RESUMEN
mtDNA is present in multiple copies in each cell derived from the expansions of those in the oocyte. Heteroplasmy, more than one mtDNA variant, may be generated by mutagenesis, paternal mtDNA leakage, and novel medical technologies aiming to prevent inheritance of mtDNA-linked diseases. Heteroplasmy phenotypic impact remains poorly understood. Mouse studies led to contradictory models of random drift or haplotype selection for mother-to-offspring transmission of mtDNA heteroplasmy. Here, we show that mtDNA heteroplasmy affects embryo metabolism, cell fitness, and induced pluripotent stem cell (iPSC) generation. Thus, genetic and pharmacological interventions affecting oxidative phosphorylation (OXPHOS) modify competition among mtDNA haplotypes during oocyte development and/or at early embryonic stages. We show that heteroplasmy behavior can fall on a spectrum from random drift to strong selection, depending on mito-nuclear interactions and metabolic factors. Understanding heteroplasmy dynamics and its mechanisms provide novel knowledge of a fundamental biological process and enhance our ability to mitigate risks in clinical applications affecting mtDNA transmission.
Asunto(s)
ADN Mitocondrial/genética , Desarrollo Embrionario/genética , Herencia Materna/genética , Mitocondrias/genética , Oogénesis/genética , Animales , Línea Celular , Embrión de Mamíferos , Femenino , Fibroblastos , Haplotipos , Masculino , Ratones , Ratones Endogámicos C57BL , OocitosRESUMEN
The mammalian epiblast is formed by pluripotent cells able to differentiate into all tissues of the new individual. In their progression to differentiation, epiblast cells and their in vitro counterparts, embryonic stem cells (ESCs), transit from naive pluripotency through a differentiation-primed pluripotent state. During these events, epiblast cells and ESCs are prone to death, driven by competition between Myc-high cells (winners) and Myc-low cells (losers). Using live tracking of Myc levels, we show that Myc-high ESCs approach the naive pluripotency state, whereas Myc-low ESCs are closer to the differentiation-primed state. In ESC colonies, naive cells eliminate differentiating cells by cell competition, which is determined by a limitation in the time losers are able to survive persistent contact with winners. In the mouse embryo, cell competition promotes pluripotency maintenance by elimination of primed lineages before gastrulation. The mechanism described here is relevant to mammalian embryo development and induced pluripotency.
Asunto(s)
Diferenciación Celular , Células Madre Pluripotentes/citología , Células Madre Pluripotentes/metabolismo , Proteínas Proto-Oncogénicas c-myc/metabolismo , Animales , Comunicación Celular , Linaje de la Célula , Proliferación Celular , Supervivencia Celular , Rastreo Celular , Células Cultivadas , Embrión de Mamíferos/citología , Gastrulación , Perfilación de la Expresión Génica , Estratos Germinativos/citología , Patrón de Herencia/genética , Ratones , Células Madre Embrionarias de Ratones/citología , Células Madre Embrionarias de Ratones/metabolismo , Factores de TiempoRESUMEN
Myc is an essential regulator of cell growth and proliferation. Myc overexpression promotes the homeostatic expansion of cardiomyocyte populations by cell competition, however whether this applies to other cardiac lineages remains unknown. The epicardium contributes signals and cells to the developing and adult injured heart and exploring strategies for modulating its activity is of great interest. Using inducible genetic mosaics, we overexpressed Myc in the epicardium and determined the differential expansion of Myc-overexpressing cells with respect to their wild type counterparts. Myc-overexpressing cells overcolonized all epicardial-derived lineages and showed increased ability to invade the myocardium and populate the vasculature. We also found massive colonization of the myocardium by Wt1Cre-derived Myc-overexpressing cells, with preservation of cardiac development. Detailed analyses showed that this contribution is unlikely to derive from Cre activity in early cardiomyocytes but does not either derive from established epicardial cells, suggesting that early precursors expressing Wt1Cre originate the recombined cardiomyocytes. Myc overexpression does not modify the initial distribution of Wt1Cre-recombined cardiomyocytes, indicating that it does not stimulate the incorporation of early expressing Wt1Cre lineages to the myocardium, but differentially expands this initial population. We propose that strategies using epicardial lineages for heart repair may benefit from promoting cell competitive ability.
Asunto(s)
Corazón/crecimiento & desarrollo , Miocardio/metabolismo , Organogénesis/genética , Proteínas Proto-Oncogénicas c-myc/genética , Animales , Diferenciación Celular/genética , Linaje de la Célula/genética , Proliferación Celular/genética , Vasos Coronarios/crecimiento & desarrollo , Vasos Coronarios/metabolismo , Vasos Coronarios/patología , Regulación del Desarrollo de la Expresión Génica , Integrasas/genética , Ratones , Miocitos Cardíacos/metabolismo , Pericardio/crecimiento & desarrollo , Pericardio/metabolismoRESUMEN
Cell-competitive interactions are widespread in nature and determine the outcome of a vast variety of biological processes. A particular class of competitive interactions takes place when alterations in intrinsic cellular properties are sensed nonautonomously by comparison between neighboring cells, resulting in the selective elimination of one cell population. This type of cell competition was first described four decades ago in developing epithelia of Drosophila. In the last 15 years, further molecular and cellular analyses have provided essential knowledge about the mechanisms, universality, and physiological relevance of cell competition. The two main phenomena triggering cell competition are alterations in cellular metabolic status and alterations in epithelial apico-basal polarity, while other reported pathways are less characterized. Cell competition plays essential roles in quality control, homeostasis, and repair of developing and adult tissues, and depending on the context, it may function as a tumor-suppressing or tumor-promoting mechanism.
Asunto(s)
Células/metabolismo , Animales , Enfermedad , Salud , Humanos , Modelos Biológicos , Transducción de SeñalRESUMEN
Heterogeneous anabolic capacity in cell populations can trigger a phenomenon known as cell competition, through which less active cells are eliminated. Cell competition has been induced experimentally in stem/precursor cell populations in insects and mammals and takes place endogenously in early mouse embryonic cells. Here, we show that cell competition can be efficiently induced in mouse cardiomyocytes by mosaic overexpression of Myc during both gestation and adult life. The expansion of the Myc-overexpressing cardiomyocyte population is driven by the elimination of wild-type cardiomyocytes. Importantly, this cardiomyocyte replacement is phenotypically silent and does not affect heart anatomy or function. These results show that the capacity for cell competition in mammals is not restricted to stem cell populations and suggest that stimulated cell competition has potential as a cardiomyocyte-replacement strategy.
Asunto(s)
Corazón/fisiología , Miocitos Cardíacos/citología , Animales , Apoptosis , Embrión de Mamíferos/metabolismo , Femenino , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Corazón/anatomía & histología , Corazón/crecimiento & desarrollo , Proteína Homeótica Nkx-2.5 , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Integrasas/genética , Integrasas/metabolismo , Proteínas con Homeodominio LIM/metabolismo , Masculino , Ratones , Miocitos Cardíacos/metabolismo , Fenotipo , Proteínas Proto-Oncogénicas c-myc/genética , Proteínas Proto-Oncogénicas c-myc/metabolismo , Recombinación Genética , Células Madre/citología , Células Madre/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
The epiblast is the mammalian embryonic tissue that contains the pluripotent stem cells that generate the whole embryo. We have established a method for inducing functional genetic mosaics in the mouse. Using this system, here we show that induction of a mosaic imbalance of Myc expression in the epiblast provokes the expansion of cells with higher Myc levels through the apoptotic elimination of cells with lower levels, without disrupting development. In contrast, homogeneous shifts in Myc levels did not affect epiblast cell viability, indicating that the observed competition results from comparison of relative Myc levels between epiblast cells. During normal development we found that Myc levels are intrinsically heterogeneous among epiblast cells, and that endogenous cell competition refines the epiblast cell population through the elimination of cells with low relative Myc levels. These results show that natural cell competition in the early mammalian embryo contributes to the selection of the epiblast cell pool.
Asunto(s)
Embrión de Mamíferos/citología , Embrión de Mamíferos/metabolismo , Estratos Germinativos/citología , Proteínas Proto-Oncogénicas c-myc/metabolismo , Animales , Apoptosis , Proliferación Celular , Células Madre Embrionarias/citología , Células Madre Embrionarias/metabolismo , Femenino , Expresión Génica , Genes myc , Estratos Germinativos/metabolismo , Masculino , Ratones , Modelos Biológicos , Mosaicismo/embriologíaRESUMEN
Grim encodes a protein required for programmed cell death in Drosophila, whose proapoptotic activity is conserved in mammalian cells. Two proapoptotic domains are relevant for Grim killing function; the N-terminal region, which induces apoptosis by disrupting inhibitor of apoptosis protein (IAP) blockage of caspase activity, and the internal GH3 domain, which triggers a mitochondrial pathway. We explored the role of these two domains in heterologous killing of mammalian cells by Grim. The GH3 domain is essential for Grim proapoptotic activity in mouse cells, whereas the N-terminal domain is dispensable. The GH3 domain is required and sufficient for Grim targeting to mitochondria and for cytochrome c release in a caspase- and N-terminal-independent, IAP-insensitive manner. These Grim GH3 activities do not require Bax or Bak function, revealing GH3 activity as the first proapoptotic stimulus able to trigger the mitochondrial death pathway in mammalian cells in the absence of multidomain proapoptotic Bcl-2 proteins.
Asunto(s)
Drosophila/metabolismo , Proteínas de la Membrana/metabolismo , Mitocondrias/patología , Proteínas Proto-Oncogénicas c-bcl-2 , Proteínas Proto-Oncogénicas/metabolismo , Secuencia de Aminoácidos , Animales , Apoptosis , Proteína Proapoptótica que Interacciona Mediante Dominios BH3 , Proteínas Portadoras/metabolismo , Muerte Celular , Supervivencia Celular , Fibroblastos/metabolismo , Vectores Genéticos , Ratones , Microscopía Fluorescente , Mitocondrias/metabolismo , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Mutación , Células 3T3 NIH , Pruebas de Precipitina , Conformación Proteica , Estructura Terciaria de Proteína , Homología de Secuencia de Aminoácido , Proteína Destructora del Antagonista Homólogo bcl-2 , Proteína X Asociada a bcl-2RESUMEN
Multicellular organisms eliminate unwanted or damaged cells by cell death, a process essential to the maintenance of tissue homeostasis. Cell death is a tightly regulated event, whose alteration by excess or defect is involved in the pathogenesis of many diseases such as cancer, autoimmune syndromes, and neurodegenerative processes. Studies in model organisms, especially in the nematode Caenorhabditis elegans, have been crucial in identifying the key molecules implicated in the regulation and execution of programmed cell death. In contrast, the study of cell death in Drosophila melanogaster, often an excellent model organism, has identified regulators and mechanisms not obviously conserved in other metazoans. Recent molecular and cellular analyses suggest, however, that the mechanisms of action of the main programmed cell death regulators in Drosophila include a canonical mitochondrial pathway.
Asunto(s)
Apoptosis , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Mitocondrias/metabolismo , Secuencia de Aminoácidos , Animales , Evolución Biológica , Proteínas de Drosophila/química , Drosophila melanogaster/citología , Modelos Biológicos , Estructura Terciaria de Proteína , Transducción de SeñalRESUMEN
Grim encodes a protein required for programmed cell death in Drosophila. The Grim N-terminus induces apoptosis by disrupting IAP blockage of caspases; however, N-terminally-deleted Grim retains pro apoptotic activity. We describe GH3, a 15 amino acid internal Grim domain absolutely required for its proapoptotic activity and sufficient to induce cell death when fused to heterologous carrier proteins. A GH3 homology region is present in the Drosophila proapoptotic proteins Reaper and Sickle. The GH3 domain and the homologous regions in Reaper and Sickle are predicted to be structured as amphipathic alpha-helixes. During apoptosis induction, Grim colocalizes with mitochondria and cytochrome c in a GH3-dependent but N-terminal- and caspase activity-independent manner. When Grim is overexpressed in vivo, both the N-terminal and the GH3 domains are equally necessary, and cooperate for apoptosis induction. The N-terminal and GH3 Grim domains thus activate independent apoptotic pathways that synergize to induce programmed cell death efficiently.
Asunto(s)
Apoptosis/fisiología , Proteínas de Drosophila/fisiología , Drosophila melanogaster/citología , Mitocondrias/fisiología , Neuropéptidos/fisiología , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Animales , Animales Modificados Genéticamente , Caspasas/fisiología , Células Cultivadas , Grupo Citocromo c/fisiología , Proteínas de Drosophila/química , Femenino , Masculino , Datos de Secuencia Molecular , Neuropéptidos/química , Fenotipo , Estructura Terciaria de Proteína , Proteínas Recombinantes de Fusión/fisiología , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Relación Estructura-ActividadRESUMEN
Tissue remodeling during embryonic development and in the adult organism relies on a subtle balance between cell growth and apoptosis. As angiogenesis involves restructuring of preexisting endothelium, we examined the role of apoptosis in new vessel formation. We show that apoptosis occurs before capillary formation but not after vessels have assembled. Using the human umbilical vein endothelial cell (HUVEC) in vitro Matrigel angiogenesis model, we show that vascular-like structure formation requires apoptotic cell death through activation of a caspase-dependent mechanism and mitochondrial cytochrome c release. Vascular-like structure formation was further blocked by caspase inhibitors such as z-VAD or Ac-DEVD-CHO, using HUVEC and human lung microvascular endothelial cells. Overexpression of anti-apoptotic human Bcl-2 or baculovirus p35 genes in HUVEC altered endothelial cell rearrangement during in vitro angiogenesis, causing impaired vessel-like structure formation. Caspase inhibitors blocked VEGF- or bFGF-induced HUVEC angiogenesis on 2- or 3-D collagen gels, respectively, confirming that apoptosis was not the result of nonspecific cell death after seeding on the matrix. In an in vivo angiogenesis assay, caspase inhibitors blocked VEGF-dependent vascular formation at the alignment step, as demonstrated histologically. This evidence indicates that endothelial cell apoptosis may be relevant for precise vascular tissue rearrangement in in vitro and in vivo angiogenesis.