RESUMEN
During embryonic development, and before functional innervation, a highly stereotypic pattern of slow- and fast-contracting primary muscle fibers is established within individual muscles of the limbs, from distinct populations of myoblasts. A difference between the fiber-type pattern found within chicken and quail pectoral muscles was exploited to investigate the contributions of somite-derived myogenic precursors and lateral plate-derived mesenchymal stroma to the establishment of muscle fiber-type patterns. Chimeric chicken/quail embryos were constructed by reciprocal transplantation of somites or lateral plate mesoderm at stages prior to muscle formation. Muscle fibers derived from quail myogenic precursors that had migrated into chicken stroma showed a quail pattern of mixed fast- and slow-contracting muscle fibers. Conversely, chicken myogenic precursors that had migrated into quail stroma showed a chicken pattern of nearly exclusive fast muscle fiber formation. These results demonstrate in vivo an intrinsic commitment to fiber-type on the part of the myoblast, independent of extrinsic signals it receives from the mesenchymal stroma in which it differentiates.
Asunto(s)
Mesodermo/fisiología , Fibras Musculares de Contracción Rápida/citología , Fibras Musculares de Contracción Lenta/citología , Transducción de Señal , Células Madre/citología , Animales , Diferenciación Celular , Embrión de Pollo , Músculos/citología , Músculos/embriología , CodornizRESUMEN
The slow myosin heavy chain 3 gene (slow MyHC3) is restricted in its expression to the atrial chambers of the heart. Understanding its regulation provides a basis for determination of the mechanisms controlling chamber-specific gene expression in heart development. The observed chamber distribution results from repression of slow MyHC3 gene expression in the ventricles. A binding site, the vitamin D response element (VDRE), for a heterodimer of vitamin D receptor (VDR) and retinoic X receptor alpha (RXR alpha) within the slow MyHC3 promoter mediates chamber-specific expression of the gene. Irx4, an Iroquois family homeobox gene whose expression is restricted to the ventricular chambers at all stages of development, inhibits AMHC1, the chick homolog of quail slow MyHC3, gene expression within developing ventricles. Repression of the slow MyHC3 gene in ventricular cardiomyocytes by Irx4 requires the VDRE. Unlike VDR and RXR alpha, Irx4 does not bind directly to the VDRE. Instead two-hybrid and co-immunoprecipitation assays show that Irx4 interacts with the RXR alpha component of the VDR/RXR alpha heterodimer and that the amino terminus of the Irx4 protein is required for its inhibitory action. These observations indicate that the mechanism of atrial chamber-specific expression requires the formation of an inhibitory protein complex composed of VDR, RXR alpha, and Irx4 that binds at the VDRE inhibiting slow MyHC3 expression in the ventricles.
Asunto(s)
Miosinas Atriales , Proteínas Aviares , Regulación de la Expresión Génica/fisiología , Proteínas de Homeodominio/metabolismo , Miocardio/metabolismo , Cadenas Pesadas de Miosina/genética , Regiones Promotoras Genéticas , Receptores de Calcitriol/metabolismo , Receptores de Ácido Retinoico/metabolismo , Factores de Transcripción/metabolismo , Animales , Sitios de Unión , Células Cultivadas , Embrión de Pollo , Clonación Molecular , Proteínas de Unión al ADN/metabolismo , Eliminación de Gen , Atrios Cardíacos/embriología , Atrios Cardíacos/metabolismo , Ventrículos Cardíacos/embriología , Ventrículos Cardíacos/metabolismo , Proteínas de Homeodominio/genética , Miosinas/genética , Reacción en Cadena de la Polimerasa , Biosíntesis de Proteínas , Codorniz , Receptores de Calcitriol/genética , Receptores de Ácido Retinoico/genética , Receptores X Retinoide , Factores de Transcripción/genéticaRESUMEN
Much of our understanding of early vertebrate embryogenesis derives from experimental work done with the chick embryo. Studies of the avian somite have played a key role in elucidating the developmental history of this important structure, the source of most muscle and bone in the organism. Here we review the development of the avian somite including morphological and molecular data on the origin of paraxial mesoderm, maturation of the segmental plate, specification and formation of somite compartments, and somite cell differentiation into cartilage and skeletal muscle.
Asunto(s)
Somitos/citología , Animales , Tipificación del Cuerpo , Cartílago/embriología , Diferenciación Celular , División Celular , Embrión de Pollo , Regulación del Desarrollo de la Expresión Génica , Hibridación in Situ , Mesodermo/citología , Microscopía Electrónica de Rastreo , Biología Molecular , Músculo Esquelético/embriología , Transducción de Señal , Somitos/metabolismo , Terminología como AsuntoRESUMEN
To study the specification of inflow structures in the heart we generated transgenic animals harboring the human alkaline phosphatase (HAP) gene driven by the proximal 840 bp of a quail SMyHC3 promoter. In transgenic mice, the SMyHC3-HAP reporter was expressed in posterior heart precursors at 8.25 dpc, in sinus venosa and in the atrium at 8.5 and 9.0 dpc, and in the atria from 10.5 dpc onwards. SMyHC3-HAP transgene expression overlapped synthesis and endogenous response to retinoic acid (RA) in the heart, as determined by antibodies directed against a key RA synthetic enzyme and by staining of RAREhsplacZ transgenic animals. A single pulse of all-trans RA administered to pregnant mice at 7.5, but not after 8.5, dpc induced cardiac dismorphology, ranging from complete absence of outflow tract and ventricles to hearts with reduced ventricles expressing both SMyHC3-HAP and ventricular markers. Blockade of RA synthesis with disulfiram inhibited RA-induced transcription and produced hearts lacking the atrial chamber. This study defines a novel marker for atrial-restricted transcription in the developing mouse heart. It also suggests that atrial-specific gene expression is controlled by localized synthesis of RA, and that exclusion of RA from ventricular precursors is essential for correct specification of the ventricles.
Asunto(s)
Fosfatasa Alcalina/genética , Cardiopatías Congénitas/inducido químicamente , Corazón/embriología , Tretinoina/metabolismo , Fosfatasa Alcalina/efectos de los fármacos , Animales , Disulfiram/farmacología , Inhibidores Enzimáticos/farmacología , Femenino , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Marcadores Genéticos , Atrios Cardíacos/embriología , Atrios Cardíacos/metabolismo , Cardiopatías Congénitas/genética , Ventrículos Cardíacos/embriología , Ventrículos Cardíacos/metabolismo , Humanos , Ratones , Ratones Transgénicos , Embarazo , Regiones Promotoras Genéticas , Elementos de Respuesta , Transducción de Señal , Transcripción Genética , Transgenes , Tretinoina/farmacologíaRESUMEN
We have used the slow myosin heavy chain (MyHC) 3 gene to study the molecular mechanisms that control atrial chamber-specific gene expression. Initially, slow MyHC 3 is uniformly expressed throughout the tubular heart of the quail embryo. As cardiac development proceeds, an anterior-posterior gradient of slow MyHC 3 expression develops, culminating in atrial chamber-restricted expression of this gene following chamberization. Two cis elements within the slow MyHC 3 gene promoter, a GATA-binding motif and a vitamin D receptor (VDR)-like binding motif, control chamber-specific expression. The GATA element of the slow MyHC 3 is sufficient for expression of a heterologous reporter gene in both atrial and ventricular cardiomyocytes, and expression of GATA-4, but not Nkx2-5 or myocyte enhancer factor 2C, activates reporter gene expression in fibroblasts. Equivalent levels of GATA-binding activity were found in extracts of atrial and ventricular cardiomyocytes from embryonic chamberized hearts. These observations suggest that GATA factors positively regulate slow MyHC 3 gene expression throughout the tubular heart and subsequently in the atria. In contrast, an inhibitory activity, operating through the VDR-like element, increased in ventricular cardiomyocytes during the transition of the heart from a tubular to a chambered structure. Overexpression of the VDR, acting via the VDR-like element, duplicates the inhibitory activity in ventricular but not in atrial cardiomyocytes. These data suggest that atrial chamber-specific expression of the slow MyHC 3 gene is achieved through the VDR-like inhibitory element in ventricular cardiomyocytes at the time distinct atrial and ventricular chambers form.
Asunto(s)
Regulación del Desarrollo de la Expresión Génica , Atrios Cardíacos/metabolismo , Cadenas Pesadas de Miosina/genética , Receptores de Calcitriol/metabolismo , Animales , Secuencia de Bases , Sitios de Unión , Células Cultivadas , Proteínas de Unión al ADN/biosíntesis , Proteínas de Unión al ADN/genética , Factor de Transcripción GATA4 , Corazón/embriología , Atrios Cardíacos/citología , Ventrículos Cardíacos/metabolismo , Datos de Secuencia Molecular , Morfogénesis , Cadenas Pesadas de Miosina/metabolismo , Regiones Promotoras Genéticas , Codorniz , Receptores de Ácido Retinoico/metabolismo , Factores de Transcripción/biosíntesis , Factores de Transcripción/genéticaRESUMEN
The quail slow myosin heavy chain 3 (slow MyHC 3) gene is expressed in the developing heart and in slow muscles of the developing limb. It is first expressed in the pulsatile cardiac tube in the embryo, and as the heart chamberizes its expression becomes restricted to the atria. To identify regulatory elements responsible for atrial-specific expression, the 5' upstream region of slow MyHC 3 gene was investigated. An atrial regulatory domain (ARD1) between -840 and -680 acts as an atrial cell-specific enhancer in primary cardiocyte cultures. ARD1 also specifies atrial-specific expression in vivo when the ARD1/heterologous promoter was introduced into developing chick embryos by a replication-competent retroviral vector. ARD1 is the first atrial cell-specific enhancer to be identified. Fine deletion and mutation analysis within ARD1 defined a 40-base pair vitamin D3 receptor-like element that controls atrial cell-specific expression of the slow MyHC 3 gene by inhibiting its expression in ventricular cardiocytes.
Asunto(s)
Atrios Cardíacos/embriología , Cadenas Pesadas de Miosina/metabolismo , Animales , Secuencia de Bases , Células Cultivadas , Embrión de Pollo , Coturnix , Cartilla de ADN/química , Elementos de Facilitación Genéticos , Técnica del Anticuerpo Fluorescente Indirecta , Regulación del Desarrollo de la Expresión Génica , Datos de Secuencia Molecular , Regiones Promotoras Genéticas , ARN Mensajero/genética , Eliminación de SecuenciaRESUMEN
We have isolated and begun characterization of the quail slow myosin heavy chain (MyHC) 3 gene, the first reported avian slow MyHC gene. Expression of slow MyHC 3 in skeletal muscle is restricted to the embryonic period of development, when the fiber pattern of future fast and slow muscle is established. In embryonic hindlimb development, slow MyHC 3 gene expression coincides with slow muscle fiber formation as distinguished by slow MyHC-specific antibody staining. In addition to expression in embryonic appendicular muscle, slow MyHC 3 is expressed continuously in the atria. Transfection of slow MyHC 3 promoter-reporter constructs into embryonic myoblasts that form slow MyHC-expressing fibers identified two regions regulating expression of this gene in skeletal muscle. The proximal promoter, containing potential muscle-specific regulatory motifs, permits expression of a reporter gene in embryonic slow muscle fibers, while a distal element, located greater than 2600 base pairs upstream, further enhances expression 3-fold. The slow muscle fiber-restricted expression of slow MyHC 3 during embryonic development, and expression of slow MyHC 3 promoter-reporter constructs in embryonic muscle fibers in vitro, makes this gene a useful marker to study the mechanism establishing the slow fiber lineage in the embryo.
Asunto(s)
Embrión no Mamífero/fisiología , Regulación del Desarrollo de la Expresión Génica , Fibras Musculares de Contracción Rápida/fisiología , Fibras Musculares de Contracción Lenta/fisiología , Músculo Esquelético/embriología , Cadenas Pesadas de Miosina/biosíntesis , Cadenas Pesadas de Miosina/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Embrión de Pollo , Pollos , Clonación Molecular , Exones , Biblioteca Genómica , Corazón/embriología , Hibridación in Situ , Datos de Secuencia Molecular , Músculo Esquelético/metabolismo , Miocardio/metabolismo , Oligodesoxirribonucleótidos , Regiones Promotoras Genéticas , Codorniz , ARN Mensajero/biosíntesis , Ratas , Homología de Secuencia de Aminoácido , Transcripción GenéticaRESUMEN
Expression of the skeletal troponin I (sTnI) gene is regulated transcriptionally in a muscle-specific fashion. We show here that the region of the sTnI gene between -160 and +61 (relative to the transcription initiation site) is able to direct expression of the bacterial chloramphenicol acetyltransferase (CAT) gene is muscle cultures at a level approximately 100 times higher than in fibroblast cultures. RNA analysis demonstrated that transcription of the CAT gene was initiated at the same site as transcription of the endogenous sTnI gene and that CAT activity levels were approximately proportional to CAT mRNA levels. Deletion analysis demonstrated that the region between nucleotides -160 and -40 contained sequences essential for full promoter activity. Surprisingly, 3' deletion analysis indicated that the first exon (-6 to +61) of the sTnI gene was also required for full activity of the sTnI promoter in skeletal muscle cells. Chimeric promoter experiments, in which segments of the sTnI and the herpes simplex virus thymidine kinase promoter were interchanged, indicated that reconstitution of a muscle-specific promoter required inclusion of both the upstream and exon I regions of the sTnI gene. Exon I, and the region immediately upstream, showed DNase protection over sequence motifs related to those found in other genes, including the tar region of human immunodeficiency virus type 1. These results demonstrate that expression of the sTnI promoter in embryonic skeletal muscle cells requires complex interaction between two separate promoter regions, one of which resides within the first 61 transcribed nucleotides of the gene.
Asunto(s)
Exones , Regulación de la Expresión Génica , Genes Reguladores , Músculos/metabolismo , Regiones Promotoras Genéticas , Transcripción Genética , Troponina/genética , Animales , Secuencia de Bases , Núcleo Celular/metabolismo , Embrión de Pollo , Deleción Cromosómica , Clonación Molecular , Genes , Datos de Secuencia Molecular , Plásmidos , Mapeo Restrictivo , Troponina IRESUMEN
The gene encoding the fast skeletal isoform of the chick troponin I (sTnI) protein has been sequenced and its organization into exons and introns established. The gene is approximately 4.5 kb in length and composed of 8 exons, the first of which contains solely 5' untranslated sequence. In addition to its major mRNA product, there is evidence that the sTnI gene encodes a second mRNA, present at low abundance levels in embryonic skeletal muscle. Sl nuclease protection and primer extension experiments indicate that the low abundance mRNA is initiated approximately 47 nucleotides upstream of the major transcriptional initiation site. Both mRNAs appear to encode identical sTnI polypeptides. A comparison of nucleotide sequence in the 5' flanking region of several muscle-specific genes, including the sTnI gene, reveals a heptanucleotide consensus sequence, 5'-CATTCCT-3', which is conserved in the 5' flanking regions of many vertebrate contractile protein genes.
Asunto(s)
Clonación Molecular , Genes , Músculos/metabolismo , Troponina/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Pollos , ADN/aislamiento & purificación , Enzimas de Restricción del ADN , Plásmidos , Troponina IRESUMEN
Portions of at least nine distinct rat amylase genes or pseudogenes have been isolated. Cloned rat genomic DNA fragments containing complete or major portions of seven of these have been examined by heteroduplex analysis and fall within two separate groups based on their degree of homology. Four gene sequences comprising one of these groups are closely related to pancreatic amylase mRNA. The other group shows significant nonhomology to both pancreatic and parotid amylase cDNAs and may represent an additional gene type(s). All of the cloned amylase gene sequences are found in rat genomic DNA. Additional amylase sequences which have not yet been cloned are also detected. Comparison of DNA from individual Sprague-Dawley rats by Southern blotting techniques indicates allelic variation at multiple amylase loci.
Asunto(s)
Amilasas/genética , Genes , Animales , Secuencia de Bases , Clonación Molecular , Enzimas de Restricción del ADN/metabolismo , Desoxirribonucleasa EcoRI , Microscopía Electrónica , Ratas , Ratas EndogámicasRESUMEN
The mRNA sequences for two rat pancreatic elastolytic enzymes have been cloned by recombinant DNA technology and their nucleotide sequences determined. Rat elastase I mRNA is 1113 nucleotides in length, plus a poly(A) tail, and encodes a preproelastase of 266 amino acids. The amino acid sequence of the predicted active form of rat elastase I is 84% homologous to porcine elastase 1. Key amino acid residues involved in determining substrate specificity of porcine elastase 1 are retained in the rat enzyme. The activation peptide of the zymogen does not appear related to that of other mammalian pancreatic serine proteases. The mRNA for elastase I is localized in the rough endoplasmic reticulum of acinar cells, as expected for the site of synthesis of an exocrine secretory enzyme. Rat elastase II mRNA is 910 nucleotides in length, plus a poly(A) tail, and encodes a preproenzyme of 271 amino acids. The amino acid sequence is more closely related to porcine elastase 1 (58% sequence identity) than to the other pancreatic serine proteases (33-39% sequence identity). Predictions of substrate preference based upon key amino acid residues that define the substrate binding cleft are consistent with the broad specificity observed for mammalian pancreatic elastase 2. The activation peptide is similar to that of the chymotrypsinogens and retains an N-terminal cysteine available to form a disulfide link to an internal conserved cysteine residue.
Asunto(s)
Clonación Molecular , Precursores Enzimáticos/genética , Páncreas/enzimología , Elastasa Pancreática/genética , ARN Mensajero , Secuencia de Aminoácidos , Animales , Secuencia de Bases , RatasRESUMEN
Rat carboxypeptidase A cDNA clones have been isolated from a cDNA library prepared from pancreatic mRNA. An almost complete mRNA sequence has been deduced that predicts a polypeptide having 78% amino acid sequence homology with bovine carboxypeptidase A. The amino acid sequence of the activation and signal peptides of the carboxypeptidase A precursor were inferred from the nucleotide sequence. The cDNA was used as a probe to identify DNA fragments containing carboxypeptidase A sequences in a bacteriophage lambda library of rat genomic DNA. Heteroduplexes revealed that the DNA coding sequence occupies 5.5 kilobases and is interrupted by nine intervening sequences. The nucleotide sequence of the 5' end of the gene and the adjacent flanking region provides information on the site of initiation of transcription and the putative control regions. There is no evident relationship between the localization of intervening sequences in the gene and functional/structural domains of the protein.