RESUMEN
MYC plays various roles in pluripotent stem cells, including the promotion of somatic cell reprogramming to pluripotency, the regulation of cell competition and the control of embryonic diapause. However, how Myc expression is regulated in this context remains unknown. The Myc gene lies within a ~ 3-megabase gene desert with multiple cis-regulatory elements. Here we use genomic rearrangements, transgenesis and targeted mutation to analyse Myc regulation in early mouse embryos and pluripotent stem cells. We identify a topologically-associated region that homes enhancers dedicated to Myc transcriptional regulation in stem cells of the pre-implantation and early post-implantation embryo. Within this region, we identify elements exclusively dedicated to Myc regulation in pluripotent cells, with distinct enhancers that sequentially activate during naive and formative pluripotency. Deletion of pluripotency-specific enhancers dampens embryonic stem cell competitive ability. These results identify a topologically defined enhancer cluster dedicated to early embryonic expression and uncover a modular mechanism for the regulation of Myc expression in different states of pluripotency.
Asunto(s)
Elementos de Facilitación Genéticos , Regulación del Desarrollo de la Expresión Génica , Células Madre Pluripotentes , Proteínas Proto-Oncogénicas c-myc , Animales , Ratones , Proteínas Proto-Oncogénicas c-myc/metabolismo , Proteínas Proto-Oncogénicas c-myc/genética , Células Madre Pluripotentes/metabolismo , Células Madre Pluripotentes/citología , Transcripción Genética , Embrión de Mamíferos/metabolismo , Células Madre Embrionarias/metabolismo , Femenino , MasculinoRESUMEN
Tracing and manipulating cells in embryos are essential to understand development. Lipophilic dye microinjections, viral transfection and iontophoresis have been key to map the origin of the progenitor cells that form the different organs in the post-implantation mouse embryo. These techniques require advanced manipulation skills and only iontophoresis, a demanding approach of limited efficiency, has been used for single-cell labelling. Here, we perform lineage tracing and local gene ablation using cell-permeant Cre recombinase (TAT-Cre) microinjection. First, we map the fate of undifferentiated progenitors to the different heart chambers. Then, we achieve single-cell recombination by titrating the dose of TAT-Cre, which allows clonal analysis of nascent mesoderm progenitors. Finally, injecting TAT-Cre to Mycnflox/flox embryos in the primitive heart tube revealed that Mycn plays a cell-autonomous role in maintaining cardiomyocyte proliferation. This tool will help researchers identify the cell progenitors and gene networks involved in organ development, helping to understand the origin of congenital defects.
Asunto(s)
Integrasas , Células Madre , Ratones , Animales , Microinyecciones , Integrasas/genética , Marcación de GenRESUMEN
Meis1 and Meis2 are homeodomain transcription factors that regulate organogenesis through cooperation with Hox proteins. Elimination of Meis genes after limb induction has shown their role in limb proximo-distal patterning; however, limb development in the complete absence of Meis function has not been studied. Here, we report that Meis1/2 inactivation in the lateral plate mesoderm of mouse embryos leads to limb agenesis. Meis and Tbx factors converge in this function, extensively co-binding with Tbx to genomic sites and co-regulating enhancers of Fgf10, a critical factor in limb initiation. Limbs with three deleted Meis alleles show proximal-specific skeletal hypoplasia and agenesis of posterior skeletal elements. This failure in posterior specification results from an early role of Meis factors in establishing the limb antero-posterior prepattern required for Shh activation. Our results demonstrate roles for Meis transcription factors in early limb development and identify their involvement in previously undescribed interaction networks that regulate organogenesis.
Asunto(s)
Tipificación del Cuerpo/genética , Proteínas de Homeodominio/genética , Esbozos de los Miembros/metabolismo , Proteína 1 del Sitio de Integración Viral Ecotrópica Mieloide/genética , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Factor 10 de Crecimiento de Fibroblastos/genética , Factor 10 de Crecimiento de Fibroblastos/metabolismo , Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismo , Proteínas de Homeodominio/metabolismo , Esbozos de los Miembros/embriología , Ratones Noqueados , Ratones Transgénicos , Modelos Genéticos , Proteína 1 del Sitio de Integración Viral Ecotrópica Mieloide/metabolismo , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Proteínas de Dominio T Box/genética , Proteínas de Dominio T Box/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Activación Transcripcional/genéticaRESUMEN
The mammalian heart contains multiple cell types that appear progressively during embryonic development. Advance in determining cardiac lineage diversification has often been limited by the unreliability of genetic tracers. Here we combine clonal analysis, genetic lineage tracing, tissue transplantation, and mutant characterization to investigate the lineage relationships between epicardium, arterial mesothelial cells (AMCs), and the coronary vasculature. We report a contribution of the second heart field (SHF) to a vasculogenic niche composed of AMCs and sub-mesothelial cells at the base of the pulmonary artery. Sub-mesothelial cells from this niche differentiate into lymphatic endothelial cells and, in close association with AMC-derived cells, contribute to and are essential for the development of ventral cardiac lymphatics. In addition, regionalized epicardial/mesothelial retinoic acid signaling regulates lymphangiogenesis, contributing to the niche properties. These results uncover a SHF vasculogenic contribution to coronary lymphatic development through a local niche at the base of the great arteries.
Asunto(s)
Diferenciación Celular , Vasos Coronarios/fisiología , Endotelio Vascular/fisiología , Corazón/fisiología , Linfangiogénesis , Vasos Linfáticos/fisiología , Pericardio/fisiología , Animales , Linaje de la Célula , Vasos Coronarios/citología , Endotelio Vascular/citología , Epitelio/fisiología , Femenino , Corazón/embriología , Vasos Linfáticos/citología , Masculino , Ratones , Pericardio/citología , Transducción de SeñalRESUMEN
During vertebrate heart development, two progenitor populations, first and second heart fields (FHF, SHF), sequentially contribute to longitudinal subdivisions of the heart tube (HT), with the FHF contributing the left ventricle and part of the atria, and the SHF the rest of the heart. Here, we study the dynamics of cardiac differentiation and morphogenesis by tracking individual cells in live analysis of mouse embryos. We report that during an initial phase, FHF precursors differentiate rapidly to form a cardiac crescent, while limited morphogenesis takes place. In a second phase, no differentiation occurs while extensive morphogenesis, including splanchnic mesoderm sliding over the endoderm, results in HT formation. In a third phase, cardiac precursor differentiation resumes and contributes to SHF-derived regions and the dorsal closure of the HT. These results reveal tissue-level coordination between morphogenesis and differentiation during HT formation and provide a new framework to understand heart development.
Asunto(s)
Corazón/embriología , Microscopía Intravital , Morfogénesis , Animales , Diferenciación Celular , Ratones , Análisis Espacio-TemporalRESUMEN
The first blood and endothelial cells of amniote embryos appear in close association in the blood islands of the yolk sac (YS). This association and in vitro lineage analyses have suggested a common origin from mesodermal precursors called hemangioblasts, specified in the primitive streak during gastrulation. Fate mapping and chimera studies, however, failed to provide strong evidence for a common origin in the early mouse YS. Additional in vitro studies suggest instead that mesodermal precursors first generate hemogenic endothelium, which then generate blood cells in a linear sequence. We conducted an in vivo clonal analysis to determine the potential of individual cells in the mouse epiblast, primitive streak, and early YS. We found that early YS blood and endothelial lineages mostly derive from independent epiblast populations, specified before gastrulation. Additionally, a subpopulation of the YS endothelium has hemogenic activity and displays characteristics similar to those found later in the embryonic hemogenic endothelium. Our results show that the earliest blood and endothelial cell populations in the mouse embryo are specified independently, and that hemogenic endothelium first appears in the YS and produces blood precursors with markers related to definitive hematopoiesis.