germ line by homologus recombination in embryonic stem cells (Braun et al., 1992b). Heterozygous mice carrying this mutation have no apparent phenotype. As these animals produce approximately only half of the amount of Myf-5 protein compared to wild-type mice, the lack of a discernable phenotype suggests that a critical threshold level of Myf-5 protein that may be required for full biological efficacy is low or does not exist. Regulation of Myf-5 activity based on its concentration and a delicate balance of positive- and negativeacting dimerization partners may not be prevalent in vivo. Homozygous Myf-5 mutation in mice results in perinatal
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Fig. 1. Homozygous Myf-5 mutant mice have a severe defect in rib formation. The ventral view of open thoraces of a neonate wild-type (A) and a homozygous Myf-5 mutant mouse (B) shows rib truncations at the costo-vertebral junctions in the mutant. While the vertebral column appears normal, all distal parts of the ribs are missing. Sections through the thoracic region of wild-type (C) and mutant (D) animals reveal the presence of intercostal muscles that appear disarranged in the mutant.
recessive lethality. These mice die shortly after birth due to respiratory failure. They exhibit a severe defect in rib formation, which leads to the loss of an intact rib cage (Fig. 1). All ribs are truncated at approximately the costo-vertebral junctions with the distal parts missing. The sternum is shortened and completely ossified. Intercostal muscles and the dorsal part of the diaphragm are present but appear disorganized, probably because orderly rib structures are missing. Other axial skeletal muscles and the muscles of the limbs appear normal in neonates. However, in somites between day 8 p.c. and approximately 11.5 p.c., when Myf5 is normally expressed, no myotomal cells expressing any of the known muscle marker genes can be detected. After day 11.5 of embryogenesis, myotomal cells as well as the premuscle masses in the limb buds begin to appear and seem to develop into quite normal skeletal muscles. These muscle cells express the myogenic HLH proteins, MyoD, myogenin and MRF4, suggesting that in vivo these genes can be activated in the absence of Myf-5. The considerable delay of the first appearance of myotomal cells is significant, as it demarcates the developmental period during which commitment of somitic cells to the myogenic fate is thought to take place. The fact that skeletal musculature develops despite this early defect in myotome formation may indicate that there is partial functional redundancy among the myogenic factors, and the later-expressed MyoD or any of the other factors may substitute for the lack of
Myf-5. It seems nevertheless surprising that precursor cells destined to become myotomal muscle retain their ability to respond to a myogenic cue for such a long time. At this early stage of the analysis, the alternative possibility remains that Myf-5 has no direct role in myogenesis in vivo but instead is crucial for the development of sclerotomal derivates, either by a cell autonomous mechanism or by inductive or permissive interactions between myotomal and sclerotomal cells that are required to establish or expand the cell compartment that gives rise to the lateral sclerotome from which ribs are made. Significantly, no other sclerotomically derived structures are affected by the Myf-5 inactivation, implying that sclerotomes are heterogeneous and only some of the cells require Myf-5 activity for further development. Additional investigations are in progress to clarify what Myf-5 does in this process. CONCLUSIONS Although the biological activities of Myf-5 determined in tissue culture systems are essentially indistinguishable from those of the other members of the MyoD family, its expression pattern during embryogenesis and its developmental role appears to be distinct. While inactivation of the Myf5 gene results in a severe phenotype, mice lacking a functional MyoD gene appear virtually normal (Rudnicki et al.,
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1992). Obviously, it will be of great interest to mutate the other members of the myogenic gene family and see the results in mice harboring double and multiple gene “knock outs”. This line of experiments will certainly help to assess the precise role and possible cooperative functions among these genes. Much to our surprise, Myf-5 turned out to be not only a myogenic factor but a gene that controls early somitogenesis, affecting more than one cell type derived from this important embryological compartment. The identification of downstream target genes that become activated by Myf-5 in early somitic cells would be of great interest in helping to understand this unexpected result. Research in the authors’ laboratory was supported by grants from the Deutsche Forschungsgemeinschaft, the European Community and the Deutsche Muskelschwundhilfe. The targeted inactivation of the Myf-5 gene in the mouse germ line was performed in collaboration with M. Rudnicki and R. Jaenisch of The Whitehead Institute, Cambridge.
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