RESUMEN
Congenital heart defects can be caused by mutations in genes that guide cardiac lineage formation. Here, we show deletion of NKX2-5, a critical component of the cardiac gene regulatory network, in human embryonic stem cells (hESCs), results in impaired cardiomyogenesis, failure to activate VCAM1 and to downregulate the progenitor marker PDGFRα. Furthermore, NKX2-5 null cardiomyocytes have abnormal physiology, with asynchronous contractions and altered action potentials. Molecular profiling and genetic rescue experiments demonstrate that the bHLH protein HEY2 is a key mediator of NKX2-5 function during human cardiomyogenesis. These findings identify HEY2 as a novel component of the NKX2-5 cardiac transcriptional network, providing tangible evidence that hESC models can decipher the complex pathways that regulate early stage human heart development. These data provide a human context for the evaluation of pathogenic mutations in congenital heart disease.
Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Redes Reguladoras de Genes , Proteína Homeótica Nkx-2.5/genética , Células Madre Embrionarias Humanas/metabolismo , Miocitos Cardíacos/metabolismo , Organogénesis/genética , Proteínas Represoras/genética , Potenciales de Acción/fisiología , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Diferenciación Celular , Línea Celular , Eliminación de Gen , Regulación del Desarrollo de la Expresión Génica , Proteína Homeótica Nkx-2.5/deficiencia , Células Madre Embrionarias Humanas/citología , Humanos , Miocardio/citología , Miocardio/metabolismo , Miocitos Cardíacos/citología , Técnicas de Placa-Clamp , Receptor alfa de Factor de Crecimiento Derivado de Plaquetas/genética , Receptor alfa de Factor de Crecimiento Derivado de Plaquetas/metabolismo , Proteínas Represoras/metabolismo , Transcripción Genética , Molécula 1 de Adhesión Celular Vascular/genética , Molécula 1 de Adhesión Celular Vascular/metabolismoRESUMEN
The study of human cardiogenesis would benefit from a detailed cell lineage fate map akin to that established for the haematopoietic lineages. Here we sought to define cell lineage relationships based on the expression of NKX2-5 and the cell surface markers VCAM1, SIRPA and CD34 during human cardiovascular development. Expression of NKX2-5(GFP) was used to identify cardiac progenitors and cardiomyocytes generated during the differentiation of NKX2-5(GFP/w) human embryonic stem cells (hESCs). Cardiovascular cell lineages sub-fractionated on the basis of SIRPA, VCAM1 and CD34 expression were assayed for differentiation potential and gene expression. The NKX2-5(pos)CD34(pos) population gave rise to endothelial cells that rapidly lost NKX2-5 expression in culture. Conversely, NKX2-5 expression was maintained in myocardial committed cells, which progressed from being NKX2-5(pos)SIRPA(pos) to NKX2-5(pos)SIRPA(pos)VCAM1(pos). Up-regulation of VCAM1 was accompanied by the expression of myofilament markers and reduced clonal capacity, implying a restriction of cell fate potential. Combinatorial expression of NKX2-5, SIRPA, VCAM1 and CD34 can be used to define discrete stages of cardiovascular cell lineage differentiation. These markers identify specific stages of cardiomyocyte and endothelial lineage commitment and, thus provide a scaffold for establishing a fate map of early human cardiogenesis.
Asunto(s)
Antígenos CD34/metabolismo , Antígenos de Diferenciación/metabolismo , Sistema Cardiovascular/crecimiento & desarrollo , Miocitos Cardíacos/citología , Células Madre Pluripotentes/citología , Células Madre Pluripotentes/metabolismo , Receptores Inmunológicos/metabolismo , Molécula 1 de Adhesión Celular Vascular/metabolismo , Diferenciación Celular/fisiología , Linaje de la Célula , Células Madre Embrionarias/citología , Células Madre Embrionarias/metabolismo , Humanos , Miocitos Cardíacos/metabolismoRESUMEN
NKX2-5 is expressed in the heart throughout life. We targeted eGFP sequences to the NKX2-5 locus of human embryonic stem cells (hESCs); NKX2-5(eGFP/w) hESCs facilitate quantification of cardiac differentiation, purification of hESC-derived committed cardiac progenitor cells (hESC-CPCs) and cardiomyocytes (hESC-CMs) and the standardization of differentiation protocols. We used NKX2-5 eGFP(+) cells to identify VCAM1 and SIRPA as cell-surface markers expressed in cardiac lineages.
Asunto(s)
Separación Celular/métodos , Células Madre Embrionarias/citología , Células Madre Embrionarias/metabolismo , Proteínas Fluorescentes Verdes/metabolismo , Proteínas de Homeodominio/metabolismo , Mioblastos Cardíacos/citología , Miocitos Cardíacos/citología , Factores de Transcripción/metabolismo , Antígenos de Diferenciación/genética , Antígenos de Diferenciación/metabolismo , Biomarcadores/análisis , Diferenciación Celular , Perfilación de la Expresión Génica , Proteína Homeótica Nkx-2.5 , Proteínas de Homeodominio/genética , Humanos , Mioblastos Cardíacos/metabolismo , Miocitos Cardíacos/metabolismo , Reacción en Cadena de la Polimerasa , Receptores Inmunológicos/genética , Receptores Inmunológicos/metabolismo , Factores de Transcripción/genética , Molécula 1 de Adhesión Celular Vascular/genética , Molécula 1 de Adhesión Celular Vascular/metabolismoRESUMEN
Macrolide-lincosamide-streptogramin B resistance is widespread, with the determinants encoding resistance to antibiotics such as erythromycin being detected in many bacterial pathogens. Resistance is most commonly mediated by the production of an Erm protein, a 23S rRNA methyltransferase. We have undertaken a mutational analysis of the Erm(B) protein from Clostridium perfringens with the objective of developing a greater understanding of the mechanism of action of this protein. A recombinant plasmid that carried the erm(B) gene was mutated by either in vitro hydroxylamine mutagenesis or passage through the mutator strain XL1-Red. Twenty-eight independently derived mutants were identified, nine of which had single point mutations in the erm(B) gene. These mutants produced stable but nonfunctional Erm(B) proteins, and all had amino acid changes within conserved methyltransferase motifs that were important for either substrate binding or catalysis. Modeling of the C. perfringens Erm(B) protein confirmed that the point mutations all involved residues important for the structure and/or function of this rRNA methyltransferase. These regions of the protein therefore represent potential targets for the rational development of methyltransferase inhibitors.
