Asunto(s)
Conducta Animal/fisiología , Proteínas de Caenorhabditis elegans/fisiología , Caenorhabditis elegans/fisiología , Modelos Animales , Contracción Muscular/fisiología , Tropomiosina/fisiología , Troponina/fisiología , Animales , Caenorhabditis elegans/química , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/química , Contracción Muscular/genética , Isoformas de Proteínas/genética , Isoformas de Proteínas/fisiología , Tropomiosina/genéticaRESUMEN
Nuclear receptor subfamily 5, group A, member 1 (NR5A1 previously known as SF-1/AD4BP) is a transcription factor involved in the development of adrenal/gonadal tissues and steroidogenic lineage cell differentiation in adult somatic stem cells. To understand the cellular signaling network that regulates NR5A1 gene expression, loss of function screening with an siRNA kinome library, and gain of function screening with an addressable full-length cDNA library representing one quarter of the human genome was carried out. The NR5A1 gene expression was activated in mesenchymal stem cells by siRNA directed against protein kinase C (PKC)-delta, erb-B3, RhoGAP (ARHGAP26), and hexokinase 2, none of which were previously known to be involved in the NR5A1 gene expression. Among these, we identified crosstalk between erb-B3 and PKC-delta signaling cascades. In addition, the gain of function studies indicated that sex-determining region Y (SRY)-box 15 (SOX15), TEA domain family member 4, KIAA1257 (a gene of unknown function), ADAM metallopeptidase with thrombospondin type 1 motif 6, Josephin domain containing 1, centromere protein, TATA box-binding protein-associated factor 5-like RNA polymerase, and inducible T-cell co-stimulator activate NR5A1 gene expression. These results provide new insights into the molecular mechanisms of NR5A1 gene expression.
Asunto(s)
Factor Esteroidogénico 1/genética , Factor Esteroidogénico 1/metabolismo , Proteínas ADAM/genética , Proteínas ADAM/metabolismo , Adenoviridae/genética , Animales , Bovinos , Línea Celular , Células Cultivadas , Proteínas Cromosómicas no Histona/genética , Proteínas Cromosómicas no Histona/metabolismo , AMP Cíclico/farmacología , ADN Complementario/genética , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Proteínas Activadoras de GTPasa/genética , Proteínas Activadoras de GTPasa/metabolismo , Biblioteca de Genes , Vectores Genéticos/genética , Hexoquinasa/genética , Hexoquinasa/metabolismo , Humanos , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/efectos de los fármacos , Células Madre Mesenquimatosas/metabolismo , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Regiones Promotoras Genéticas/genética , Proteína Quinasa C-delta/genética , Proteína Quinasa C-delta/metabolismo , ARN Interferente Pequeño/genética , Receptor ErbB-3/genética , Receptor ErbB-3/metabolismo , Factores de Transcripción SOX/genética , Factores de Transcripción SOX/metabolismo , Factores de Transcripción de Dominio TEA , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Cardiogenesis can be induced in vitro in ES cells, though it is difficult to distinguish cardiac-specific genes, since embryoid bodies simultaneously differentiate into multiple lineages. In the present study, transient serum removal during culture greatly enhanced cardiogenesis, and reduced generation of endothelial and hematopoietic cells. Using DNA microarray analysis of 24 differentiated sample cultures including cardiogenesis-enhanced cells, we successfully selected genes up-regulated in embryoid bodies that had undergone cardiogenic differentiation. Besides contractile protein genes, cardiac transcriptional regulatory genes, such as Nkx2-5, Gata4/5, Mef2c, and Myocd, were primary constituents of the first 100 genes chosen as cardiogenesis-associated genes. Further, whole mount in situ hybridization analysis of 13 genes containing non-characterized ones confirmed that most of them were specifically expressed in the heart region of mouse embryos from E9.5-10.5. Based on our results, we consider that the present profiling method may be useful to identify novel genes important for cardiac development.
Asunto(s)
Células Madre Embrionarias/fisiología , Corazón/embriología , Análisis de Secuencia por Matrices de Oligonucleótidos , Animales , Apoptosis , Diferenciación Celular , Línea Celular , Medio de Cultivo Libre de Suero , Células Madre Embrionarias/citología , Regulación de la Expresión Génica , Cardiopatías Congénitas/genética , Humanos , Mutación , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Transcripción/metabolismo , Transcripción Genética , TransfecciónRESUMEN
Endocardial cells and cardiomyocytes differentiate from the cardiogenic mesoderm at about the same time during development. Although in vitro embryonic stem (ES) cell systems have been used to study the differentiation of various types of cell lineages, including cardiomyocytes, smooth muscle cells, and vascular endothelial cells, differentiation of endocardial cells, or endocardiogenesis, has not been well reported, because of a lack of specific molecular markers. In our search for cardiogenesis-associated genes expressed in embryoid bodies, we found several genes expressed in the heart region of mouse embryos, but not in cardiomyocytes. To identify the cell types expressing these genes, CD31(+) cells were taken from mouse embryos on embryonic day (E)8.5 and E9.5 and sorted, then their transcripts were analyzed using quantitative RT-PCR analyses. In those embryos, Gata4 and Nfatc1, as well as newly identified Cgnl1 and Dok4 were found to be preferentially expressed in endocardial cells, but not in yolk sac endothelial cells, while Cdh5 and Kdr were expressed in both cardiac and yolk sac endothelial cells. Immunohistochemical analyses of embryoid bodies revealed that some CD31(+) cells co-expressing Gata4 and Nfatc1 were located in close proximity to cardiomyocytes. These results suggest that embryoid bodies express endocardial specific genes and likely generate endocardial cells along with cardiomyocytes. Further, they indicate that these new marker genes are useful to study the origin and induction of endocardial cells, and identify other endocardial markers.
Asunto(s)
Células Madre Embrionarias/citología , Células Madre Embrionarias/fisiología , Endocardio/crecimiento & desarrollo , Endocardio/metabolismo , Factor de Transcripción GATA4/metabolismo , Perfilación de la Expresión Génica/métodos , Factores de Transcripción NFATC/metabolismo , Animales , Biomarcadores , Diferenciación Celular , Células Cultivadas , Endocardio/citología , Regulación del Desarrollo de la Expresión Génica/fisiología , RatonesRESUMEN
Gene duplication is a major genetic event that can produce multiple protein isoforms. Comparative sequence and functional analysis of related gene products can provide insights into protein family evolution. To characterize the Caenorhabditis elegans troponin I family, we analyzed gene structures, tissue expression patterns and RNAi phenotypes of four troponin I isoforms. Tissue expression patterns were determined using lacZ/gfp/rfp reporter gene assays. The tni-1, tni-2/unc-27 and tni-3 genes, each encoding a troponin I isoform, are uniquely expressed in body wall, vulval and anal muscles but at different levels; tni-4 was expressed solely in the pharynx. Expressing tni-1 and -2 gene RNAi caused motility defects similar to unc-27 (e155) mutant, a tni-2 null allele. The tni-3 RNAi expression produced egg laying defects while the tni-4 RNAi caused arrest at gastrulation. Overlay analyses were used to assay interactions between the troponin I and two troponin C isoforms. The three body wall troponin I isoforms interacted with body wall and pharyngeal troponin C isoforms; TNI-4 interacted only with pharyngeal troponin C. Our results suggest the body wall genes have evolved following duplication of the pharynx gene and provide important data about gene duplication and functional differentiation of nematode troponin I isoforms.
Asunto(s)
Caenorhabditis elegans/genética , Troponina C/metabolismo , Troponina I/genética , Secuencia de Aminoácidos , Animales , Caenorhabditis elegans/metabolismo , Datos de Secuencia Molecular , Isoformas de Proteínas , Interferencia de ARN , Homología de Secuencia , Troponina C/genética , Troponina I/metabolismoRESUMEN
Wnt signaling plays a crucial role in the control of morphogenesis in several tissues. Herein, we describe the role of Wnt11 during cardiac differentiation of embryonic stem cells. First, we examined the expression profile of Wnt11 during the course of differentiation in embryoid bodies, and then compared its expression in retinoic acid-treated embryoid bodies with that in untreated. In differentiating embryoid bodies, Wnt11 expression rose along with that of Nkx2.5 expression and continued to increase. When the embryoid bodies were treated with retinoic acid, Wnt11 expression decreased in parallel with the decreased expression of cardiac genes. Further, treatment of embryoid bodies with medium containing Wnt11 increased the expression of cardiac marker genes. Based on these results, we propose that Wnt11 plays an important role for cardiac development by embryoid bodies, and may be a key regulator of cardiac muscle cell proliferation and differentiation during heart development.