Cell competition acts as a purifying selection to eliminate cells with mitochondrial defects during early mouse development

Abstract

Cell competition is emerging as a quality control mechanism that eliminates unfit cells in a wide range of settings from development to the adult. However, the nature of the cells normally eliminated by cell competition and what triggers their elimination remains poorly understood. Here we have performed single cell transcriptional profiling of early mouse embryos and find that the cells eliminated show the hallmarks of cell competition, are mis-patterned and have mitochondrial defects. We demonstrate that mitochondrial defects are common to a range of different loser cell types and that manipulating mitochondrial function is sufficient to trigger competition. Importantly, we show that in the embryo loser epiblast cells display mitochondrial DNA mutations and that even small changes in mitochondrial DNA sequence can influence the competitive ability of the cell.Our results therefore suggest that cell competition is a purifying selection that optimises metabolic output prior to gastrulation. Running title: Cell competition and mitochondrial selectionDuring the early stages of mammalian development, the cellular and molecular landscape is 2 profoundly remodelled. As embryonic cells approach gastrulation, when the precursors of all 3 embryonic tissues are specified, they need to rewire the transcriptional, epigenetic, metabolic and 4 signalling networks that govern cell identity (Kojima et al., 2014). These changes are accompanied 5 by a marked acceleration in the proliferation rate (Snow, 1977) and need to be orchestrated with 6 the different morphogenetic processes that re-shape the embryo (reviewed in (Stower and 7 Srinivas, 2018). The scale of this remodelling creates the potential for the emergence of abnormal 8 cells that need to be removed to prevent them from contributing to the soma or germline during 9 development. This requirement implies that there must be stringent cell fitness quality control 10 mechanisms acting around the time of gastrulation. One such control has been postulated to be 11 cell competition, a fitness sensing mechanism eliminating cells that, although viable, are less fit 12 than their neighbours (reviewed in (Bowling et al., 2019; Diaz-Diaz and Torres, 2019; Madan et 13 al., 2018). During cell competition, the cells that are eliminated are generically termed losers, while 14 the fitter cells that survive are referred to as winners. 15Cell competition has been primarily studied in Drosophila, where it was first described in the cell RNA sequencing (scRNA-seq). To ensure we can capture the eliminated cells, as we have 70 done before (Bowling et al., 2018), we isolated embryos at E5.5 and cultured them for 16 hours in 71 the presence of a caspase inhibitors (CI) or vehicle (DMSO) ( Figure 1A and Figure S1A). 72Unsupervised clustering of the scRNA-seq data revealed five clusters: two corresponding to extra-73 embryonic tissues (visceral endoderm and extra-embryonic ectoderm) and three that expressed 74 epiblast marker genes (Figure 1B-C and Figure S1B-D). Interestingly, cells from CI...

P53 and PUMA in Pluripotent Cell Competition

“…P53 (Bondar and Medzhitov, 2010;Dejosez, 2013), mTOR (Bowling et al, 2018) or autophagy (Sancho et al, 2013). Alteration of mitochondrial dynamics through DRP1/MFN2 proteins (Lima et al, 2020) or the activation of stress responses as oxidative stress (Kucinski et al, 2017) can also trigger CC.…”

“…We next moved to study P53, PUMA and MYC expression in vivo. We used 6.5dpc mouse embryos given that MYC-driven CC, as well as other CC models, have been described at this stage (Bowling et al, 2018;Clavería et al, 2013;Díaz-Díaz et al, 2017;Lima et al, 2020;Sancho et al, 2013).…”

“…To account for clonal variation, including possible off-target mutations, we used for each gene at least three mutant and three WT sister clones. According to the literature referring to similar Competition assays, these experiments were performed in differentiating conditions (Dejosez et al, 2013;Lima et al, 2020;Sancho et al, 2013).…”

“…CC induced by BMP-signalling deficient cells occurs in differentiating conditions but this does not take place if naïve pluripotency is enforced in 2i condition (Lima et al, 2020;Sancho et al, 2013). Regarding P53-induced CC, Dejosez et al, showed that p53 -/cells seem to outcompete WT cells in the early mouse embryo and in ESCs in differentiating conditions, although it is not clear whether this is due to an autonomous behaviour or to CC.…”

“…CC has been described to eliminate stressed cells in different models. For instance, in the epiblast, loser cells exhibit different stress indicators including altered mitochondrial function or high P53 expression (Díaz-Díaz et al, 2017;Lima et al, 2020;Zhang et al, 2017). Moreover, in the adult, CC eliminates cells displaying oxidative stress, proteotoxic stress or active P53 response in Drosophila (Bondar and Medzhitov, 2010;Kucinski et al, 2017;Wagstaff et al, 2016;Zhang et al, 2017;Baumgartnert el al., 2021).…”