RESUMEN
Although studies of the differentiation from mouse embryonic stem (ES) cells to vascular endothelial cells (ECs) provide an excellent model for investigating the molecular mechanisms underlying vascular development, temporal dynamics of gene expression and chromatin modifications have not been well studied. Herein, using transcriptomic and epigenomic analyses based on H3K4me3 and H3K27me3 modifications at a genome-wide scale, we analysed the EC differentiation steps from ES cells and crucial epigenetic modifications unique to ECs. We determined that Gata2, Fli1, Sox7 and Sox18 are master regulators of EC that are induced following expression of the haemangioblast commitment pioneer factor, Etv2. These master regulator gene loci were repressed by H3K27me3 throughout the mesoderm period but rapidly transitioned to histone modification switching from H3K27me3 to H3K4me3 after treatment with vascular endothelial growth factor. SiRNA knockdown experiments indicated that these regulators are indispensable not only for proper EC differentiation but also for blocking the commitment to other closely aligned lineages. Collectively, our detailed epigenetic analysis may provide an advanced model for understanding temporal regulation of chromatin signatures and resulting gene expression profiles during EC commitment. These studies may inform the future development of methods to stimulate the vascular endothelium for regenerative medicine.
Asunto(s)
Células Endoteliales/metabolismo , Epigénesis Genética , Factor de Transcripción GATA2/genética , Histonas/genética , Células Madre Embrionarias de Ratones/metabolismo , Proteína Proto-Oncogénica c-ets-1/genética , Factores de Transcripción SOXF/genética , Animales , Diferenciación Celular , Linaje de la Célula/genética , Células Endoteliales/citología , Factor de Transcripción GATA2/antagonistas & inhibidores , Factor de Transcripción GATA2/metabolismo , Histonas/metabolismo , Ratones , Células Madre Embrionarias de Ratones/citología , Análisis de Secuencia por Matrices de Oligonucleótidos , Cultivo Primario de Células , Isoformas de Proteínas/antagonistas & inhibidores , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteína Proto-Oncogénica c-ets-1/antagonistas & inhibidores , Proteína Proto-Oncogénica c-ets-1/metabolismo , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Factores de Transcripción SOXF/antagonistas & inhibidores , Factores de Transcripción SOXF/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
The origin and developmental mechanisms underlying coronary vessels are not fully elucidated. Here we show that myocardium-derived angiopoietin-1 (Ang1) is essential for coronary vein formation in the developing heart. Cardiomyocyte-specific Ang1 deletion results in defective formation of the subepicardial coronary veins, but had no significant effect on the formation of intramyocardial coronary arteries. The endothelial cells (ECs) of the sinus venosus (SV) are heterogeneous population, composed of APJ-positive and APJ-negative ECs. Among these, the APJ-negative ECs migrate from the SV into the atrial and ventricular myocardium in Ang1-dependent manner. In addition, Ang1 may positively regulate venous differentiation of the subepicardial APJ-negative ECs in the heart. Consistently, in vitro experiments show that Ang1 indeed promotes venous differentiation of the immature ECs. Collectively, our results indicate that myocardial Ang1 positively regulates coronary vein formation presumably by promoting the proliferation, migration and differentiation of immature ECs derived from the SV.
Asunto(s)
Angiopoyetina 1/metabolismo , Vasos Coronarios/embriología , Células Madre Embrionarias/fisiología , Corazón/embriología , Miocardio/metabolismo , Angiopoyetina 1/genética , Animales , Diferenciación Celular/fisiología , Quimera , Cartilla de ADN/genética , Vectores Genéticos/genética , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Inmunohistoquímica , Hibridación in Situ , Ratones , Reacción en Cadena de la Polimerasa , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
Specific gene knockout and rescue experiments are powerful tools in developmental and stem cell biology. Nevertheless, the experiments require multiple steps of molecular manipulation for gene knockout and subsequent rescue procedures. Here we report an efficient and single step strategy to generate gene knockout-rescue system in pluripotent stem cells by promoter insertion with CRISPR/Cas9 genome editing technology. We inserted a tetracycline-regulated inducible gene promoter (tet-OFF/TRE-CMV) upstream of the endogenous promoter region of vascular endothelial growth factor receptor 2 (VEGFR2/Flk1) gene, an essential gene for endothelial cell (EC) differentiation, in mouse embryonic stem cells (ESCs) with homologous recombination. Both homo- and hetero-inserted clones were efficiently obtained through a simple selection with a drug-resistant gene. The insertion of TRE-CMV promoter disrupted endogenous Flk1 expression, resulting in null mutation in homo-inserted clones. When the inserted TRE-CMV promoter was activated with doxycycline (Dox) depletion, Flk1 expression was sufficiently recovered from the downstream genomic Flk1 gene. Whereas EC differentiation was almost completely perturbed in homo-inserted clones, Flk1 rescue with TRE-CMV promoter activation restored EC appearance, indicating that phenotypic changes in EC differentiation can be successfully reproduced with this knockout-rescue system. Thus, this promoter insertion strategy with CRISPR/Cas9 would be a novel attractive method for knockout-rescue experiments.
Asunto(s)
Proteínas Asociadas a CRISPR/genética , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/genética , Técnicas de Inactivación de Genes/métodos , Células Madre Pluripotentes/metabolismo , Regiones Promotoras Genéticas/genética , Animales , Proteínas Asociadas a CRISPR/metabolismo , Diferenciación Celular/genética , Línea Celular , Citomegalovirus/genética , Doxiciclina/farmacología , Células Madre Embrionarias/citología , Células Madre Embrionarias/metabolismo , Células Endoteliales/citología , Células Endoteliales/metabolismo , Expresión Génica/efectos de los fármacos , Immunoblotting , Ratones , Modelos Genéticos , Mutagénesis Insercional , Células Madre Pluripotentes/citología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Tetraciclina/farmacología , Receptor 2 de Factores de Crecimiento Endotelial Vascular/genética , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Ets family protein Etv2 (also called ER71 or Etsrp) is a key factor for initiation of vascular and blood development from mesodermal cells. However, regulatory mechanisms and inducing signals for Etv2 expression have been largely unknown. Previously, we revealed that cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA) signaling enhanced differentiation of vascular progenitors into endothelial cells (ECs) and hematopoietic cells (HPCs) using an embryonic stem cell (ESC) differentiation system. Here, we show that PKA activation in an earlier differentiation stage can trigger EC/HPC differentiation through Etv2 induction. We found Etv2 was markedly upregulated by PKA activation preceding EC and HPC differentiation. We identified two cAMP response element (CRE) sequences in the Etv2 promoter and 5'-untranslated region and confirmed that CRE-binding protein (CREB) directly binds to the CRE sites and activates Etv2 transcription. Expression of a dominant negative form of CREB completely inhibited PKA-elicited Etv2 expression and induction of EC/HPCs from ESCs. Furthermore, blockade of PKA significantly inhibited Etv2 expression in ex vivo whole-embryo culture using Etv2-Venus knockin mice. These data indicated that PKA/CREB pathway is a critical regulator for the initiation of EC/HPC differentiation via Etv2 transcription. This early-stage molecular linkage between a triggering signal and transcriptional cascades for differentiation would provide novel insights in vascular and blood development and cell fate determination.
Asunto(s)
Diferenciación Celular , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Células Endoteliales/citología , Células Madre Hematopoyéticas/citología , Proteína Proto-Oncogénica c-ets-1/metabolismo , Transducción de Señal , Regiones no Traducidas 5'/genética , Animales , Secuencia de Bases , Línea Celular , Embrión de Mamíferos/citología , Embrión de Mamíferos/metabolismo , Células Madre Embrionarias/citología , Células Madre Embrionarias/enzimología , Células Endoteliales/metabolismo , Activación Enzimática , Células Madre Hematopoyéticas/metabolismo , Ratones , Modelos Biológicos , Datos de Secuencia Molecular , Regiones Promotoras Genéticas/genética , Proteína Proto-Oncogénica c-ets-1/genética , ARN Interferente Pequeño/metabolismo , Técnicas de Cultivo de Tejidos , Transcripción GenéticaRESUMEN
Molecular mechanisms controlling arterial-venous specification have not been fully elucidated. Previously, we established an embryonic stem cell differentiation system and demonstrated that activation of cAMP signaling together with VEGF induces arterial endothelial cells (ECs) from Flk1(+) vascular progenitor cells. Here, we show novel arterial specification machinery regulated by Notch and beta-catenin signaling. Notch and GSK3beta-mediated beta-catenin signaling were activated downstream of cAMP through phosphatidylinositol-3 kinase. Forced activation of Notch and beta-catenin with VEGF completely reconstituted cAMP-elicited arterial EC induction, and synergistically enhanced target gene promoter activity in vitro and arterial gene expression during in vivo angiogenesis. A protein complex with RBP-J, the intracellular domain of Notch, and beta-catenin was formed on RBP-J binding sites of arterial genes in arterial, but not venous ECs. This molecular machinery for arterial specification leads to an integrated and more comprehensive understanding of vascular signaling.