PROJECT SUMMARY/ABSTRACT A fundamental question in cell biology is how cells measure and maintain their characteristic sizes. We use two systems in Drosophila development to study cell size control that provide a natural system for uncoupling growth and division: embryogenesis and oogenesis. The early embryo is an extremely large cell that undergoes rapid divisions without cell growth, while the oocyte uses polyploid nurse cells to grow to a massive size without dividing. In the embryo, the final cell size is determined by the nucleus to cytoplasm ratio (N/C ratio). The N/C ratio controls a major developmental transition known as the mid-blastula transition (MBT) where the cell cycle stops and zygotic transcription initiates. Recently, we discovered a surprising mechanism for N/C-ratio sensing in the pre-MBT embryo. Hyper-abundant maternally provided histone H3 acts as a competitive inhibitor of the DNA-damage checkpoint kinase, Chk1, to prevent cell cycle slowing. As more and more nuclei are generated by the successive divisions the pool of “free” (ie-not chromatin-incorporated) H3 is imported into the increasing numbers of nuclei and then incorporated into chromatin thereby releasing Chk1 inhibition to allow cell cycle slowing once a threshold N/C ratio is reached. In oogenesis, polyploid nurse cells generate the maternal supply of materials required for the egg and “dump” their contents into the oocyte to achieve the correct volume. Histone biogenesis appears to play a role in regulating progression through oogenesis as well, though the molecular mechanism is unclear. Over the next five years, work in this R35 MIRA proposal will: 1) interrogate the molecular mechanisms by which maternally provided H3 contributes to cell size sensing at the MBT; 2) understand how the N/C ratio affects nuclear and chromatin composition leading up to the MBT; and 3) extend the lab’s current models of cell size sensing to the growing egg chamber. These projects will further the long-term goal of understanding cell size and cell cycle control in a diverse array of tissue types and developmental timepoints. The resulting insights will expand our understanding of fundamental processes shared by most living cells but that are obscured in other model systems by the tight coupling between cell size and cell cycle progression.