RESUMEN
Reported divergent responses of coral growth and skeletal microstructure to the nutrient environment complicate knowledge-based management of water quality in coral reefs. By re-evaluating published results considering the taxonomy of the studied corals and the N:P stoichiometry of their nutrient environment, we could resolve some of the major apparent contradictions. Our analysis suggests that Acroporids behave differently to several other common genera and show distinct responses to specific nutrient treatments. We hypothesised that both the concentrations of dissolved inorganic N and P in the water and their stoichiometry shape skeletal growth and microstructure. We tested this hypothesis by exposing Acropora polystoma fragments to four nutrient treatments for > 10 weeks: high nitrate/high phosphate (HNHP), high nitrate/low phosphate (HNLP), low nitrate/high phosphate (LNHP) and low nitrate/low phosphate (LNLP). HNHP corals retained high zooxanthellae densities and their linear extension and calcification rates were up to ten times higher than in the other treatments. HNLP and LNLP corals bleached through loss of symbionts. The photochemical efficiency (Fv/Fm) of residual symbionts in HNLP corals was significantly reduced, indicating P-starvation. Micro-computed tomography (µCT) of the skeletal microstructure revealed that reduced linear extension in nutrient limited or nutrient starved conditions (HNLP, LNHP, LNLP) was associated with significant thickening of skeletal elements and reduced porosity. These changes can be explained by the strongly reduced linear extension rate in combination with a smaller reduction in the calcification rate. Studies using increased skeletal density as a proxy for past thermal bleaching events should consider that such an increase in density may also be associated with temperature-independent response to the nutrient environment. Furthermore, the taxonomy of corals and seawater N:P stoichiometry should be considered when analysing and managing the impacts of nutrient pollution. Supplementary Information: The online version contains supplementary material available at 10.1007/s00338-022-02223-0.
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No disponible
Asunto(s)
Animales , Ratones , Células Madre/inmunología , Esclerosis Amiotrófica Lateral/fisiopatología , Células Satélite del Músculo Esquelético/inmunología , Modelos Animales de EnfermedadRESUMEN
Pax genes have important roles in the regulation of stem cell behavior, leading to tissue differentiation. In the case of skeletal muscle, Pax3 and Pax7 perform this function both during development and on regeneration in the adult. The myogenic determination gene Myf5 is directly activated by Pax3, leading to the formation of skeletal muscle. Fgfr4 is also a direct Pax3 target and Sprouty1, which encodes an intracellular inhibitor of fibroblast growth factor (FGF) signaling, is under Pax3 control. Orchestration of FGF signaling, through Fgfr4/Sprouty1, modulates the entry of cells into the myogenic program, thus controling the balance between stem cell self-renewal and tissue differentiation. This and other aspects of Pax3/7 function in regulating the behavior of skeletal muscle stem cells are discussed.
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Mioblastos Esqueléticos/citología , Mioblastos Esqueléticos/metabolismo , Factor de Transcripción PAX7/metabolismo , Factores de Transcripción Paired Box/metabolismo , Células Madre/citología , Células Madre/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Células Madre Adultas/citología , Células Madre Adultas/metabolismo , Animales , Diferenciación Celular , Movimiento Celular , Proliferación Celular , Supervivencia Celular , Desarrollo Embrionario , Células Madre Embrionarias/citología , Células Madre Embrionarias/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Modelos Biológicos , Células Madre Multipotentes/citología , Células Madre Multipotentes/metabolismo , Desarrollo de Músculos , Factor 5 Regulador Miogénico/genética , Factor 5 Regulador Miogénico/metabolismo , Factor de Transcripción PAX3 , Factor de Transcripción PAX7/genética , Factores de Transcripción Paired Box/genética , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Receptor Tipo 4 de Factor de Crecimiento de Fibroblastos/genética , Receptor Tipo 4 de Factor de Crecimiento de Fibroblastos/metabolismo , Transducción de SeñalRESUMEN
The class VI POU domain family member known as Emb in the mouse (rat Brn5 or human mPOU/TCFbeta1) is present in vivo as a protein migrating at about 80 kDa on western blots, considerably larger than that predicted (about 42 kDa) from previously cloned coding sequences. By RT-PCR and 5' RACE strategies a full-length Emb sequence, Emb FL, is now identified. Shorter sequences encoding the -COOH terminal, and an -NH(2) terminal isoform, EmbN, were also isolated. Comparisons of Emb coding sequences between species, including the full-length zebra fish, POU(c), are presented, together with a compilation of the multiple transcripts produced by alternative splicing and the presence of different transcriptional start and stop sites, from the Emb gene.
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Proteínas de Unión al ADN/genética , Factores de Transcripción/genética , Empalme Alternativo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Western Blotting , Línea Celular , Línea Celular Tumoral , Clonación Molecular , ADN Complementario/química , ADN Complementario/genética , Proteínas de Unión al ADN/metabolismo , Genes/genética , Humanos , Datos de Secuencia Molecular , Factores del Dominio POU , Isoformas de Proteínas/genética , Ratas , Alineación de Secuencia , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Factores de Transcripción/metabolismo , Transcripción Genética/genéticaRESUMEN
Expression of the mouse cardiac actin gene depends on a distal enhancer (-7 kbp) which has been shown, in transgenic mice, to direct expression to embryonic skeletal muscle. The presence of this distal sequence is also associated with reproducible expression of cardiac actin transgenes. In differentiated skeletal muscle cells, activity of the enhancer is driven by an E box, binding MyoD family members, and by a 3' AT-rich sequence which is in the location of a DNase I-hypersensitive site. This sequence does not bind MEF2 proteins, or other known muscle transcription factors, directly. Oct1 and Emb, a class VI POU domain protein, bind to consensus sites on the DNA, and it is the binding of Emb which is important for activity. Emb binds as a major complex with MEF2D and the histone transacetylase p300. The form of Emb present in this complex and as a major form in muscle cell extracts is longer (80 kDa) than that previously described. These results demonstrate the importance of this novel complex in the transcriptional regulation of the cardiac actin gene and suggest a potential role in chromatin remodeling associated with muscle gene activation.
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Acetiltransferasas/metabolismo , Actinas/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas de Unión al ADN/metabolismo , Elementos de Facilitación Genéticos , Regulación de la Expresión Génica , Histonas/metabolismo , Miocardio/metabolismo , Factores de Transcripción/metabolismo , Actinas/metabolismo , Animales , Secuencia de Bases , Línea Celular , Huella de ADN , Histona Acetiltransferasas , Factores de Transcripción MEF2 , Sustancias Macromoleculares , Ratones , Ratones Endogámicos C3H , Datos de Secuencia Molecular , Factores Reguladores Miogénicos , Alineación de Secuencia , Activación Transcripcional , Factores de Transcripción p300-CBPRESUMEN
Development of the arterial pole of the heart is a critical step in cardiogenesis, yet its embryological origin remains obscure. We have analyzed a transgenic mouse line in which beta-galactosidase activity is observed in the embryonic right ventricle and outflow tract of the heart and in contiguous splanchnic and pharyngeal mesoderm. The nlacZ transgene has integrated upstream of the fibroblast growth factor 10 (Fgf10) gene and comparison with the expression pattern of Fgf10 in pharyngeal mesoderm indicates transgene control by Fgf10 regulatory sequences. Dil labeling shows a progressive movement of cells from the pharyngeal arch region into the growing heart tube between embryonic days 8.25 and 10.5. These data suggest that arterial pole myocardium originates outside the classical heart field.
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Factores de Crecimiento de Fibroblastos/genética , Corazón/embriología , Mesodermo/metabolismo , Faringe/embriología , Animales , Carbocianinas/metabolismo , Factor 10 de Crecimiento de Fibroblastos , Colorantes Fluorescentes/metabolismo , Corazón/fisiología , Hibridación in Situ , Mesodermo/citología , Ratones , Ratones Transgénicos , Miocardio/citología , Miocardio/metabolismo , Faringe/metabolismo , Transgenes , beta-Galactosidasa/genética , beta-Galactosidasa/metabolismoRESUMEN
Research in the past year has added to our understanding of the signalling systems that specify myogenic identity in the embryo and of the regulation and roles of MyoD family members. New insights into the movement of muscle precursor cells include the demonstration that Lbx1 is essential for their migration from the somite to some but not all sites of muscle formation elsewhere. Later in development, ras as well as calcineurin signalling is now implicated in the definition of slow versus fast fibre types. The myogenic identity of precursor cells in the adult depends on Pax7, the orthologue of Pax3 which is required for early myogenesis; this finding is of major importance for muscle regeneration and the active field of stem cell research.
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Proteínas de Unión al ADN , Músculo Esquelético/embriología , Transducción de Señal/fisiología , Transactivadores , Animales , Movimiento Celular/fisiología , Regulación de la Expresión Génica , Proteínas Musculares/genética , Músculo Esquelético/citología , Músculo Esquelético/metabolismo , Factor 5 Regulador Miogénico , VertebradosRESUMEN
Myoblast transplantation is a potential therapeutic approach for the genetic modification of host skeletal muscle tissue. To be considered an effective, long-lived method of delivery, however, it is essential that at least a proportion of the transplanted cells also retain their proliferative potential. We sought to investigate whether transplanted neonatal myoblasts can contribute to the satellite cell compartment of adult skeletal muscle by using the Myf5nlacZ/+ mouse. The Myf5nlacZ/+ mouse has nlacZ targeted to the Myf5 locus resulting in beta-galactosidase activity in quiescent satellite cells. Following transplantation, beta-galactosidase-labelled nuclei were detected in host muscles, showing that donor cells had been incorporated. Significantly, beta-galactosidase-positive, and therefore donor-derived, satellite cells were detected. When placed in culture, beta-galactosidase marked myogenic cells emanated from the parent fibre. These observations demonstrate that cell transplantation not only results in the incorporation of donor nuclei into the host muscle syncytia, but also that the donor cells can become functional satellite cells. The Myf5nlacZ/+ mouse therefore provides a novel and specific marker for determining the contribution of transplanted cells to the satellite cell pool.
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Trasplante de Células/métodos , Terapia Genética/métodos , Músculo Esquelético/embriología , Músculo Esquelético/trasplante , Distrofia Muscular de Duchenne/terapia , Animales , Diferenciación Celular , Núcleo Celular/enzimología , Ratones , Ratones Endogámicos mdx , Microscopía Fluorescente , Modelos Animales , Músculo Esquelético/citología , Distrofia Muscular de Duchenne/patología , beta-Galactosidasa/genéticaRESUMEN
Abstract Transcriptional differences between left and right cardiac chambers are revealed by an nlacZ reporter transgene controlled by regulatory sequences of the MLC3F gene, which is expressed in the left ventricle (LV), atrioventricular canal (AVC), and right atrium (RA). To examine the role of left-right signalling in the acquisition of left and right chamber identity, we have investigated MLC3F transgene expression in iv mutant mice. iv/iv mice exhibit randomised direction of heart looping and an elevated frequency of associated laterality defects, including atrial isomerism. At fetal stages, 3F-nlacZ-2E transgene expression remains confined to the morphological LV, AVC, and RA in L-loop hearts, although these appear on the opposite side of the body. In cases of morphologically distinguishable right atrial appendage isomerism, both atrial appendages show strong transgene expression. Conversely, specimens with morphological left atrial appendage isomerism show only weak expression in both atrial appendages. The earliest left-right atrial differences in the expression of the 3F-nlacZ-2E transgene are observed at E8.5. DiI labelling experiments confirmed that transcriptional regionalisation of the 3F-nlacZ-2E transgene at this stage reflects future atrial chamber identity. In some iv/iv embryos at E8.5, the asymmetry of 3F-nlacZ-2E expression was lost, suggesting atrial isomerism at the transcriptional level prior to chamber formation. These data suggest that molecular specification of left and right atrial but not ventricular chambers is dependent on left-right axial cues.
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Cadenas Ligeras de Miosina/biosíntesis , Cadenas Ligeras de Miosina/genética , Animales , Galactósidos/metabolismo , Genes Reporteros , Atrios Cardíacos/metabolismo , Inmunohistoquímica , Hibridación in Situ , Indoles/metabolismo , Isomerismo , Ratones , Ratones Mutantes , Ratones Transgénicos , Miocardio/metabolismo , Transducción de Señal , Factores de Tiempo , Transcripción Genética , Transgenes , Función Ventricular , beta-Galactosidasa/metabolismoAsunto(s)
Células Madre , Adulto , Animales , Diferenciación Celular , Embrión de Mamíferos/citología , Humanos , InvestigaciónRESUMEN
Skeletal muscle is one of a several adult post-mitotic tissues that retain the capacity to regenerate. This relies on a population of quiescent precursors, termed satellite cells. Here we describe two novel markers of quiescent satellite cells: CD34, an established marker of hematopoietic stem cells, and Myf5, the earliest marker of myogenic commitment. CD34(+ve) myoblasts can be detected in proliferating C2C12 cultures. In differentiating cultures, CD34(+ve) cells do not fuse into myotubes, nor express MyoD. Using isolated myofibers as a model of synchronous precursor cell activation, we show that quiescent satellite cells express CD34. An early feature of their activation is alternate splicing followed by complete transcriptional shutdown of CD34. This data implicates CD34 in the maintenance of satellite cell quiescence. In heterozygous Myf5(nlacZ/+) mice, all CD34(+ve) satellite cells also express beta-galactosidase, a marker of activation of Myf5, showing that quiescent satellite cells are committed to myogenesis. All such cells are positive for the accepted satellite cell marker, M-cadherin. We also show that satellite cells can be identified on isolated myofibers of the myosin light chain 3F-nlacZ-2E mouse as those that do not express the transgene. The numbers of satellite cells detected in this way are significantly greater than those identified by the other three markers. We conclude that the expression of CD34, Myf5, and M-cadherin defines quiescent, committed precursors and speculate that the CD34(-ve), Myf5(-ve) minority may be involved in maintaining the lineage-committed majority.
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Antígenos CD34/aislamiento & purificación , Proteínas de Unión al ADN , Proteínas Musculares/aislamiento & purificación , Músculo Esquelético/citología , Células Madre/citología , Transactivadores , Animales , Diferenciación Celular , Linaje de la Célula , Ratones , Ratones Transgénicos , Fibras Musculares Esqueléticas/citología , Músculo Esquelético/embriología , Factor 5 Regulador Miogénico , Fragmentos de Péptidos/aislamiento & purificación , ARN Mensajero/aislamiento & purificación , RegeneraciónRESUMEN
During heart development, 2 fast-conducting regions of working myocardium balloon out from the slow-conducting primary myocardium of the tubular heart. Three regions of primary myocardium persist: the outflow tract, atrioventricular canal, and inflow tract, which are contiguous throughout the inner curvature of the heart. The contribution of the inflow tract to the definitive atrial chambers has remained enigmatic largely because of the lack of molecular markers that permit unambiguous identification of this myocardial domain. We now report that the genes encoding atrial natriuretic factor, myosin light chain (MLC) 3F, MLC2V, and Pitx-2, and transgenic mouse lines expressing nlacZ under the control of regulatory sequences of the mouse MLC1F/3F gene, display regionalized patterns of expression in the atrial component of the developing mouse heart. These data distinguish 4 broad transcriptional domains in the atrial myocardium: (1) the atrioventricular canal that will form the smooth-walled lower atrial rim proximal to the ventricles; (2) the atrial appendages; (3) the caval vein myocardium (systemic inlet); and (4) the mediastinal myocardium (pulmonary inlet), including the atrial septa. The pattern of expression of Pitx-2 reveals that each of these transcriptional domains has a distinct left and right component. This study reveals for the first time differential gene expression in the systemic and pulmonary inlets, which is not shared by the contiguous atrial appendages and provides evidence for multiple molecular compartments within the atrial chambers. Furthermore, this work will allow the contribution of each of these myocardial components to be studied in congenitally malformed hearts, such as those with abnormal venous return.
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Regulación del Desarrollo de la Expresión Génica , Atrios Cardíacos/embriología , Miocardio/metabolismo , Proteínas Nucleares , Transcripción Genética , Envejecimiento/metabolismo , Animales , Antígenos de Diferenciación/biosíntesis , Factor Natriurético Atrial/genética , Factor Natriurético Atrial/metabolismo , Genes Reporteros/genética , Atrios Cardíacos/citología , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Operón Lac , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Miocardio/citología , Cadenas Ligeras de Miosina/genética , Cadenas Ligeras de Miosina/metabolismo , Especificidad de Órganos/genética , Factores de Transcripción Paired Box , Ratas , Ratas Wistar , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Transgenes , Proteína del Homeodomínio PITX2RESUMEN
The myogenic factor Myf5 plays a key role in muscle cell determination, in response to signalling cascades that lead to the specification of muscle progenitor cells. We have adopted a YAC transgenic approach to identify regulatory sequences that direct the complex spatiotemporal expression of this gene during myogenesis in the mouse embryo. Important regulatory regions with distinct properties are distributed over 96 kb upstream of the Myf5 gene. The proximal 23 kb region directs early expression in the branchial arches, epaxial dermomyotome and in a central part of the myotome, the epaxial intercalated domain. Robust expression at most sites in the embryo where skeletal muscle forms depends on an enhancer-like sequence located between -58 and -48 kb from the Myf5 gene. This element is active in the epaxial and hypaxial myotome, in limb muscles, in the hypoglossal chord and also at the sites of Myf5 transcription in prosomeres p1 and p4 of the brain. However later expression of Myf5 depends on a more distal region between -96 and -63 kb, which does not behave as an enhancer. This element is necessary for expression in head muscles but strikingly only plays a role in a subset of trunk muscles, notably the hypaxially derived ventral body muscles and also those of the diaphragm and tongue. Transgene expression in limb muscle masses is not affected by removal of the -96/-63 region. Epaxially derived muscles and some hypaxial muscles, such as the intercostals and those of the limb girdles, are also unaffected. This region therefore reveals unexpected heterogeneity between muscle masses, which may be related to different facets of myogenesis at these sites. Such regulatory heterogeneity may underlie the observed restriction of myopathies to particular muscle subgroups.
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Proteínas de Unión al ADN , Proteínas Musculares/genética , Músculo Esquelético/embriología , Factores Reguladores Miogénicos/genética , Secuencias Reguladoras de Ácidos Nucleicos , Transactivadores , Animales , Tipificación del Cuerpo , Cromosomas Artificiales de Levadura , Regulación del Desarrollo de la Expresión Génica , Biblioteca Genómica , Cabeza/embriología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Factor 5 Regulador Miogénico , SomitosRESUMEN
Isoform diversity in striated muscle is largely controlled at the level of transcription. In this review we will concentrate on studies concerning transcriptional regulation of the alkali myosin light chain 1F/3F gene. Uncoupled activity of the MLC1F and 3F promoters, together with complex patterns of transcription in developing skeletal and cardiac muscle, combine to make analysis of this gene particularly intriguing. In vitro and transgenic studies of MLC1F/3F regulatory elements have revealed an array of cis-acting modules that each drive a subset of the expression pattern of the two promoters. These cis-acting regulatory modules, including the MLC1F and 3F promoter regions and two skeletal muscle enhancers, control tissue-specificity, cell or fibre-type specificity, and the spatiotemporal regulation of gene expression, including positional information. How each of these regulatory modules acts and how their individual activites are integrated to coordinate transcription at this locus are discussed.
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Regulación de la Expresión Génica , Variación Genética , Músculo Esquelético/fisiología , Cadenas Ligeras de Miosina/genética , Animales , Secuencia de Bases , Humanos , Datos de Secuencia Molecular , Fibras Musculares Esqueléticas/fisiología , Músculo Esquelético/embriología , Transcripción Genética , TransgenesRESUMEN
Many cardiac genes are broadly expressed in the early heart and become restricted to the atria or ventricles as development proceeds. Additional transcriptional differences between left and right compartments of the embryonic heart have been described recently, in particular for a number of transgenes containing cardiac regulatory elements. We now demonstrate that three myosin genes which become transcriptionally restricted to the atria between embryonic day (E) 12.5 and birth, alpha-myosin heavy chain (MHC), myosin light chain (MLC) 1A and MLC2A, are coordinately downregulated in the compact myocardium of the left ventricle before that of the right ventricle. alpha-MHC protein also accumulates in the right, but not left, compact ventricular myocardium during this period, suggesting that this transient regionalization contributes to fktal heart function. dHAND and eHAND, basic helix-loop-helix transcription factors known to be expressed in the right and left ventricles respectively at E10. 5, remain regionalized between E12.5 and E14.5. Downregulation of alpha-MHC, MLC1A, and MLC2A in iv/iv embryos, which have defective left/right patterning, initiates in the morphological left (systemic) ventricle regardless of its anatomical position on the right or left hand side of the heart. This points to the importance of left/right ventricular differences in sarcomeric gene expression patterns during fktal cardiogenesis and indicates that these differences originate in the embryo in response to anterior-posterior patterning of the heart tube rather than as a result of cardiac looping. Dev Dyn 2000;217:75-85.
Asunto(s)
Función Atrial , Regulación del Desarrollo de la Expresión Génica , Miosinas/biosíntesis , Miosinas/genética , Función Ventricular , Animales , Regulación hacia Abajo , Atrios Cardíacos/embriología , Ventrículos Cardíacos/embriología , Secuencias Hélice-Asa-Hélice , Ratones , Isoformas de Proteínas/biosíntesis , Isoformas de Proteínas/genética , Factores de Transcripción/fisiologíaRESUMEN
Myf5 is a key basic Helix-Loop-Helix transcription factor capable of converting many non-muscle cells into muscle. Together with MyoD it is essential for initiating the skeletal muscle programme in the embryo. We previously identified unexpected restricted domains of Myf5 transcription in the embryonic mouse brain, first revealed by Myf5-nlacZ(+/)(-) embryos (Tajbakhsh, S. and Buckingham, M. (1995) Development 121, 4077-4083). We have now further characterized these Myf5 expressing neurons. Retrograde labeling with diI, and the use of a transgenic mouse line expressing lacZ under the control of Myf5 regulatory sequences, show that Myf5 transcription provides a novel axonal marker of the medial longitudinal fasciculus (mlf) and the mammillotegmental tract (mtt), the earliest longitudinal tracts to be established in the embryonic mouse brain. Tracts projecting caudally from the developing olfactory system are also labelled. nlacZ and lacZ expression persist in the adult brain, in a few ventral domains such as the mammillary bodies of the hypothalamus and the interpeduncular nucleus, potentially derived from the embryonic structures where the Myf5 gene is transcribed. To investigate the role of Myf5 in the brain, we monitored Myf5 protein accumulation by immunofluorescence and immunoblotting in neurons transcribing the gene. Although Myf5 was detected in muscle myotomal cells, it was absent in neurons. This would account for the lack of myogenic conversion in brain structures and the absence of a neural phenotype in homozygous null mutants. RT-PCR experiments show that the splicing of Myf5 primary transcripts occurs correctly in neurons, suggesting that the lack of Myf5 protein accumulation is due to regulation at the level of mRNA translation or protein stability. In the embryonic neuroepithelium, Myf5 is transcribed in differentiated neurons after the expression of neural basic Helix-Loop-Helix transcription factors. The signalling molecules Wnt1 and Sonic hedgehog, implicated in the activation of Myf5 in myogenic progenitor cells in the somite, are also produced in the viscinity of the Myf5 expression domain in the mesencephalon. We show that cells expressing Wnt1 can activate neuronal Myf5-nlacZ gene expression in dissected head explants isolated from E9.5 embryos. Furthermore, the gene encoding the basic Helix-Loop-Helix transcription factor mSim1 is expressed in adjacent cells in both the somite and the brain, suggesting that signalling molecules necessary for the activation of mSim1 as well as Myf5 are present at these different sites in the embryo. This phenomenon may be widespread and it remains to be seen how many other potentially potent regulatory genes, in addition to Myf5, when activated do not accumulate protein at inappropriate sites in the embryo.
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Encéfalo/embriología , Proteínas de Unión al ADN , Regulación del Desarrollo de la Expresión Génica/genética , Proteínas Musculares/genética , Transactivadores , Proteínas de Pez Cebra , Animales , Axones/metabolismo , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico , Encéfalo/metabolismo , Carbocianinas , Línea Celular , Técnica del Anticuerpo Fluorescente , Marcadores Genéticos , Proteínas Hedgehog , Secuencias Hélice-Asa-Hélice/genética , Humanos , Hibridación in Situ , Operón Lac , Ratones , Ratones Transgénicos , Proteínas Musculares/metabolismo , Factor 5 Regulador Miogénico , Proteínas/genética , Proteínas Proto-Oncogénicas/genética , Proteínas Represoras/genética , Factores de Transcripción/genética , Proteínas Wnt , Proteína Wnt1RESUMEN
Axial structures (neural tube/notochord) and surface ectoderm activate myogenesis in the mouse embryo; their action can be reproduced, at least in part, by several molecules such as Sonic hedgehog and Wnts. Recently, soluble Wnt antagonists have been identified. Among those examined only Frzb1 was found to be expressed in the presomitic mesoderm and newly formed somites and thus its possible role in regulating myogenesis was investigated in detail. When presomitic mesoderm or newly formed somites were cultured with axial structures and surface ectoderm on a feeder layer of C3H10T1/2 cells expressing Frzb1, myogenesis was abolished or severely reduced in presomitic mesoderm and the three most recently formed somites. In contrast, no effect was observed on more mature somites. Inhibition of myogenesis did not appear to be associated with increased cell death since the final number of cells in the explants grown in the presence of Frzb1 was only slightly reduced in comparison with controls. In order to examine the possible function of Frzb1 in vivo, we developed a method based on the overexpression of the soluble antagonist by transient transfection of WOP cells with a Frzb1 expression vector and injection of transfected cells into the placenta of pregnant females before the onset of maternofoetal circulation. Frzb1, secreted by WOP cells, accumulated in the embryo and caused a marked reduction in size of caudal structures. Myogenesis was strongly reduced and, in the most severe cases, abolished. This was not due to a generalized toxic effect since only several genes downstream of the Wnt signaling pathway such as En1, Noggin and Myf5 were downregulated; in contrast, Pax3 and Mox1 expression levels were not affected even in embryos exhibiting the most severe phenotypes. Taken together, these results suggest that Wnt signals may act by regulating both myogenic commitment and expansion of committed cells in the mouse mesoderm.