DESCRIPTION (provided by applicant): Decades of research have demonstrated the importance of serotonin (5-HT) signaling for the appropriate development and survival of neuronal cell populations throughout the central and enteric nervous systems. Expression of 5-HT4 receptor is necessary for neural crest (NC)-derived enteric neurons to differentiate, proliferate, and renew during postnatal development. In addition, 5-HT1 receptor expression promotes development of thalamocortical neurons in the fetal forebrain in both mice and humans. Despite thorough investigation of 5-HT signaling in these areas, there have been very few studies inspecting the role of 5-HT in development of other peripheral nervous system components. Yet, there are several lines of evidence pointing to a fundamental role for 5-HT signaling in the development and maintenance of the autonomic innervation of the lower urinary tract (LUT). Public gene expression databases indicate expression of the 5-HT3 receptor in the fetal mouse LUT. Our own preliminary work validates and extends this finding by identifying expression of the 5HT3A and 5-HT3B receptor subunits in NC-derived neuronal progenitors. The physiological relevance of 5-HT signaling in the LUT is confirmed by knock-in studies in mice. Mutant mice expressing a hypersensitive form of the 5-HT3A receptor gene, Htr3a vs/vs, display a hypertrophic bladder phenotype in conjunction with urinary incontinence. Finally, intriguing clinical data has shown profound success in treating stress and urge urinary incontinence in postmenopausal women using serotonin and norepinephrine reuptake inhibitors (SNRIs). These findings prompt an investigation of the role of 5-HT signaling in the development and maintenance of LUT innervation. The hypothesis that 5-HT signaling promotes the survival, differentiation, and balance of lineages derived from NC-derived progenitors innervating the LUT will be thoroughly tested in three specific aims. In Aim 1, the spatial, temporal and cell-type specific patterns of 5-HT3 receptor subunit expression during the development and maturation of LUT autonomic innervation will be determined via in situ hybridization and immunohistochemical localization. In Aim 2, the role of the 5-HT3A receptor in the neural development of the LUT will be defined. Migratory patterns, survival and balance of neuronal lineages in the context of Htr3a loss will be investigated using an Htr3a knockout (Htr3a-/-) mouse model crossed to a transgenic Htr3a-EGFP fluorescent reporter. In Aim 3, alterations in gene expression during fetal LUT neurogenesis that occur as a result of Htr3a loss will be determined by flow-cytometric isolation of NC-derived neuronal progenitors from the LUT. Gene expression within Htr3a positive populations will be quantified by RNA-Seq analysis and differential gene expression validated by quantitative PCR. Elucidating the processes of neurogenesis in the development of the LUT is critical for identifying disease mechanisms and developing novel therapeutic strategies for the treatment of deficits of autonomic innervation and urinary incontinence.

The aim of this proposal is to use mouse model systems to define the role of serotonin in neurogenesis within autonomic ganglia of the lower urinary tract. The results of the experiments proposed will fill large gaps in our knowledge about serotonin's role in the development of peripheral innervation outside the central and enteric nervous systems. Our findings will define mechanistic processes that are essential to peripheral neurogenesis and will advance efforts to compensate for deficiencies of lower urinary tract function such as the urinary incontinence that occurs during ageing or as a consequence of peripheral nerve damage.