Project Summary/Abstract Sexually reproducing animals perform innate sexually dimorphic behaviors that are important for their reproductive success. Sex differentiation of the brain and behavior is established through the action of master regulators that work downstream of sex determination factors. In mammals, nuclear hormone receptors fill this role; in insects, neural sex differentiation is mediated by the male-specific transcription factor Fruitless (FruM). The importance of these master regulators in sexual differentiation of the brain and behavior is well established. However, how a subset of neurons become specified for their expression and function is poorly understood. To explore this, we use the Drosophila melanogaster as a model. In both sexes, fruitless is transcribed in a subset of neurons that wire together into sexually differentiated circuits regulating social behaviors. However, FruM protein is only translated in the male. Most D. melanogaster central brain neurons are born from progenitor cells that produce neurons in a pair-wise fashion, producing two distinct hemilineages. Diverse fruitless neurons are contained in at least 60 different hemilineages that make up the D. melanogaster central brain along with sister cells that do not express fruitless. This suggests that hemilineage identity is an important factor for the specification of fruitless transcription and for the impact of Fruitless protein. Using scRNA-seq to study fruitless expressing neurons in the context of their neuronal hemilineages, I identified the nuclear receptor dissatisfaction as being enriched in the entire hemilineage that contains the fruitless-expressing mAL neurons (Medially located, just above Antennal Lobe), including sister neurons that do not express fruitless. Interestingly, dissatisfaction has previously been shown to be important for sex-specific behaviors and my preliminary data also shows it is important for the overall development of this hemilineage. Here, we will test how Dissatisfaction impacts development of this hemilineage and how this sex-shared factor impacts the sex differentiation process in circuits which control social behaviors. The work proposed in this application will elucidate how a neuron’s lineage origin interacts with the sex differentiation process to specify its circuit function during development. This F99/K00 proposal describes the training and mentorship the applicant will acquire to help transition her to an independent research position at an academic institution. The applicant will receive the necessary training and mentorship from her sponsor and co-sponsor to successfully complete the experiments outlined in this proposal and to transition her to a strong postdoctoral lab. She will also have additional support from her thesis committee members, the wider neuroscience University of Michigan community and from experts from other institutions. This proposal also outlines additional training in scientific research...