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ABSTRACT: The transcriptional regulatory network that controls the determination and differentiation of skeletal muscle cells in the embryo has at its core the four Myogenic Regulatory Factors (MRFs), Myf5, MyoD, Mrf4 and Myogenin. These bHLH transcription factors act by binding, as obligate heterodimers with the ubiquitously expressed E proteins, to the E-box sequence CANNTG. While all skeletal muscle cells have the same underlying function their progenitors arise at many sites in the embryo and it has become apparent that the upstream activators of the cascade differ in these various populations so that it can be switched on by a variety of inductive signals, some of which act by initiating transcription, some by maintaining it. The application of genome wide approaches has provided important new information as to how the MRFs function to activate the terminal differentiation programme and some of these data provide significant mechanistic insights into questions which have exercised the field for many years. We also consider the emerging roles played by miRNAs in the regulation of both upstream activators and terminal differentiation genes. This article is protected by copyright. All rights reserved.
FEBS Journal 06/2013; · 3.79 Impact Factor
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ABSTRACT: The specification of the skeletal muscle lineage during craniofacial development is dependent on the activity of MYF5 and MYOD, two members of the myogenic regulatory factor family. In the absence of MYF5 or MYOD there is not an overt muscle phenotype, whereas in the double Myf5;MyoD knockout branchiomeric myogenic precursors fail to be specified and skeletal muscle is not formed. The transcriptional regulation of Myf5 is controlled by a multitude of regulatory elements acting at different times and anatomical locations, with at least five operating in the branchial arches. By contrast, only two enhancers have been implicated in the regulation of MyoD. In this work, we characterize an enhancer element that drives Myf5 expression in the branchial arches from 9.5 days post-coitum and show that its activity in the context of the entire locus is dependent on two highly conserved E-boxes. These binding sites are required in a subset of Myf5-expressing cells including both progenitors and those which have entered the myogenic pathway. The correct levels of expression of Myf5 and MyoD result from activation by musculin and TCF21 through direct binding to specific enhancers. Consistent with this, we show that in the absence of musculin the timing of activation of Myf5 and MyoD is not affected but the expression levels are significantly reduced. Importantly, normal levels of Myf5 expression are restored at later stages, which might explain the absence of particular muscles in the Msc;Tcf21 double-knockout mice.
Development 03/2012; 139(5):958-67. · 6.60 Impact Factor
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ABSTRACT: The transcriptional regulation of the Mrf4/Myf5 locus depends on a multitude of enhancers that, in equilibria with transcription balancing sequences and the promoters, regulate the expression of the two genes throughout embryonic development and in the adult. Transcription in a particular set of muscle progenitors can be driven by the combined outputs of several enhancers that are not able to recapitulate the entire expression pattern in isolation, or by the action of a single enhancer the activity of which in isolation is equivalent to that within the context of the locus. We identified a new enhancer element of this second class, ECR111, which is highly conserved in all vertebrate species and is necessary and sufficient to drive Myf5 expression in ventro-caudal and ventro-rostral somitic compartments in the mouse embryo. EMSA analyses and data obtained from binding-site mutations in transgenic embryos show that a binding site for a TEA Domain (TEAD) transcription factor is essential for the function of this new enhancer, while ChIP assays show that at least two members of the family of transcription factors bind to it in vivo.
Developmental Biology 07/2011; 355(2):372-80. · 4.07 Impact Factor
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ABSTRACT: In order to fully describe the expression pattern of the transcription factor FoxO1, we have screened the ES cell genetrap repository databases and obtained a clone that contains the ß-geo reporter gene inserted within intron 1 of FoxO1. We then used the ES cell clone to generate a new mouse strain (B6;129P2- Foxo1(Gt(AD0086)Wtsi/JJC)), which expresses ß-geo according to the endogenous FoxO1 pattern, and collected embryo stages from 7.0dpc to 18.5dpc. We show that the expression of FoxO1 is highly dynamic, starting in the neuroepithelium and then extending into the developing vasculature, including all early stages of heart formation. There is a dramatic switch of expression at 11.5dpc in which most vascular expression is abolished and replaced by skeletal muscle expression. In addition FoxO1 is also expressed in several epithelial structures including the olfactory and otic systems, the cornea and at different levels of the gut depending on developmental stage. At later foetal stages, FoxO1 is upregulated again in the same tissues were it is active during early development, including skeletal muscle, vascular system and neuroepithelium.
Gene Expression Patterns 03/2011; 11(5-6):299-308. · 2.02 Impact Factor
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ABSTRACT: During embryonic development the integration of numerous synergistic signalling pathways turns a single cell into a multicellular organism with specialized cell types and highly structured, organized tissues. To achieve this, cells must grow, proliferate, differentiate and die according to their spatiotemporal position. Unravelling the mechanisms by which a cell adopts the correct fate in response to its local environment remains one of the fundamental goals of biological research. In vertebrates skeletal myogenesis is coordinated by the activation of the myogenic regulatory factors (MRFs) in response to signals that are interpreted by their associated regulatory elements in different precursor cells during development. The MRFs trigger a cascade of transcription factors and downstream structural genes, ultimately resulting in the generation of one of the fundamental histotypes. In this review we discuss the regulation of the different MRFs in relation to their position in the myogenic cascade, the changes in the general transcriptional machinery during muscle differentiation and the emerging importance of miRNA regulation in skeletal myogenesis.
Experimental Cell Research 11/2010; 316(18):3014-8. · 3.58 Impact Factor
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ABSTRACT: During development, gene activation is stringently regulated to restrict expression only to the correct cell type and correct developmental stage. Here, we present mechanistic evidence that suggests DNA methylation contributes to this regulation by suppressing premature gene activation. Using the mouse Myogenin promoter as an example of the weak CpG island class of promoters, we find that it is initially methylated but becomes demethylated as development proceeds. Full hypersensitive site formation of the Myogenin promoter requires both the MEF2 and SIX binding sites, but binding to only one site can trigger the partial chromatin opening of the nonmethylated promoter. DNA methylation markedly decreases hypersensitive site formation that now occurs at a detectable level only when binding to both MEF2 and SIX binding sites is possible. This suggests that the probability of activating the methylated promoter is low until two of the factors are coexpressed within the same cell. Consistent with this, the single-cell analysis of developing somites shows that the coexpression of MEF2A and SIX1, which bind the MEF2 and SIX sites, correlates with the fraction of cells that demethylate the Myogenin promoter. Taken together, these studies imply that DNA methylation helps to prevent inappropriate gene activation until sufficient activating factors are coexpressed.
Molecular and cellular biology 05/2010; 30(15):3805-15. · 6.06 Impact Factor
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ABSTRACT: We have analysed the expression during mouse development of the four member Lingo/LERN gene family which encodes type 1 transmembrane proteins containing 12 extracellular leucine rich repeats, an immunoglobulin C2 domain and a short intracellular tail. Each family member has a distinct pattern of expression in the mouse embryo as is the case for the related NLRR, FLRT and LRRTM gene families. Lingo1/LERN1 is expressed in the developing trigeminal, facio-acoustic and dorsal root ganglia. An interesting expression pattern is also observed in the somites with expression localising to the inner surface of the dermomyotome in the ventro-caudal lip. Further expression is seen in lateral cells of the hindbrain and midbrain, lateral cells in the motor horn of the neural tube, the otic vesicle epithelium and epithelium associated with the developing gut. Lingo3/LERN2 is expressed in a broad but specific pattern in many tissues across the embryo. Lingo2/LERN3 is seen in a population of cells lying adjacent to the epithelial lining of the olfactory pit while Lingo4/LERN4 is expressed in the neural tube in a subset of progenitors adjacent to the motor neurons. Expression of all Lingo/LERN genes increases as the embryo develops but is low in the adult with only Lingo1/LERN1 and Lingo2/LERN3 being detectable in adult brain.
Gene Expression Patterns 02/2008; 8(2):79-86. · 2.02 Impact Factor
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ABSTRACT: The linked Mrf4 and Myf5 genes encode two transcription factors essential for the determination and differentiation of skeletal muscle in the embryo. The locus is controlled by a multitude of interdigitated enhancers that activate gene expression at different times and in precisely defined progenitor cell populations. Manipulation of the enhancer-promoter composition of the locus reveals a novel mechanism for the regulation of such a gene cluster. Enhancers, promoters, and a new class of elements we call transcription balancing sequences, which can act as cryptic promoters, exist in a series of equilibria to ensure that enhancers and promoters together produce the highly dynamic and exquisitely specific expression patterns of the two genes. The proposed model depends upon nonproductive interactions between enhancers and both minimal and cryptic promoters, and is distinct from those developed for the beta-globin and Hox clusters. Moreover, it provides an explanation for the unexpected phenotypes of the three Mrf4 knockout alleles.
Genes & Development 02/2008; 22(2):265-76. · 11.66 Impact Factor
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ABSTRACT: Mrf4 (Myf6) is a member of the basic helix-loop-helix (bHLH) myogenic regulatory transcription factor (MRF) family, which also contains Myod, Myf5 and myogenin. Mrf4 is implicated in commitment of amniote cells to skeletal myogenesis and is also abundantly expressed in many adult muscle fibres. The specific role of Mrf4 is unclear both because mrf4 null mice are viable, suggesting redundancy with other MRFs, and because of genetic interactions at the complex mrf4/myf5 locus. We report the cloning and expression of an mrf4 gene from zebrafish, Danio rerio, which shows conservation of linkage to myf5. Mrf4 mRNA accumulates in a subset of terminally differentiated muscle fibres in parallel with myosin protein in the trunk and fin. Although most, possibly all, trunk muscle expresses mrf4, the level of mRNA is dynamically regulated. No expression is detected in muscle precursor cell populations prior to myosin accumulation. Moreover, mrf4 expression is not detected in head muscles, at least at early stages. As fish mature, mrf4 expression is pronounced in the region of slow muscle fibres.
Gene Expression Patterns 09/2007; 7(7):738-45. · 2.02 Impact Factor
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ABSTRACT: We have analysed the expression during mid-gestation mouse development of the four member LRRTM gene family which encodes type 1 transmembrane proteins containing 10 extracellular leucine rich repeats and a short intracellular tail. Each family member has a developmentally regulated pattern of expression distinct from all other members. LRRTM1 is expressed in the neural tube, otic vesicle, apical ectodermal ridge, forebrain and midbrain up to a sharp central boundary. LRRTM2 is expressed in a subset of progenitors in the neural tube. LRRTM3 is expressed in a half somite wide stripe in the presomitic mesoderm adjacent to the boundary with the most recently formed somite. Additional expression is seen in the neural tube, forebrain and hindbrain. LRRTM4 is expressed in the limb mesenchyme, neural tube, caudal mesoderm and in three distinct regions of the head. Later expression occurs in a subset of the developing sclerotome. Each family member has a unique expression domain within the neural tube.
Gene Expression Patterns 02/2007; 7(1-2):23-9. · 2.02 Impact Factor
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ABSTRACT: Within the mammalian genome, there are many multimember gene families that encode membrane proteins with extracellular leucine rich repeats which are thought to act as cell adhesion or signalling molecules. We previously showed that the members of the NLRR gene family are expressed in a developmentally restricted manner in the mouse with NLRR-1 being expressed in the developing myotome. The FLRT gene family shows a similar genomic layout and predicted protein secondary structure to the NLRRs so we analysed expression of the three FLRT genes during mouse development. FLRTs are glycosylated membrane proteins expressed at the cell surface which localise in a homophilic manner to cell-cell contacts expressing the focal adhesion marker vinculin. Each member of the FLRT family has a distinct, highly regulated expression pattern, as was seen for the NLRR family. FLRT3 has a provocative expression pattern during somite development being expressed in regions of the somite where muscle precursor cells migrate from the dermomyotome and move into the myotome, and later in myotomal precursors destined to migrate towards their final destination, for example, those that form the ventral body wall. FLRT3 is also expressed at the midbrain/hindbrain boundary and in the apical ectodermal ridge, regions where FGF signalling is known to be important, suggesting that the role for FLRT3 in FGF signalling identified in Xenopus is conserved in mammals. FLRT1 is expressed at brain compartmental boundaries and FLRT2 is expressed in a subset of the sclerotome, adjacent to the region that forms the syndetome, suggesting that interaction with FGF signalling may be a general property of FLRT proteins. We confirmed this by showing that all FLRTs can interact with FGFR1 and FLRTs can be induced by the activation of FGF signalling by FGF-2. We conclude that FLRT proteins act as regulators of FGF signalling, being induced by the signal and then able to interact with the signalling receptor, in many tissues during mouse embryogenesis. This process may, in part, be dependent on homophilic intercellular interactions between FLRT molecules.
Developmental Biology 10/2006; 297(1):14-25. · 4.07 Impact Factor
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ABSTRACT: During vertebrate embryogenesis, the somites form by segmentation of the trunk mesoderm, lateral to the neural tube, in an anterior to posterior direction. Analysis of differential gene expression during somitogenesis has been problematic due to the limited amount of tissue available from early mouse embryos. To circumvent these problems, we developed a modified differential display PCR technique that is highly sensitive and yields products that can be used directly as in situ hybridisation probes. Using this technique, we isolated NLRR-1 as a gene expressed in the myotome of developing somites but not in the presomitic mesoderm. Detailed expression analysis showed that this gene was expressed in the skeletal muscle precursors of the myotome, branchial arches and limbs as well as in the developing nervous system. Somitic expression occurs in the earliest myoblasts that originate from the dorsal lip in a pattern reminiscent of the muscle determination gene Myf5, but not at the ventral lip, indicating that NLRR-1 is expressed in a subset of myotome cells. The NLRR genes comprise a three-gene family encoding glycosylated transmembrane proteins with external leucine-rich repeats, a fibronectin domain, an immunoglobulin domain and short intracellular tails capable of mediating protein-protein interaction. Analysis of NLRR-3 expression revealed regulated expression in the neural system in developing ganglia and motor neurons. NLRR-2 expression appears to be predominately confined to the adult. The regulated embryonic expression and cellular location of these proteins suggest important roles during mouse development in the control of cell adhesion, movement or signalling.
Developmental Biology 06/2005; 281(2):145-59. · 4.07 Impact Factor
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ABSTRACT: The myogenic regulatory factor Myf5 is integral to the initiation and control of skeletal muscle formation. In adult muscle, Myf5 is expressed in satellite cells, stem cells of mature muscle, but not in the myonuclei that sustain the myofibre. Using the Myf5(nlacZ/+) mouse, we now show that Myf5 is also constitutively expressed in muscle spindles-stretch-sensitive mechanoreceptors, while muscle denervation induces extensive reactivation of the Myf5 gene in myonuclei. To identify the elements involved in the regulation of Myf5 in adult muscle, we analysed reporter gene expression in a transgenic bacterial artificial chromosome (BAC) deletion series of the Mrf4/Myf5 locus. A BAC carrying 140 kb upstream of the Myf5 transcription start site was sufficient to drive all aspects of Myf5 expression in adult muscle. In contrast, BACs carrying 88 and 59 kb upstream were unable to drive consistent expression in satellite cells, although expression in muscle spindles and reactivation of the locus in myonuclei were retained. Therefore, as during development, multiple enhancers are required to generate the full expression pattern of Myf5 in the adult. Together, these observations show that elements controlling adult Myf5 expression are genetically separable and possibly distinct from those that control Myf5 during development. These studies are a first step towards identifying cognate transcription factors involved in muscle stem cell regulation.
Developmental Biology 10/2004; 273(2):454-65. · 4.07 Impact Factor
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ABSTRACT: Myf5, the skeletal muscle determination gene, is first expressed in the dorso-medial aspect of the somite under the control of an element we have called the early epaxial enhancer. It has subsequently been reported that this enhancer is a direct target of Shh signaling mediated by Gli transcription factors (Gustafsson et al. 2002). We here demonstrate that activation of Myf5 expression depends on neither Shh function nor an intact Gli binding site, although the Gli site is necessary for continuation of expression. We suggest that the discrepancy is due to the existence of specific interactions between the enhancer and the Myf5 promoter.
Genes & Development 01/2004; 17(23):2870-4. · 11.66 Impact Factor
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Juliette Hadchouel,
Jaime J Carvajal,
Philippe Daubas,
Lola Bajard,
Ted Chang,
Didier Rocancourt,
David Cox,
Dennis Summerbell,
Shahragim Tajbakhsh, Peter W J Rigby,
Margaret Buckingham
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ABSTRACT: Myf5 is the first myogenic regulatory factor to be expressed in the mouse embryo and it determines the entry of cells into the skeletal muscle programme. A region situated between -58 kb and -48 kb from the gene directs Myf5 transcription at sites where muscles will form. We now show that this region consists of a number of distinct regulatory elements that specifically target sites of myogenesis in the somite, limbs and hypoglossal cord, and also sites of Myf5 transcription in the central nervous system. Deletion of these sequences in the context of the locus shows that elements within the region are essential, and also reveals the combinatorial complexity of the transcriptional regulation of Myf5. Both within the -58 kb to -48 kb region and elsewhere in the locus, multiple sequences are present that direct transcription in subdomains of a single site during development, thus revealing distinct phases of myogenesis when subpopulations of progenitor cells enter the programme of skeletal muscle differentiation.
Development 09/2003; 130(15):3415-26. · 6.60 Impact Factor
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ABSTRACT: Hox genes are key determinants of anteroposterior patterning of animal embryos, and spatially restricted expression of these genes is crucial to this function. In this study, we demonstrate that expression of Hoxb4 in the paraxial mesoderm of the mouse embryo is transcriptionally regulated in several distinct phases, and that multiple regulatory elements interact to maintain the complete expression domain throughout embryonic development. An enhancer located within the intron of the gene (region C) is sufficient for appropriate temporal activation of expression and the establishment of the correct anterior boundary in the paraxial mesoderm (somite 6/7). However, the Hoxb4 promoter is required to maintain this expression beyond 8.5 dpc. In addition, sequences within the 3' untranslated region (region B) are necessary specifically to maintain expression in somite 7 from 9.0 dpc onwards. Neither the promoter nor region B can direct somitic expression independently, indicating that the interaction of regulatory elements is crucial for the maintenance of the paraxial mesoderm domain of Hoxb4 expression. We further report that the domain of Hoxb4 expression is restricted by regulating transcript stability in the paraxial mesoderm and by selective translation and/or degradation of protein in the neural tube. Moreover, the absence of Hoxb4 3'-untranslated sequences from transgene transcripts leads to inappropriate expression of some Hoxb4 transgenes in posterior somites, indicating that there are sequences within region B that are important for both transcriptional and post-transcriptional regulation.
Development 07/2003; 130(12):2717-28. · 6.60 Impact Factor
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BioTechniques 01/2003; 33(6):1206-8. · 2.67 Impact Factor
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ABSTRACT: Vertebrate myogenesis is controlled by four transcription factors known as the myogenic regulatory factors (MRFs): Myf5, Mrf4, myogenin and MyoD. During mouse development Myf5 is the first MRF to be expressed and it acts by integrating multiple developmental signals to initiate myogenesis. Numerous discrete regulatory elements are involved in the activation and maintenance of Myf5 gene expression in the various muscle precursor populations, reflecting the diversity of the signals that control myogenesis. Here we focus on the enhancer that recapitulates the first phase of Myf5 expression in the epaxial domain of the somite, in order to identify the subset of cells that first transcribes the gene and therefore gain insight into molecular, cellular and anatomical facets of early myogenesis. Deletion of this enhancer from a YAC reporter construct that recapitulates the Myf5 expression pattern demonstrates that this regulatory element is necessary for expression in the early epaxial somite but in no other site of myogenesis. Importantly, Myf5 is subsequently expressed in the epaxial myotome under the control of other elements located far upstream of the gene. Our data suggest that the inductive signals that control Myf5 expression switch rapidly from those that impinge on the early epaxial enhancer to those that impinge on the other enhancers that act later in the epaxial somite, indicating that there are significant changes in either the signalling environment or the responsiveness of the cells along the rostrocaudal axis. We propose that the first phase of Myf5 epaxial expression, driven by the early epaxial enhancer in the dermomyotome, is necessary for early myotome formation, while the subsequent phases are associated with cytodifferentiation within the myotome.
Development 11/2002; 129(19):4571-80. · 6.60 Impact Factor
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ABSTRACT: The development of skeletal muscle in vertebrate embryos is controlled by a transcriptional cascade involving the four myogenic regulatory factors. In the somites of the mouse embryo the order of expression is thought to be Myf5, Myogenin, Mrf4 and MyoD. We have re-examined the expression pattern of Mrf4 and show that in the hypaxial domain of thoracic somites (the somitic bud) Mrf4 expression precedes or is contemporaneous with that of Myf5, suggesting that this transcription factor plays a hitherto unsuspected role in myogenesis.
Mechanisms of Development 10/2002; 117(1-2):331-5. · 2.83 Impact Factor
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ABSTRACT: Understanding how boundaries and domains of Hox gene expression are determined is critical to elucidating the means by which the embryo is patterned along the anteroposterior axis. We have performed a detailed analysis of the mouse Hoxb4 intron enhancer to identify upstream transcriptional regulators. In the context of an heterologous promoter, this enhancer can establish the appropriate anterior boundary of mesodermal expression but is unable to maintain it, showing that a specific interaction with its own promoter is important for maintenance. Enhancer function depends on a motif that contains overlapping binding sites for the transcription factors NFY and YY1. Specific mutations that either abolish or reduce NFY binding show that it is crucial for enhancer activity. The NFY/YY1 motif is reiterated in the Hoxb4 promoter and is known to be required for its activity. As these two factors are able to mediate opposing transcriptional effects by reorganizing the local chromatin environment, the relative levels of NFY and YY1 binding could represent a mechanism for balancing activation and repression of Hoxb4 through the same site.
Development 09/2002; 129(16):3887-99. · 6.60 Impact Factor
