RESUMO
Skeletal myogenesis in vertebrates is initiated at different sites of skeletal muscle formation during development, by activation of specific control elements of the myogenic regulatory genes. In the mouse embryo, Myf5 is the first myogenic determination gene to be expressed and its spatiotemporal regulation requires multiple enhancer sequences, extending over 120â kb upstream of the Mrf4-Myf5 locus. An enhancer, located at -57/-58â kb from Myf5, is responsible for its activation in myogenic cells derived from the hypaxial domain of the somite, that will form limb muscles. Pax3 and Six1/4 transcription factors are essential activators of this enhancer, acting on a 145-bp core element. Myogenic progenitor cells that will form the future muscle masses of the limbs express the factors necessary for Myf5 activation when they delaminate from the hypaxial dermomyotome and migrate into the forelimb bud, however they do not activate Myf5 and the myogenic programme until they have populated the prospective muscle masses. We show that Msx1 and Meox2 homeodomain-containing transcription factors bind in vitro and in vivo to specific sites in the 145-bp element, and are implicated in fine-tuning activation of Myf5 in the forelimb. Msx1, when bound between Pax and Six sites, prevents the binding of these key activators, thus inhibiting transcription of Myf5 and consequent premature myogenic differentiation. Meox2 is required for Myf5 activation at the onset of myogenesis via direct binding to other homeodomain sites in this sequence. Thus, these homeodomain factors, acting in addition to Pax3 and Six1/4, fine-tune the entry of progenitor cells into myogenesis at early stages of forelimb development.
RESUMO
Dorsal spinal neurogenesis is orchestrated by the combined action of signals secreted from the roof plate organizer and a downstream transcriptional cascade. Within this cascade, Msx1 and Msx2, two homeodomain transcription factors (TFs), are induced earlier than bHLH neuralizing TFs. Whereas bHLH TFs have been shown to specify neuronal cell fate, the function of Msx genes remains poorly defined. We describe dramatic alterations of neuronal patterning in Msx1/Msx2 double-mutant mouse embryos. The most dorsal spinal progenitor pool fails to express the bHLH neuralizing TF Atoh1, which results in a lack of Lhx2-positive and Barhl2-positive dI1 interneurons. Neurog1 and Ascl1 expression territories are dorsalized, leading to ectopic dorsal differentiation of dI2 and dI3 interneurons. In proportion, the amount of Neurog1-expressing progenitors appears unaffected, whereas the number of Ascl1-positive cells is increased. These defects occur while BMP signaling is still active in the Msx1/Msx2 mutant embryos. Cell lineage analysis and co-immunolabeling demonstrate that Atoh1-positive cells derive from progenitors expressing both Msx1 and Msx2. In vitro, Msx1 and Msx2 proteins activate Atoh1 transcription by specifically interacting with several homeodomain binding sites in the Atoh1 3' enhancer. In vivo, Msx1 and Msx2 are required for Atoh1 3' enhancer activity and ChIP experiments confirm Msx1 binding to this regulatory sequence. These data support a novel function of Msx1 and Msx2 as transcriptional activators. Our study provides new insights into the transcriptional control of spinal cord patterning by BMP signaling, with Msx1 and Msx2 acting upstream of Atoh1.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/metabolismo , Fator de Transcrição MSX1/metabolismo , Medula Espinal/metabolismo , Animais , Sequência de Bases , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Sítios de Ligação , Proteínas Morfogenéticas Ósseas/metabolismo , Diferenciação Celular/genética , Embrião de Mamíferos/metabolismo , Elementos Facilitadores Genéticos/genética , Interneurônios/citologia , Interneurônios/metabolismo , Camundongos , Dados de Sequência Molecular , Mutação/genética , Ligação Proteica/genética , Transdução de Sinais/genética , Medula Espinal/embriologia , Células-Tronco/metabolismoRESUMO
The Myf5 gene plays an important role in myogenic determination during mouse embryo development. Multiple genomic regions of the Mrf4-Myf5 locus have been characterised as enhancer sequences responsible for the complex spatiotemporal expression of the Myf5 gene at the onset of myogenesis. These include an enhancer sequence, located at -111 kb upstream of the Myf5 transcription start site, which is responsible of Myf5 activation in ventral somitic domains (Ribas et al., 2011. Dev. Biol. 355, 372-380). We show that the -111 kb-Myf5 enhancer also directs transgene expression in some limb muscles, and is active at foetal as well as embryonic stages. We have carried out further characterisation of the regulation of this enhancer and show that the paired-box Pax3 transcription factor binds to it in vitro as in vivo, and that Pax binding sites are essential for its activity. This requirement is independent of the previously reported regulation by TEAD transcription factors. Six1/4 which, like Pax3, are important upstream regulators of myogenesis, also bind in vivo to sites in the -111 kb-Myf5 enhancer and modulate its activity. The -111 kb-Myf5 enhancer therefore shares common functional characteristics with another Myf5 regulatory sequence, the hypaxial and limb 145 bp-Myf5 enhancer, both being directly regulated in vivo by Pax3 and Six1/4 proteins. However, in the case of the -111 kb-Myf5 enhancer, Six has less effect and we conclude that Pax regulation plays a major role in controlling this aspect of the Myf5 gene expression at the onset of myogenesis in the embryo.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/metabolismo , Desenvolvimento Muscular , Fator Regulador Miogênico 5/genética , Fator Regulador Miogênico 5/fisiologia , Fatores de Transcrição Box Pareados/genética , Fatores de Transcrição Box Pareados/fisiologia , Transativadores/metabolismo , Animais , Sequência de Bases , Sítios de Ligação , Fator II de Transcrição COUP/metabolismo , Elementos Facilitadores Genéticos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microscopia de Fluorescência/métodos , Modelos Biológicos , Modelos Genéticos , Dados de Sequência Molecular , Fator de Transcrição PAX3 , Plasmídeos/metabolismo , Homologia de Sequência do Ácido NucleicoRESUMO
How muscle diversity is generated in the vertebrate body is poorly understood. In the limb, dorsal and ventral muscle masses constitute the first myogenic diversification, as each gives rise to distinct muscles. Myogenesis initiates after muscle precursor cells (MPCs) have migrated from the somites to the limb bud and populated the prospective muscle masses. Here, we show that Sonic hedgehog (Shh) from the zone of polarizing activity (ZPA) drives myogenesis specifically within the ventral muscle mass. Shh directly induces ventral MPCs to initiate Myf5 transcription and myogenesis through essential Gli-binding sites located in the Myf5 limb enhancer. In the absence of Shh signaling, myogenesis is delayed, MPCs fail to migrate distally, and ventral paw muscles fail to form. Thus, Shh production in the limb ZPA is essential for the spatiotemporal control of myogenesis and coordinates muscle and skeletal development by acting directly to regulate the formation of specific ventral muscles.
Assuntos
Extremidades/embriologia , Proteínas Hedgehog/metabolismo , Desenvolvimento Muscular/genética , Músculo Esquelético/embriologia , Mioblastos/citologia , Animais , Diferenciação Celular , Proliferação de Células , Sobrevivência Celular , Células Cultivadas , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Proteínas Hedgehog/genética , Botões de Extremidades/citologia , Botões de Extremidades/embriologia , Camundongos , Camundongos Transgênicos , Músculo Esquelético/citologia , Músculo Esquelético/metabolismo , Mioblastos/metabolismo , Transdução de SinaisRESUMO
The LIM homeodomain gene Islet-1 (ISL1) encodes a transcription factor that has been associated with the multipotency of human cardiac progenitors, and in mice enables the correct deployment of second heart field (SHF) cells to become the myocardium of atria, right ventricle and outflow tract. Other markers have been identified that characterize subdomains of the SHF, such as the fibroblast growth factor Fgf10 in its anterior region. While functional evidence of its essential contribution has been demonstrated in many vertebrate species, SHF expression of Isl1 has been shown in only some models. We examined the relationship between human ISL1 and FGF10 within the embryonic time window during which the linear heart tube remodels into four chambers. ISL1 transcription demarcated an anatomical region supporting the conserved existence of a SHF in humans, and transcription factors of the GATA family were co-expressed therein. In conjunction, we identified a novel enhancer containing a highly conserved ISL1 consensus binding site within the FGF10 first intron. ChIP and EMSA demonstrated its direct occupation by ISL1. Transcription mediated by ISL1 from this FGF10 intronic element was enhanced by the presence of GATA4 and TBX20 cardiac transcription factors. Finally, transgenic mice confirmed that endogenous factors bound the human FGF10 intronic enhancer to drive reporter expression in the developing cardiac outflow tract. These findings highlight the interest of examining developmental regulatory networks directly in human tissues, when possible, to assess candidate non-coding regions that may be responsible for congenital malformations.
Assuntos
Fator 10 de Crescimento de Fibroblastos/genética , Coração/embriologia , Proteínas com Homeodomínio LIM/metabolismo , Organogênese/genética , Fatores de Transcrição/metabolismo , Transcrição Gênica , Animais , Sequência de Bases , Biologia Computacional , Ensaio de Desvio de Mobilidade Eletroforética , Elementos Facilitadores Genéticos/genética , Éxons/genética , Feminino , Fator 10 de Crescimento de Fibroblastos/metabolismo , Fator de Transcrição GATA4/genética , Fator de Transcrição GATA4/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Genes Reporter , Membro Posterior/embriologia , Membro Posterior/metabolismo , Humanos , Íntrons/genética , Proteínas com Homeodomínio LIM/genética , Camundongos , Camundongos Transgênicos , Dados de Sequência Molecular , Miocárdio/citologia , Miocárdio/metabolismo , Gravidez , Ligação Proteica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas com Domínio T/metabolismo , Fatores de Transcrição/genéticaRESUMO
Myf5 is a key myogenic determination factor, specifically present at sites of myogenesis. Surprisingly, during mouse development, this gene is also transcribed in restricted areas of the central nervous system, although the Myf5 protein is not detectable. We have investigated the regulation of Myf5 expression in the central nervous system. Using both in ovo electroporation in the chick embryo and transgenesis in the mouse, we show that regulatory sequences that direct neuronal Myf5 transcription are present in a distal element located between -55 and -54.3 Kb from the Myf5 gene. An Oct6/Tst1 binding site is required for embryonic brain expression, and in the Oct6 mutant mouse embryo, Myf5 transcripts are no longer detectable in the brain. The Wnt-beta catenin signalling pathway is also implicated. Finally we show that post-transcriptional regulation of Myf5 gene expression involves miRNA repression acting through the Myf5-3'UTR.
Assuntos
Encéfalo , Fator Regulador Miogênico 5/genética , Transcrição Gênica , Regiões 3' não Traduzidas , Animais , Sítios de Ligação , Encéfalo/citologia , Encéfalo/crescimento & desenvolvimento , Embrião de Galinha , Eletroporação , Camundongos , Camundongos Transgênicos , MicroRNAs/fisiologia , Desenvolvimento Muscular/genética , Elementos Reguladores de Transcrição , Fatores de Transcrição , Proteínas Wnt/metabolismoRESUMO
Myf5, a member of the myogenic regulatory factor family, plays a major role in determining myogenic cell fate at the onset of skeletal muscle formation in the embryo. Spatiotemporal control of its expression during development requires multiple enhancer elements spread over >100 kb at the Myf5 locus. Transcription in embryonic limbs is regulated by a 145-bp element located at -57.5 kb from the Myf5 gene. In the present study we show that Myf5 expression is severely impaired in the limb buds of Six1(-/-) and Six1(-/-)Six4(-/+) mouse mutants despite the presence of myogenic progenitor cells. The 145-bp regulatory element contains a sequence that binds Six1 and Six4 in electromobility shift assays in vitro and in chromatin immunoprecipitation assays with embryonic extracts. We further show that Six1 is able to transactivate a reporter gene under the control of this sequence. In vivo functionality of the Six binding site is demonstrated by transgenic analysis. Mutation of this site impairs reporter gene expression in the limbs and in mature somites where the 145-bp regulatory element is also active. Six1/4 therefore regulate Myf5 transcription, together with Pax3, which was previously shown to be required for the activity of the 145-bp element. Six homeoproteins, which also directly regulate the myogenic differentiation gene Myogenin and lie genetically upstream of Pax3, thus control hypaxial myogenesis at multiple levels.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Proteínas de Homeodomínio/fisiologia , Fator Regulador Miogênico 5/biossíntese , Fator Regulador Miogênico 5/genética , Transativadores/fisiologia , Animais , Sequência de Bases , Células Cultivadas , Embrião de Galinha , Proteínas de Homeodomínio/genética , Botões de Extremidades , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Dados de Sequência Molecular , Fator Regulador Miogênico 5/metabolismo , Fator de Transcrição PAX3 , Fatores de Transcrição Box Pareados/fisiologia , Transativadores/deficiência , Transativadores/genéticaRESUMO
The transcription factors Pax3 and Pax7 are important regulators of myogenic cell fate, as demonstrated by genetic manipulations in the mouse embryo. Pax3 lies genetically upstream of MyoD and has also been shown recently to directly control Myf5 transcription in derivatives of the hypaxial somite, where it also plays an important role in ensuring cell survival. Both Pax3 and Pax7 are expressed in myogenic progenitor cells derived from the central dermomyotome that make a major contribution to skeletal muscle growth. In Pax3/Pax7 double mutants, the myogenic determination genes, Myf5 and MyoD, are not activated in these cells which become incorporated into other tissues or die. This again demonstrates the dual function of Pax factors in regulating the entry of progenitor cells into the myogenic programme and in ensuring their survival. Pax3 expression marks cells in the dermomyotome that either become myogenic or downregulate Pax3 and assume another cell fate. The latter include the smooth muscle cells of the dorsal aorta that share a common clonal origin with the skeletal muscle of the myotome, thus illustrating the initial multipotency of Pax3 expressing cells.
Assuntos
Diferenciação Celular/fisiologia , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Células-Tronco Mesenquimais/fisiologia , Músculos/embriologia , Fator de Transcrição PAX7/metabolismo , Fatores de Transcrição Box Pareados/metabolismo , Animais , Sobrevivência Celular/fisiologia , Células-Tronco Mesenquimais/metabolismo , Camundongos , Proteína MyoD/metabolismo , Fator Regulador Miogênico 5/metabolismo , Fator de Transcrição PAX3 , Fator de Transcrição PAX7/genética , Fatores de Transcrição Box Pareados/genéticaRESUMO
We address the molecular control of myogenesis in progenitor cells derived from the hypaxial somite. Null mutations in Pax3, a key regulator of skeletal muscle formation, lead to cell death in this domain. We have developed a novel allele of Pax3 encoding a Pax3-engrailed fusion protein that acts as a transcriptional repressor. Heterozygote mouse embryos have an attenuated mutant phenotype, with partial conservation of the hypaxial somite and its myogenic derivatives, including some hindlimb muscles. At these sites, expression of Myf5 is compromised, showing that Pax3 acts genetically upstream of this myogenic determination gene. We have characterized a 145-base-pair (bp) regulatory element, at -57.5 kb from Myf5, that directs transgene expression to the mature somite, notably to myogenic cells of the hypaxial domain that form ventral trunk and limb muscles. A Pax3 consensus site in this sequence binds Pax3 in vitro and in vivo. Multimers of the 145-bp sequence direct transgene expression to sites of Pax3 function, and an assay of its activity in the chick embryo shows Pax3 dependence. Mutation of the Pax3 site abolishes all expression controlled by the 145-bp sequence in transgenic mouse embryos. We conclude that Pax3 directly regulates Myf5 in the hypaxial somite and its derivatives.
Assuntos
Extremidades/embriologia , Desenvolvimento Muscular , Fator Regulador Miogênico 5/metabolismo , Fatores de Transcrição Box Pareados/fisiologia , Células-Tronco/metabolismo , Animais , Embrião de Galinha , Feminino , Triagem de Portadores Genéticos , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Desenvolvimento Muscular/genética , Fator de Transcrição PAX3 , Fatores de Transcrição Box Pareados/genética , Fenótipo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Somitos/metabolismoRESUMO
We studied the immunological basis for the very potent encephalitogenicity of myelin/oligodendrocyte glycoprotein (MOG), a minor component of myelin in the CNS that is widely used to induce experimental autoimmune encephalomyelitis (EAE). For this purpose, we generated a mutant mouse lacking a functional mog gene. This MOG-deficient mouse presents no clinical or histological abnormalities, permitting us to directly assess the role of MOG as a target autoantigen in EAE. In contrast to WT mice, which developed severe EAE following immunization with whole myelin, MOG-deficient mice had a mild phenotype, demonstrating that the anti-MOG response is a major pathogenic component of the autoimmune response directed against myelin. Moreover, while MOG transcripts are expressed in lymphoid organs in minute amounts, both MOG-deficient and WT mice show similar T and B cell responses against the extracellular domain of MOG, including the immunodominant MOG 35-55 T cell epitope. Furthermore, no differences in the fine specificity of the T cell responses to overlapping peptides covering the complete mouse MOG sequence were observed between MOG+/+ and MOG-/- mice. In addition, upon adoptive transfer, MOG-specific T cells from WT mice and those from MOG-deficient mice are equally pathogenic. This total lack of immune tolerance to MOG in WT C57BL/6 mice may be responsible for the high pathogenicity of the anti-MOG immune response as well as the high susceptibility of most animal strains to MOG-induced EAE.
Assuntos
Tolerância Imunológica , Glicoproteína Associada a Mielina/genética , Glicoproteína Associada a Mielina/fisiologia , Animais , Linfócitos B/imunologia , Northern Blotting , Western Blotting , Encéfalo/metabolismo , Divisão Celular , Encefalomielite Autoimune Experimental/genética , Encefalomielite Autoimune Experimental/imunologia , Feminino , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Microscopia Eletrônica , Modelos Genéticos , Proteínas da Mielina , Bainha de Mielina/metabolismo , Glicoproteína Mielina-Oligodendrócito , Peptídeos/química , Fenótipo , Reação em Cadeia da Polimerase , Linfócitos T/imunologia , Linfócitos T/metabolismo , Fatores de Tempo , Distribuição TecidualRESUMO
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.
Assuntos
Proteínas de Ligação a DNA , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Desenvolvimento Muscular/fisiologia , Proteínas Musculares/genética , Transativadores , Animais , Membro Posterior/metabolismo , Camundongos , Camundongos Transgênicos , Desenvolvimento Muscular/genética , Fator Regulador Miogênico 5 , Somitos/metabolismoRESUMO
During embryogenesis, skeletal muscle forms in the vertebrate limb from progenitor cells originating in the somites. These cells delaminate from the hypaxial edge of the dorsal part of the somite, the dermomyotome, and migrate into the limb bud, where they proliferate, express myogenic determination factors and subsequently differentiate into skeletal muscle. A number of regulatory factors involved in these different steps have been identified. These include Pax3 with its target c-met, Lbx1 and Mox2 as well as the myogenic determination factors Myf5 and MyoD and factors required for differentiation such as Myogenin, Mrf4 and Mef2 isoforms. Mutants for genes such as Lbx1 and Mox2, expressed uniformly in limb muscle progenitors, reveal unexpected differences between fore and hind limb muscles, also indicated by the differential expression of Tbx genes. As development proceeds, a secondary wave of myogenesis takes place, and, postnatally, satellite cells become located under the basal lamina of adult muscle fibres. Satellite cells are thought to be the progenitor cells for adult muscle regeneration, during which similar genes to those which regulate myogenesis in the embryo also play a role. In particular, Pax3 as well as its orthologue Pax7 are important. The origin of secondary/fetal myoblasts and of adult satellite cells is unclear, as is the relation of the latter to so-called SP or stem cell populations, or indeed to potential mesangioblast progenitors, present in blood vessels. The oligoclonal origin of postnatal muscles points to a small number of founder cells, whether or not these have additional origins to the progenitor cells of the somite which form the first skeletal muscles, as discussed here for the embryonic limb.
Assuntos
Indução Embrionária/fisiologia , Extremidades/embriologia , Músculo Esquelético/embriologia , Vertebrados/embriologia , Animais , Proteínas de Ligação a DNA/genética , Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Botões de Extremidades , Camundongos , Morfogênese/fisiologia , Fatores de Regulação Miogênica/genética , Regeneração/genética , Células-Tronco/fisiologiaRESUMO
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.