PROJECT SUMMARY Neural crest progenitors are essential for development of peripheral ganglia in the autonomic nervous system. Much is known about processes that control differentiation of neural crest progenitors for many autonomic ganglia. However, very little is known about formation of cell lineages within pelvic ganglia. Regulatory processes that control neurogenesis, differentiation, diversification, and maturation of pelvic autonomic neurons are undefined. Pelvic ganglia play essential roles in initiating bladder contraction and mediating efficient emptying. Thus, studies of pelvic neurons will inform understanding of urinary tract conditions that affect a large portion of the population. In humans, pelvic autonomic neurons are scattered primarily in the inferior hypogastric plexus, while in rodents pelvic neurons are aggregated into major pelvic ganglion situated alongside the lower urinary tract (LUT). Our prior LUT studies in mice identified expression of the transcription factor Pax3 in fetal and postnatal pelvic ganglia when pelvic autonomic neurons are differentiating and maturing. Because Pax3 is widely expressed during development, mutations in this gene are typically lethal due to neural tube defects. We generated novel lines of mice that have loss of Pax3 in neural crest lineages. These animals are postnatal viable and exhibit deficits of bladder wall innervation with altered voiding patterns. Neural-crest restricted Pax3 mutants offer an exciting opportunity to identify key regulatory aspects of pelvic ganglia formation and determine how deficits of pelvic autonomic neurons relate to LUT dysfunction. In this study we focus on postnatal stages of pelvic ganglia maturation to test the following hypotheses: 1 – Pax3 is essential for producing the normal allocation of neuron types as the mouse major pelvic ganglion matures postnatally. 2 – neural crest-specific loss of Pax3 reduces total numbers of pelvic autonomic neurons at maturity. Use of single-cell sequencing and high-resolution large-scale microscopy will be applied to assess the final composition of pelvic ganglia in postnatal Pax3 mutants compared to normal littermates. Research outcomes will provide greater understanding of how alterations in pelvic innervation contribute to LUT dysfunction.