Periodic Reporting for period 3 - StemCellHabitat (Metabolic and Timed Control of Stem Cell Fate in the Developing Animal) Reporting period: 2021-02-01 to 2022-07-31 Summary of the context and overall objectives of the project Stem cells are undifferentiated cells capable of dividing several times to self-renew and to generate more specialized cells essential for tissue, organ and ultimately whole organism formation. Stem cells exist not only in embryos but also in adults, where they are mainly involved in tissue homeostasis and repair. Stem cells thus harbor a great potential for regenerative medicine as they are potentially great sources of new specialized cells. Although several aspects of stem cell biology are understood it is still not fully known how stem cells are directed to generate specific differentiated cells or how to efficiently regulate stem cell proliferation. This project aims at studying how stem cells are normally regulated during animal development according to their spatial, temporal and metabolic identity to determine their proliferation and the type of differentiated cells formed. To answer these questions, this project uses Drosophila melanogaster, an animal complex enough to be similar to higher eukaryotes and yet simple enough to dissect the mechanistic details of cell regulation and its impact on the organism. Drosophila has several stem cell populations all dynamically regulated during development and is thus a fantastic model to study stem cells. Using a multidisciplinary approach combining genetics, cell type/age sorting, multi-omics analysis, fixed and 3D-live stem cell imaging and metabolite dynamics, this project proposes an integrative approach to investigate how stem cells are regulated in the developing animal. Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far During the first half of this project we have found that developmental cues, in particular from hormone signaling are essential triggers to instruct neural stem cells to stop proliferating and therefore to terminate neurogenesis. We have importantly found that hormonal signalling is mainly required to regulate systemic development which includes remodelling of other organs that then signal the brain and neural stem cells.At the cell autonomous level, we have found that neural stem cell differentiation requires differential regulation of basal transcription. We have also identified many novel candidate regulators of neural stem cell fate and neuronal formation that will now be further analysed.Stem cell fate has been described to be intricately connected with energy metabolism. Although energy metabolism occurs in several cellular compartments, it is in the mitochondria that an important fraction of all metabolic reactions occurs, including oxidative phosphorylation. We have explored how the metabolic regulation of stem cell fate is coordinated with mitochondrial activity and dynamics. We have found that mitochondrial dynamics in the Drosophila ovary regulates germ stem cell number, cell fate and female fertility. Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far) We have interestingly discovered that neural stem cells in Drosophila have the intrinsic capacity to proliferate indefinitely as long as they are kept in the animal but insensitive to developmental cues. This opens interesting research avenues in terms of their practical usage.We have also found many novel regulators of neural stem cell fate and neural fate commitment, which we will now explore. It is our expectation to have a global molecular characterisation of the transcriptional and metabolic mechanisms involved in fate regulation during brain development. Developing Drosophila Brain-Neural stem cells in red, Neurons in blue, Neural lineages in green.