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DRP1-mediated regulation of mitochondrial dynamics determines the apoptotic response upon embryonic differentiation

Barbara Pernaute, Juan Miguel Sánchez Nieto, Salvador Pérez-Montero, Aida di Gregorio, Ana Lima, Katerina Lawlor, Sarah Bowling, Gianmaria Liccardi, Alejandra Tomás, Pascal Meier, Guy A. Rutter, Ivana Barbaric, Tristan A. Rodríguez

Preprint posted on November 08, 2019 https://www.biorxiv.org/content/10.1101/835751v1.full

Higher sensitivity to apoptosis driven by mitochondrial dynamics: Lowering of the apoptotic threshold in the post-implantation embryo is regulated by changes in mitochondrial shape and connectivity.

Selected by Katarzyna Kuzmicz-Kowalska

Background

In developing mammalian embryos, the size of the embryo is tightly regulated especially around the time of gastrulation. At this stage, the number of cells rapidly expands by a factor of about 100 (Snow 1977) and cells undergo cell fate changes as they exit pluripotency. In the wildtype embryo, up to 35% of cells are eliminated upon gastrulation (Bowling et al., 2018).  Removed cells are typically less fit, mis-specified or carrying mutations (Bowling et al., 2019, Sancho et al., 2013, Diaz- Diaz et al., 2017, Claveria et al., 2013), suggesting that this increase in cell death serves as a quality control mechanism. Previous studies have shown that post-implantation epiblast cells are more susceptible to apoptosis-inducing signals than cells of pre-implantation embryos (Heyer et al., 2000, Pernaute et al., 2014). However, the cause of this switch in sensitivity to cell death and the underlying molecular mechanism have not been identified.

Experimental system

The authors investigated how the sensitivity to apoptosis changes upon gastrulation. They used naïve pluripotent mouse embryonic stem cells (ESCs) and primed pluripotent epiblast stem cells (EpiSCs) to model pre- and post- implantation embryonic cells, respectively. They showed that upon exposure to different sources of stress these cells recapitulate the specific apoptotic response observed in vivo.

Key findings

Higher sensitivity to apoptosis of EpiSCs is not caused by differences in relative expression levels of regulators of the mitochondrial apoptotic pathway

The authors first demonstrated that the mitochondrial branch of apoptotic pathways is more active in EpiSCs than ESCs, which prompted them to investigate the underlying mechanism. The activity of the mitochondrial pathway depends on the balance between pro- and anti-apoptotic proteins from the BCL-2 family. The authors found that only one of the anti-apoptotic proteins, BCL-2, was elevated in EpiSCs, but this increase could also be explained as an adaptation to high cell death levels. Furthermore, the induction of cell death with small molecule compound ABT-737, which inhibits the activity of the BCL-2 protein, did not affect the balance between mitochondrial regulators. These results suggest that differences in the relative expression levels of proteins from the BCL-2 family are not driving the increased susceptibility of EpiSCs to apoptosis.

Mitochondrial dynamics changes upon exit from naïve pluripotency

An alternative mechanism called ‘mitochondrial dynamics’, which relies on changes in mitochondrial shape resulting from fission and fusion events, has been implicated in controlling the mitochondrial apoptosis pathway. In developing mammalian embryos the shape of mitochondria rapidly changes from round to elongated around the time of implantation (Lima et al., 2018).  Here the authors found that, like in the embryo, the mitochondria change their shape and connectivity between ESCs and EpiSCs. The authors further showed that differences in activity and levels of DRP1 protein, a key regulator of mitochondrial fission, are responsible for this shift in mitochondrial dynamics using CRISPR-generated Drp1 knock-out ESCs.

The levels of DRP1 regulate the apoptotic threshold in pluripotent cells

To address the role of DRP1 in the regulation of cell death sensitivity the authors investigated apoptosis levels in Drp1 knock-out cells in response to different stimuli. Loss of Drp1 was sufficient to lower the apoptotic threshold of ESCs. Conversely, overexpression of Drp1 during ESC differentiation reduced their sensitivity to induction of cell death. In both experiments, perturbations of Drp1 levels did not cause any changes in pluripotency state of ESCs.

Altogether, the preprint proposes a new mechanism whereby the dynamic reshaping of mitochondrial networks controls the switch in the sensitivity to apoptosis upon gastrulation.

Why did I like this paper?

I chose this preprint because it provides a new interesting mechanism through which cells can quickly adapt to the environmental signals driving the embryo-shaping wave of cell elimination. In recent years, the triggers for cell competition and markers of less-fit cells at the time of gastrulation have been discussed, but the intracellular network that governs the apoptotic response has not been fully understood.

In my opinion, the authors very thoroughly looked into the different parameters of mitochondrial function, I especially liked the analysis of mitochondrial circularity. I also appreciated how the in vitro data were able to so closely recapitulate the embryonic development.

Open questions/Questions to the authors

1.While the exact molecular mechanism underlying the role of DRP1 in regulating the apoptotic response remains unclear, the authors proposed that changes in occurrence of fission and the corresponding changes in mitochondrial shape might affect the ability of BAX protein to bind and permeabilize the outer mitochondrial membrane. The results from the analysis of BAX expression levels seem to support this hypothesis as in ESCs the overall levels of BAX in whole cell lysate (WCL) are higher than in EpiSCs. It would be interesting to see if the levels of BAX in WCL and mitochondrial fractions are affected upon Drp1 perturbations.

2. It is interesting that although the apoptotic threshold is lowered in EpiSCs and post-implantation embryos, not all of the cells ‘commit’ cell death. The results show that mitochondrial dynamics seems to be rather homogenous between the cells. If it is indeed the case, then how can this be reconciled with the heterogenous cell death response? Do the authors think that the shape of the mitochondria and fusion/fission events can also have an effect on cell cycle progression in these cells?

References

Bowling, S., Lawlor, K. & Rodriguez, T.A. Cell competition: the winners and losers of fitness selection. Development 146 (2019).

Bowling, S., Di Gregorio, A., Sancho, M., Pozzi, S., Aarts, M., Signore, M., Schneider, M., Martinez-Barbera, JP., Gil, J., Rodríguez, TA. Nat Commun. 9(1):1763 (2018).

Clavería, C., Giovinazzo, G., Sierra, R., Torres, M. Myc-driven endogenous cell competition in the early mammalian embryo. Nature. 500, 39–44 (2013).

Díaz-Díaz, C., Fernandez de Manuel, L., Jimenez-Carretero, D., Montoya, MC., Clavería, C., Torres, M. Dev Cell. 42, 585-599 (2017).

Heyer, B.S., MacAuley, A., Behrendtsen, O. & Werb, Z. Hypersensitivity to DNA damage leads to increased apoptosis during early mouse development. Genes Dev 14, 2072-2084 (2000).

Lima, A., Burgstaller, J., Sanchez-Nieto, J.M. & Rodriguez, T.A. The Mitochondria and the Regulation of Cell Fitness During Early Mammalian Development. Curr Top Dev Biol 128, 339-363 (2018)

Pernaute, B., Spruce, T., Smith, KM., Sánchez-Nieto, JM., Manzanares, M., Cobb, B., Rodríguez, TA. MicroRNAs control the apoptotic threshold in primed pluripotent stem cells through regulation of BIM. Genes Dev 28, 1873-1878 (2014).

Sancho, M., Di-Gregorio, A., George, N., Pozzi, S., Sánchez, JM., Pernaute, B., Rodríguez, TA. Competitive Interactions Eliminate Unfit Embryonic Stem Cells at the Onset of Differentiation. Developmental Cell. 26, 19–30 (2013).

Snow, M.H.L. Gastrulation in the mouse: growth and regionalization of the epiblast. J. Embryol. exp. Morph. 42, 293-303 (1977).

 

Tags: cell death, gastrulation, mitochondria, mouse embryo

Posted on: 12th February 2020

doi: https://doi.org/10.1242/prelights.16890

Read preprint (No Ratings Yet)




  • Author's response

    Tristan A. Rodríguez, Barbara Pernaute, Juan Miguel Sánchez Nieto and Salvador Pérez-Montero shared

    Q1: While the exact molecular mechanism underlying the role of DRP1 in regulating the apoptotic response remains unclear, the authors proposed that changes in occurrence of fission and the corresponding changes in mitochondrial shape might affect the ability of BAX protein to bind and permeabilize the outer mitochondrial membrane. The results from the analysis of BAX expression levels seem to support this hypothesis as in ESCs the overall levels of BAX in whole cell lysate (WCL) are higher than in EpiSCs. It would be interesting to see if the levels of BAX in WCL and mitochondrial fractions are affected upon Drp1 perturbations.

    This is a very good question, as understanding how DRP1 affects the apoptotic response is key. We are currently thinking of three different possibilities. The first two relate to the rounded mitochondrial shape of ESCs due to DRP1 induced fission.  It is possible, as you indicate and has been previously shown (Renault et al., Mol. Cell 2015), that the rounded mitochondria of ESCs could reduce the ability of BAX to permeabilize the mitochondrial membrane. To test this, as you suggest, it would be interesting to see if mutation of Drp1 affects BAX levels/activity. Alternatively, the rounded mitochondria of ESCs could protect these cells from apoptosis by decreasing mitochondria-ER contact sites, and therefore slowing down the transfer of ER derived pro-apoptotic signals. The final possibility relates to the known importance of DRP1 for mitophagy. It is possible that ESCs have higher mitophagy levels that differentiating cells, and that this increases their apoptotic threshold by allowing an efficient clean-up of damaged mitochondria. We would haste to add, that these are not mutually exclusive possibilities.

    Q2: It is interesting that although the apoptotic threshold is lowered in EpiSCs and post-implantation embryos, not all of the cells ‘commit’ cell death. The results show that mitochondrial dynamics seems to be rather homogenous between the cells. If it is indeed the case, then how can this be reconciled with the heterogenous cell death response? Do the authors think that the shape of the mitochondria and fusion/fission events can also have an effect on cell cycle progression in these cells?

    Again, this is a very interesting point. What we think is that the changes in DRP1 activity facilitates the removal of damaged cells, rather than committing normal cells to death. In this way, cells that may be moderately damaged would not be removed in the naïve pluripotent state, but if similar levels of damage occur upon the onset of differentiation this causes their rapid elimination. We hypothesize that one reason for this may be to do with the proliferation rates of pre and post-implantation embryos. In pre-implantation embryos the cell cycle length is in the order of 16 hours, but in the early post-implantation epiblast it can be between 3-6 hours, allowing for the rapid replacement of damaged cells and therefore making this replacement more cost-effective.

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