RESUMEN
The ETS factor ETV2 (aka ER71) is essential for the generation of the blood and vascular system, as ETV2 deficiency leads to a complete block in blood and endothelial cell formation and embryonic lethality in the mouse. However, the ETV2-mediated gene regulatory network and signaling governing hematopoietic and endothelial cell development are poorly understood. Here, we map ETV2 global binding sites and carry out in vitro differentiation of embryonic stem cells, and germ line and conditional knockout mouse studies to uncover mechanisms involved in the hemangiogenic fate commitment from mesoderm. We show that ETV2 binds to enhancers that specify hematopoietic and endothelial cell lineages. We find that the hemangiogenic progenitor population in the developing embryo can be identified as FLK1(high)PDGFRα(-). Notably, these hemangiogenic progenitors are exclusively sensitive to ETV2-dependent FLK1 signaling. Importantly, ETV2 turns on other Ets genes, thereby establishing an ETS hierarchy. Consequently, the hematopoietic and endothelial cell program initiated by ETV2 is maintained partly by other ETS factors through an ETS switching mechanism. These findings highlight the critical role that transient ETV2 expression plays in the regulation of hematopoietic and endothelial cell lineage specification and stability.
Asunto(s)
Células Sanguíneas/citología , Células Sanguíneas/metabolismo , Diferenciación Celular , Células Endoteliales/citología , Células Endoteliales/metabolismo , Factores de Transcripción/metabolismo , Animales , Sitios de Unión , Diferenciación Celular/genética , Linaje de la Célula/genética , Inmunoprecipitación de Cromatina , Femenino , Regulación del Desarrollo de la Expresión Génica , Redes Reguladoras de Genes , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/metabolismo , Secuenciación de Nucleótidos de Alto Rendimiento , Inmunofenotipificación , Masculino , Ratones , Ratones Noqueados , Motivos de Nucleótidos , Especificidad de Órganos/genética , Posición Específica de Matrices de Puntuación , Unión Proteica , Receptores de Factores de Crecimiento Endotelial Vascular/metabolismo , Transducción de Señal , Factores de Transcripción/genética , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismo , Factores de Crecimiento Endotelial Vascular/metabolismoRESUMEN
The fetal liver kinase 1 (FLK-1)(+) hemangioblast can generate hematopoietic, endothelial, and smooth muscle cells (SMCs). ER71/ETV2, GATA2, and SCL form a core transcriptional network in hemangioblast development. Transient coexpression of these three factors during mesoderm formation stage in mouse embryonic stem cells (ESCs) robustly enhanced hemangioblast generation by activating bone morphogenetic protein (BMP) and FLK-1 signaling while inhibiting phosphatidylinositol 3-kinase, WNT signaling, and cardiac output. Moreover, etsrp, gata2, and scl inhibition converted hematopoietic field of the zebrafish anterior lateral plate mesoderm to cardiac. FLK-1(+) hemangioblasts generated by transient coexpression of the three factors (ER71-GATA2-SCL [EGS]-induced FLK-1(+)) effectively produced hematopoietic, endothelial, and SMCs in culture and in vivo. Importantly, EGS-induced FLK-1(+) hemangioblasts, when codelivered with mesenchymal stem cells as spheroids, were protected from apoptosis and generated functional endothelial cells and SMCs in ischemic mouse hindlimbs, resulting in improved blood perfusion and limb salvage. ESC-derived, EGS-induced FLK-1(+) hemangioblasts could provide an attractive cell source for future hematopoietic and vascular repair and regeneration.
Asunto(s)
Células Madre Embrionarias/citología , Hemangioblastos/citología , Miocitos Cardíacos/metabolismo , Factores de Transcripción/metabolismo , Pez Cebra/metabolismo , Animales , Células Cultivadas , Femenino , Regulación de la Expresión Génica , Hemangioblastos/metabolismo , Miembro Posterior/irrigación sanguínea , Miembro Posterior/lesiones , Mesodermo/metabolismo , Ratones , Datos de Secuencia Molecular , Neovascularización Fisiológica , Transducción de Señal , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Two distinct types of Flk-1(+) mesoderm, hemangiogenic and cardiogenic, are thought to contribute to blood, vessel, and cardiac cell lineages. However, our understanding of how Flk-1(+) mesoderm is specified is currently limited. In the present study, we investigated whether ER71, an Ets transcription factor essential for hematopoietic and endothelial cell lineage development, could modulate the hemangiogenic or cardiogenic outcome of the Flk-1(+) mesoderm. We show that Flk-1(+) mesoderm can be divided into Flk-1(+)PDGFRα(-) hemangiogenic and Flk-1(+)PDGFRα(+) cardiogenic mesoderm. ER71-deficient embryonic stem cells produced only the Flk-1(+)PDGFRα(+) cardiogenic mesoderm, which generated SMCs and cardiomyocytes. Enforced ER71 expression in the wild-type embryonic stem cells skewed toward the Flk-1(+)PDGFRα(-) mesoderm formation, which generated hematopoietic and endothelial cells. Whereas hematopoietic and endothelial cell genes were positively regulated by ER71, cardiac and Wnt signaling pathway genes were negatively regulated by ER71. We show that ER71 could inhibit Wnt signaling in VE-cadherin-independent as well as VE-cadherin-dependent VE-cadherin/ß-catenin/Flk-1 complex formation. Enforced ß-catenin could rescue cardiogenic mesoderm in the context of ER71 overexpression. In contrast, ER71-deficient Flk-1(+) mesoderm displayed enhanced Wnt signaling, which was reduced by ER71 re-introduction. We provide the molecular basis for the antagonistic relationship between hemangiogenic and cardiogenic mesoderm specification by ER71 and Wnt signaling.