Mitochondrial Dynamics in the Control of the Pluripotent States

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At the beginning of development of an embryo, cells start off with the potential to give rise to all the cell types and tissues that will form the newborn organism. This potential is called pluripotency, and those cells that are pluripotent hold great promise for regenerative medicine. This promise is not only due to their future clinical applications in cellular therapies, but also because now patient specific pluripotent cells can be generated for "disease-in-a-dish" approaches to understand human diseases. Stem cell therapies are being developed for diseases ranging from diabetes to neurodegenerative disorders and a stem cell based mechanism is thought to underlie different forms of cancer. Over the last few years considerable efforts have been invested into identifying the signalling and gene regulatory networks that regulate the pluripotent state and control the first steps of differentiation, when cells start to acquire a lineage specific identity. These studies have identified the key molecular features that provide pluripotent cell identity. In contrast to this, we know relatively little about the cell biology changes that accompany exit from the pluripotent state, for example how the different organelles of the cell adapt to the different environments they encounter during the first stages of differentiation. Of particular interest are mitochondria, that are not only the powerhouse of the cell, but also regulate a diverse range of cellular processes, including the ability of the cell to respond to cell death signals, their potential to differentiate, as well as how they respond to the different signalling pathways that regulate cell identity and function.We have identified that during the first stages of differentiation, cells undergo a dramatic change in the morphology of their mitochondria, suggesting that mitochondrial dynamics change during exit of pluripotency. Additionally we have found that the first stages of differentiation, cells alter their metabolism and their response to cell death stimuli, becoming hypersensitive to death signals. In this proposal we will investigate how mitochondrial dynamics impact of these changes. We will do this by using a combination of experiments performed in pluripotent mouse stem lines and mouse embryos, that allow us to readily determine the in vivo relevance of our data, as well as human embryonic stem cell models, that provide us with insight of the conservation in human of our findings. Specifically we will do three things.In the first place we will study in detail, at the ultrastructural level, the precise changes that occur in mitochondrial morphology as cells exit the pluripotent state. We will do this by analysing the distribution of mitochondrial proteins in pluripotent and differentiating cells, as well as by imaging mitochondrial dynamics upon exit of pluripotency. We will then manipulate mitochondrial dynamics and study how this affects cell identity. For this we will study three cellular processes that are likely to be affected by mitochondrial changes, the cells metabolism, it's ability to respond to cell death stimuli, and its potential to differentiate into the different lineages that will form the embryo. Finally, we will exploit the significant knowledge of the signalling pathways that maintain pluripotency and that drive differentiation, to identify novel signalling inputs that regulate mitochondrial dynamics and function in stem cells. Together, these experiments will provide a unique insight into an essential, but understudied aspect of early embryonic development, the dynamics of the mitochondria, as well as addressing how they impact on the process of differentiation.