Cristina Clavería

Myc-driven endogenous cell competition in the early mammalian embryo

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.

Inhibition of programmed cell death impairs in vitro vascular-like structure formation and reduces in vivo angiogenesis

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 caspasedependent mechanism and mitochondrial cytochrome c release. Vascular‐like structure formation was further blocked by caspase inhibitors such as z‐VAD or AcD‐EVD‐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.—Segura, I., Serrano, A., González de Buitrago, G., González, M. A., Abad, J. L., Clavería, C., Gómez, L., Bernad, A., Martínez‐A, C., Riese, H. H. Inhibition of programmed cell death impairs in vitro vascular‐like structure formation and reduces in vivo angiogenesis. FASEB J. 16, 833–841 (2002)

GH3, a novel proapoptotic domain in Drosophila Grim, promotes a mitochondrial death pathway.

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 α‐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.

Drosophila grim induces apoptosis in mammalian cells

Genetic studies have shown that grim is a central genetic switch of programmed cell death in Drosophila; however, homologous genes have not been described in other species, nor has its mechanism of action been defined. We show here that grim expression induces apoptosis in mouse fibroblasts. Cell death induced by grim in mammalian cells involves membrane blebbing, cytoplasmic loss and nuclear DNA fragmentation. Grim‐induced apoptosis is blocked by both natural and synthetic caspase inhibitors. We found that grim itself shows caspase‐dependent proteolytic processing of its C‐terminus in vitro. Grim‐induced death is antagonized by bcl‐2 in a dose‐dependent manner, and neither Fas signalling nor p53 are required for grim pro‐apoptotic activity. Grim protein localizes both in the cytosol and in the mitochondria of mouse fibroblasts, the latter location becoming predominant as apoptosis progresses. These results show that Drosophila grim induces death in mammalian cells by specifically acting on mitochondrial apoptotic pathways executed by endogenous caspases. These findings advance our knowledge of the mechanism by which grim induces apoptosis and show the conservation through evolution of this crucial programmed cell death pathway.

Cell Competition Promotes Phenotypically Silent Cardiomyocyte Replacement in the Mammalian Heart

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.

Mitochondrial apoptotic pathways induced by Drosophila programmed cell death regulators.

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.

Erratum: Myc overexpression enhances epicardial contribution to the developing heart and promotes extensive expansion of the cardiomyocyte population.

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