SUMMARY The mammalian target of rapamycin complex 1 (mTORC1) signaling pathway regulates cell size and growth and is frequently mutated in disease, including in a class of neurodevelopmental disorders known as “mTORopathies.” One such disorder, Tuberous Sclerosis Complex (TSC), affects nearly 1 in every 6,000 newborns and is characterized by the growth of benign tumors throughout the body. TSC is caused by an inactivating mutation in the genes that encode the negative regulators of mTORC1, leading to protein loss of function, hyperactivation of mTORC1, and increased cell proliferation through phosphorylation of ribosomal protein S6 (p-S6) and eukaryotic translation initiation factor 4E-binding protein 1 (p-4EBP1). Twenty percent of TSC patients develop a large tumor that preferentially presents near the ventral region of the ventricular- subventricular zone (V-SVZ), the largest neural stem cell niche in the adult brain. Recent studies of neural stem cells in the V-SVZ revealed variable transcriptional and functional capabilities corresponding to a cell’s position along the dorsoventral axis of the V-SVZ, including differential activation of mTORC1 and susceptibility to TSC tumor formation. Further, different populations of neural stem cells in the niche are mitotically active at different times throughout pre- and postnatal neural development. Postnatally, mTORC1 has been shown to be important in regulating neural stem cell quiescence, but mTORC1 signaling in the prenatal V-SVZ and its effect on quiescence in embryonic neural stem cells has not been investigated. The goal of this project is to determine the developmental stage when differential mTORC1 activity emerges along the dorsoventral axis of the V-SVZ and the extent to which dysregulated mTORC1 signaling alters cell fate. The central hypothesis of this project is that levels of mTORC1-dependent p-4EBP1, but not p-S6, determine both a prenatal neural stem cell's mitotic activity and susceptibility to TSC tumor development. To test this hypothesis, an inducible mouse model and pharmacologic agents will be used to manipulate mTORC1 signaling during embryogenesis. To map the emergence of differences in mTORC1 activity in healthy and disease states, per-cell levels of mTORC1-dependent phosphorylation events will be quantified via imaging and flow cytometry analyses of embryonic neural stem cells. To compare results across species and platforms, mTORC1-dependent signaling will be quantified in cerebral organoid models grown from induced pluripotent stem cells derived from TSC patients. Through this project, I will build upon my prior training to evaluate cell signaling and differentiation in human and mouse models of disease. Results of this work will determine the role of mTORC1 in regulating prenatal neural stem cell fate in health and disease. Clinically, this work will provide a greater understanding of the dysregulated signaling mechanisms that lead to perinatal development of tumors in the...