Project Summary/Abstract The equilibrium between stem cell self-renewal and differentiation is a cornerstone of tissue health. Stem cells must maintain healthy, heterogeneous stem cell pools throughout the lifetime of the animal, while also producing the differentiated daughter cells necessary for optimal tissue function. Controlled shifts mediated by changes in the specific signals that promote self-renewal versus differentiation may be leveraged for tissue repair after injury or prevention of aging symptoms. In contrast, continuous imbalance can lead to aberrant states such as tumor formation when self-renewal is favored, or stem cell loss when differentiation is the primary outcome. Defining the molecular mechanisms that determine stem cell fate is therefore a pressing need. Here, we investigate the possibility that epithelial Follicle Stem Cells (FSCs) in the fly ovary utilize classical neurotransmitter signaling to dictate self-renewal versus differentiation fate decisions. In recent work, we demonstrated that axon-like projections extend from FSCs in response to feeding, forming interactive webs that span the niche. Disruption of projection growth and interactions via mutation of the axon regulators still life (sif, TIAM-1), and sickie (NAV2) leads to developmental defects and, importantly, disrupts the balance of cell fate markers that instruct self-renewal or differentiation. Projection growth depends on Hedgehog (Hh) signaling, with the transcriptional activator Cubuitus Interruptus (Ci) necessary for extension. Reasoning that Ci transcriptional targets might thus be critical for mediating communication between FSC projections and their targets (other FSCs or germ cells), we defined the gene expression changes that occur in FSCs at 3 hours timepoints after feeding. The proposed work focused on a highly enriched set of genes with known functions at neuromuscular junction synapses, and the model that FSC fate is determined by the balance of GABA versus glutamate signaling. The idea that non-neuronal stem cells communicate via neurotransmitter signaling may be applicable to other stem cell systems, emphasizing that our precise molecular dissection of the spatio-temporal function of candidate regulators may uncover a new fundamental mechanism for regulation of stem cell fate.