E Michelle Southard-Smith - US grants

DESCRIPTION (provided by application): Hirschsprung disease (HSCR) is a complex genetic disorder. The primary clinical feature, aganglionic megacolon, arises as a consequence of anomalies in development of the enteric nervous system and subsequent absence of enteric ganglia. Family members carrying identical gene mutations often exhibit differences in penetrance and length of gut lacking enteric neurons. This variation suggests that - like many human genetic diseases - multiple genes modulate HSCR penetrance and severity. Mouse models of HSCR have successfully identified genes that participate in enteric nervous system pathology. At present five genes are known to contribute to the etiology of human HSCR cases. Four additional genes can contribute to aganglionosis based on phenotypes in the mouse models. Collectively, these genes account for no more than 30 percent of total HSCR cases. The significant challenge remains to identify additional genes and genetic interactions that account for the remaining spectrum of HSCR cases. Four of the known HSCR susceptibility loci are up-regulated in neural crest stem cells (NCSC) indicating that HSCR is a consequence of defects in NCSC function. SoxW is highly expressed in NCSC and is deficient in a subset of HSCR cases. Sox10[unreadable][unreadable]m mice recapitulate aganglionosis and other extra-intestinal autonomic deficits observed in HSCR patients. Our prior studies of SoxW have established that genetic background impacts severity of intestinal aganglionosis. We have successfully localized five modifier loci of Sox1(P[unreadable]m that affect penetrance and severity of aganglionosis. Two of the genes that underlie these modifier loci have been definitively identified. The goal of the proposed studies is gain a more complete understanding of the genetic underpinnings of aganglionosis by identifying additional genes and the gene interactions that impact this disease. The experimental strategy will increase our understanding of pathways essential for enteric development and pathogenesis of gastrointestinal dysmotility syndromes. Aim 1 will use genome-wide analysis of a large F2 cohort and an extended FT backcross pedigree of Sox10[unreadable][unreadable]m mice to identify refined intervals of aganglionosis modifiers and define genetic interactions that impact severity and penetrance of this phenotype. Aim 2 will define Sox10[unreadable][unreadable]m phenotype variation across multiple inbred strains and identify shared haplotypes that alter aganglionosis by quantifying extent of enteric deficits in FT progeny derived from Sox10[unreadable]om congenic lines. Genome-wide haplotype association will identify genetic intervals of previously undetected Sox70 modifiers shared between strains. Aim 3 will establish mechanisms of SoxW modifier interaction by transcriptional profiling of enteric NCSC in SoxWP[unreadable]m congenic lines during critical stages of neural crest migration within the developing gastrointestinal tract. Our analysis will reveal the complex genetic architecture of HSCR, establish how signaling pathways in NCSC interact to produce aganglionosis at the organismal level, and identify potential therapeutic targets in Gl dysmotility syndromes.

DESCRIPTION (provided by the applicant): Hirschsprung disease (HSCR) is a complex genetic disorder whose primary clinical feature is the absence of intrinsic ganglia in the distal intestine. Defects of sympathetic and parasympathetic function have also been described in HSCR patients. Mutations in any one of several genes can give rise to the peripheral nervous system (PNS) deficits of this disorder. Incomplete penetrance and variable severity of PNS defects, exhibited by HSCR family members carrying identical gene mutations, suggests that genetic background modulates disease severity. Sox10dom mice display all the features of aganglionosis and extra-intestinal autonomic deficits described in HSCR patients. Sox10dom mice have been extremely valuable for defining the role of the Sox 10 transcription factor in development of neural crest (NC) that contributes to the PNS. Our analysis of Sox10dom mice in F1-hybrid and congenic strains demonstrates that genetic background impacts the severity of aganglionosis. The phenotypic variation we observe mimics that seen in human HSCR sibs, and suggests that modifier loci influence development of Sox10 derivatives in mouse and man. In the proposed experiments, we will test the hypothesis that multiple modifier loci contribute to the severity of autonomic defects in Sox10dom mutants. To systematically localize all genomic regions that modulate the severity of aganglionosis, we will perform a genome survey of Sox10dom intercross progeny. Selective genotyping of progeny at the phenotypic extremes will be performed to establish linkage to loci that influence HSCR severity. Our preliminary analyses in candidate gene approaches have identified a major modifier of enteric development, and suggest that additional modifiers contribute to the severity of NC defects. We will use extended pedigrees of Sox10dom mice for fine-mapping through intervals of candidate loci and modifiers detected by genome survey. Intervals that associate with severity of phenotype will be investigated in our pedigrees with genotyping panels, derived from mouse genome sequence that can be posted as a shared resource for the genetics community. To investigate the relevance of enteric NC modifiers to autonomic phenotypes outside the gut, we will perform a comparative analysis of sympathetic and parasympathetic function in Sox10dom congenic lines. These studies will contribute to our knowledge of the genetic basis for variation in development and disease of autonomic phenotypes in the PNS.

DESCRIPTION (provided by applicant): Congenital gastrointestinal motility disorders arise as a consequence of anomalies in development of the enteric nervous system (ENS). While progress in defining the regulatory molecules and signaling pathways that govern ENS development has come from studies of spontaneous and engineered mouse mutants, our understanding of neuronal and glial lineage divergence in this system is still rudimentary. Human and mouse mutations demonstrate that Sox10 is critical for the neural crest (NC) progenitors that populate the ENS during development as haploinsufficiency or dominant negative forms of this transcription factor lead to aganglionic megacolon. However, little is known regarding the specific ENS cell types that derive from Sox10 precursors. Fundamental insight into the processes of ENS development and the complex cell interactions that can produce motility disorders may arise from detailed analysis of Sox10 NC derivatives. To evaluate the role of Sox10 within the context of normal ENS ontogeny, conventional gene disruption analyses that result in haploinsufficiency must be avoided. We propose to develop and apply transgenic approaches that harness Sox10 regulatory regions in conjunction with detailed immunohistochemical studies to determine the relationship of Sox10 to cell lineage divergence in the ENS. By comparative genomic sequence analysis we have identified potential cis-regulatory regions scattered throughout the Sox10 locus. Our initial analysis of 5' flanking sequence indicates that regulatory elements either within introns or at a distance from the gene must be required in vivo to recapitulate normal Sox10 expression. To generate tools for investigation of cell lineage pathways in the ENS, we will use BAC modification to localize the regulatory regions that confer expression of Sox10 in enteric NC. Modified Sox10 BACs driving expression of inducible CRE reporters will be deployed in transgenic mice as lineage tracing tools to investigate the relationship of Sox10 to divergence of neurons and glia in the ENS. Coupled with Sox10 immunohistochemistry (IHC) during embryonic migration and differentiation of enteric NC, these studies will construct a detailed picture of ENS lineage divergence. Our analysis will pioneer exploration of cell lineage pathways in the ENS and provide essential reagents for future functional analysis of Sox10 and other genes with potential relevance for ENS development. This proposal represents a distinct divergence from the PI's main research focus and qualifies for consideration under the "Innovative and Exploratory Research in Digestive Diseases and Nutrition" Program Announcement.

[unreadable] DESCRIPTION (provided by applicant): Neural innervation determines appropriate contractility of the detrusor muscle in the bladder. When appropriately innervated, the bladder is compliant and can hold variable volumes of urine at low pressures. Failure of the bladder to modulate internal pressures results in transfer of high pressures to the kidney with subsequent glomerular damage; thus, patients with neurogenic bladder disease are at high risk for renal failure and renal transplantation. Spina bifida is the most common etiology of pediatric neurogenic bladder. The association between neural tube defects and bladder dysfunction as well as the neural crest (NC) origin of sympathetic and parasympathetic inputs that innervate the bladder implies that NC derivatives are essential participants in normal bladder development. Anatomical and immunohistochemical studies of bladder innervation have identified neural components and some of the neurotransmitter subtypes that are present in late fetal and postnatal development. However, a comprehensive understanding of bladder cell types that derive from NC is lacking. Identification of NC lineages and genes that control their differentiation within the bladder is of significant relevance to understanding the etiology and potential treatments for neurogenic bladder. In vitro neural crest stem cells give rise to neurons, glia and myofibroblasts. A critical question that remains to be addressed is whether only bladder innervation is NC-derived or if neural crest stem cells also contribute to additional lineages in this organ. Because deficiencies of bladder smooth muscle have been reported in fetuses with myelomeningocele, the relationship between NC and smooth muscle differentiation is of particular interest. We will test the hypothesis that multiple lineages within the bladder are NC-derived by analysis of engineered mouse models throughout ontogeny of the bladder. We have implemented BAG recombination methods that enable tracing of neural crest stem cells expressing SoxlO. Aim 1 will establish a temporal profile of NC migration into the bladder and define co-localization of lineage markers in the bladder with SoxlO transgene expression. Aim 2 will comprehensively identify NC- derived cell types in the bladder by implementing Cre-/oxP fate mapping. In Aim 3 we will capture neural crest stem cells from the developing bladder to define temporal changes in developmental potential and identify transcriptional profiles specific to sacral NC. Aim 4 will define mechanisms of altered NC development in myelodysplastic bladders of mouse spina bifida models. Our analysis will pioneer exploration of NC lineages pathways in the bladder, identify candidate genes for future analysis of disease alleles in neurogenic bladder patients and begin to define mechanisms of altered NC development in spina bifida mouse models. The proposed studies directly address multiple needs for basic research in bladder development as stated in the "Strategic Plan for Pediatric Urology" and are responsive to the objectives of the "Basic Research in the Bladder and Lower Urinary Tract" Program Announcement. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): This application addresses broad Challenge Area (11) Regenerative Medicine and Specific Challenge Topic 11-DK-103 Organ Innervation. Anatomical and immunohistochemical studies of bladder innervation have identified neural components and some of the neurotransmitter subtypes that are present in late fetal and postnatal development. The association between neural tube defects and bladder dysfunction as well as the neural crest (NC) origin of sympathetic and parasympathetic inputs that innervate the bladder indicates that NC derivatives are essential participants in normal bladder development. Yet, our understanding of how bladder innervation develops and the signaling pathways that control migration of NC-derived progenitors into the bladder are still rudimentary. Moreover, fundamental information about development of innervation associated with the urethral sphincters that control release of urine from the bladder is lacking. Identifying trophic molecules and signaling mechanisms that control migration of NC into the lower urinary tract (LUT) and regulate the differentiation of these progenitors upon arrival is an essential prelude to therapeutic approaches seeking to restore LUT neural components damaged by surgical interventions or innervate tissue-engineered bladder scaffolds in patients. Until recently, isolation of NC-derived cells from the bladder wall to discern their patterns of gene expression was not feasible. Now we have developed unique transgenic lines of mice to enable imaging, isolation and characterization of these multipotent progenitors. As a result we have established the developmental timing and routes of migration taken by neural progenitors that populate the lower urinary tract. Our studies reveal critical transition points in this developmental process at which NC progenitors traffic into the urogenital sinus and then pause in their migration, as if awaiting signaling events that draw them into specific locations. Multiple examples of differential expression between NC populations have been reported that specifically traffic these progenitors into distinct areas of the developing embryo. It is possible that signaling molecules essential to development of other aspects of the peripheral nervous system may also play a role in development of innervation in the LUT. However it is already well known that axial level along the neural tube is a major factor in determining the migratory properties, developmental potential and ability of NC-derived lineages to colonize different regions of the embryo. Thus, it is highly likely that sacral NC progenitors that populate the LUT and give rise to pelvic ganglia and neural components of the bladder and urethra express a unique complement of receptors and signaling molecules that guide the phases of their migration. We propose to capture NC-derived progenitors at discrete transition points during their migration into the LUT and define their gene expression profiles. This approach will circumvent the technical challenge of the scarcity and dispersed distribution of NC-derived progenitors within tissues has previously hampered efforts to identify their gene expression signatures within organs. Comparative bioinformatics between NC cell populations isolated from distinct transition phases during LUT innervation will rapidly identify signaling pathways that participate in development of the resulting neural components. To provide temporal and spatial information relevant to LUT innervation we will generate in situ hybridizations of candidate genes that are likely effectors of LUT innervation based on their roles in other aspects of peripheral nervous system development. Gene expression patterns derived by in situ hybridization will complement the microarray work and identify key trophic factors and guidance molecules during the critical stages when NC progenitors traffic into the LUT. Our overall goal is to establish a gene expression resource that will provide rational for clinical efforts to restore function to damaged pelvic neural elements or tissue- engineered bladder substitutes. The proposed studies will generate a comprehensive resource of gene expression patterns that can be mined by investigators interested in urogenital tract development and disease. Our efforts will propel the field of LUT innervation development forward and make a significant impact on biomedical research that seeks to restore bladder innervation across a range of clinical settings, from pediatric patients with spina bifida to patients suffering pelvic neural damage secondary to surgical manipulations. PUBLIC HEALTH RELEVANCE: The studies proposed aim to identify essential genes that control development of nerves in the lower urinary tract that regulate bladder control and sexual function. These studies are important for understanding how these nerves normally develop and for deriving technologies that will restore neural function in urogenital birth defects or after pelvic surgery. This proposal is in response to the broad Challenge grant area of Regenerative medicine and meets multiple needs for basic research in development lower urinary tract innervation.

DESCRIPTION (provided by applicant): Purchase of an Episcopic Fluorescence image Capture System (EFIC) from Vashaw Scientific is proposed. This instrument will serve the research programs of four major users distributed among several departments on campus. The development of new EFIC projects is also anticipated from additional investigators across the strong, largely NIH-funded life sciences community at Vanderbilt. The ability to pursue EFIC imaging studies has not previously been possible for the vast majority of investigators at our institution because the only other instrument with these capabilities in the United States is located in the laboratory of Dr. Cecilia Lo at the National Heart Lung and Blood Institute on the main NIH campus in Bethesda, Maryland. Dr. Lo has generously worked with us to establish preliminary data for two of the major users on this application (Southard-Smith &Baldwin). Use of an off-site facility on a collaborative basis is sufficient for obtaining preliminary data to demonstrate feasibility and value of this methodology but cannot support specific well-defined research projects even for a limited number of investigators. Moreover, some samples are best imaged immediately after tissue harvest. As a consequence, the requisite coordination of embryos, embedding, transportation, and immediate sectioning becomes prohibitive for routine off site analysis. The availability of a local EFIC facility combined with a ready mechanism to advertise the resource and image potential to interested colleagues through the Program in Developmental Biology that spans multiple departments will have a broad positive impact on NIH-sponsored basic and translational research at our institution. The availability of an EFIC system will mean a dramatic leap forward in the ability of medical and biological researchers at Vanderbilt University to comprehensively address questions about anatomical distributions of specific cell types and morphological alterations that arise in disease states. The availability of EFIC on campus will significantly enhance the productivity of the research groups in the Vanderbilt life sciences community, which is largely devoted to studies of human health and development of therapeutic strategies to combat disease. PUBLIC HEALTH RELEVANCE: Installation of an Episcopic Fluorescence Image Capture (EFIC) system will allow scientists to visualize in 3-D the complex shape changes that occur during formation of organ systems like the heart, urogenital tract and skeleton. The ability of the EFIC system to image based on either tissue auto fluorescence or gene expression patterns makes it feasible to investigate the effects of specific gene changes or environmental insults on the developing embryo. Understanding the outcomes of such insults will lead to greater understanding of the causes and alternate therapies for birth defects and human disease.

DESCRIPTION (provided by applicant): Gliogenesis and maintenance of glial cell types are critical to development and function of the nervous system. Sox10 is a developmental transcription factor that is essential for development of multiple glial lineages including oligodendrocytes in the central nervous system as well as neural crest-derived Schwann cells, satellite glia and enteric nervous system neurons and glia in the periphery. Investigating Sox10 function in these distinct populations has been difficult because simple gene knockouts cause complete loss of gene expression in early neural crest progenitors resulting in embryonic lethality. Efforts to temporally induce loss of Sox10 have been hampered by kinetics of mRNA and protein decay. In the context of the R03 mechanism we propose generation of a COnditional INducible (COIN) dominant negative allele of Sox10 in mice as a novel tool for analysis of gene function. Specific Aim 1 will generate mice bearing a COIN cassette in the Sox10 locus that upon Cre action results in expression of a fluorescently tagged dominant negative Sox10 isoform. Specific Aim 2 will define the effects of the Sox10COIN allele on oligodendrocyte and enteric neural crest-derived lineages before and after COIN inversion. The ability to conditionally disrupt Sox10 expression and function in distinct populations will significantly impact the field by opening avenues for analysis of developmental mechanisms that are relevant for directed differentiation of progenitors cells to treat central and peripheral neuropathies.

Our goal is to reveal developmental and genetic mechanisms that control formation of cell types from the neural crest (NC) in the lower urinary tract (LUT). NC progenitor cells form pelvic autonomic ganglia and peripheral glia along nerve fibers that innervate all aspects of this organ system. Appropriate innervation of the bladder and urethra are required for normal bladder contractility and urinary continence. Failure to regulate normal urine pressures within the bladder, as occurs in neurogenic bladder, predisposes to kidney failure when high pressures damage glomeruli. In children, Spina bifida (SB) and spinal dysraphism are the most common causes of neurogenic bladder dysfunction, with patients exhibiting deficits of bladder wall nerves and muscle. The association between neural tube defects and bladder dysfunction, as well as the NC origin of sympathetic and parasympathetic inputs that innervate the bladder, implies that NC derivatives are essential participants in normal bladder development. In vitro neural crest stem cells are capable of generating neurons, glia and myofibroblasts. However, a fate map of the LUT with cellular resolution that relates mature NC-derived cell types to structures within this organ system does not yet exist. Thus it remains to be seen whether only pelvic autonomic neurons and glia are NC-derived or if neural crest stem cells also contribute other essential cell types to the LUT. Alternatively, it is possible that NC-derived progenitors exert inductive effects on developing muscle in the LUT analogous to inductive mechanisms in cardiac and thymus development. We will test the hypothesis that multiple lineages within the bladder are NC- derived and are required for normal bladder development through analysis of engineered mouse models. Aim 1 will derive a comprehensive fate map of NC-derived lineages in the LUT. Aim 2 will define mechanisms of altered NC development in a mouse SB model that mimics LUT deficiencies seen in SB patients. Aim 3 will target temporal and tissue specific ablation of Pax3 to establish lineage requirements for this gene in directing NC progenitors as they populate the bladder. Our analysis will pioneer exploration of NC lineages in the bladder and identify altered developmental processes in a mouse model of SB that are relevant for understanding the etiology of bladder disease in SB patients.

DESCRIPTION (provided by applicant): Recurrent pain of the bladder, pelvis or urogenital floor is a defining feature of chronic pelvic pain syndromes including Interstitial Cystitis and Chronic Prostatitis. The causative pathology of these disorders is not known despite efforts to identify disease markers. The cell bodies of pain- sensing neurons, nociceptors, that innervate the lower urinary tract reside in the dorsal root ganglia amidst many other sensory neurons and can develop inappropriate activity following inflammation and infection. How these processes lead to altered sensitivity of nociceptors and set the stage for chronic pain is not completely understood. Throughout the nervous system serotonin (5-HT) exerts a wide range of effects on neurogenesis, differentiation, and maintenance of neurons and can act as an inflammatory neuromodulator. Signaling through serotonin receptors (5-HTRs) has been shown to modulate nociception and contributes to sensitization of the sensory neurons. The effects of signaling through 5-HTRs in the lower urinary tract have not been investigated in detail because even the cell-type specific distribution of these receptors has not been evaluated in this system. This research proposal aims to generate fundamental knowledge that will increase understanding of nociceptive processes in the bladder and urethra by identifying the distribution of 5-HTRs in pelvic innervation during development and maturation of the lower urinary tract. Transcriptional profiles of serotonergic lumbar and sacral sensory neurons will be derived to obtain a molecular fingerprint of the pelvic sensory neurons that express 5-HTRs. The results will establish a framework so that role of 5- HT signaling in the urogenital tract may be related to other nociceptors. To further elaborate links between 5-HT signaling and nociception, we will generate transgenic reporter mice that express multi-cistronic fluorescent reporter proteins in nociceptive sensory neurons. Multi- spectral transgene expression will enable simultaneous localization of cell nuclei, splasma membrane, and fluctuations in intracellular calcium within nociceptors. These reporters will enable direct imaging of cellular responses to pharmacological interventions and environmental assaults. The information gained will advance future studies of chronic visceral pain in the lower urinary tract and thus meet the criteria for inclusion in the Nociceptive GenitoUrinary Development Molecular Anatomy Project (nGUDMAP).

DESCRIPTION (provided by applicant): Gliogenesis and maintenance of glial cell types are critical to development and function of the nervous system. Sox10 is a transcription factor that is essential for development of multiple glial lineages including oligodendrocytes in the central nervous system as well as neural crest- derived Schwann cells, satellite glia and enteric nervous system neurons and glia in the periphery. Regulatory regions from the Sox10 gene have previously been used to drive expression of single fluorophore transgenes for tracking the migration of glial progenitors and the distribution of mature glial cell types. These first generaton tools have been valuable but have not permitted concurrent imaging of cell nucleus, morphology, or signaling between individual cells. Calcium signaling is a fundamental cellular mechanism by which cells transmit intracellular signals in response to extrinsic stimuli or transmit signals to adjacent cells and is essential for many aspects of glial cell development and maintenance. Studies of these processes have been primarily investigated in cell culture systems that are amenable to loading with fluorescent dyes or transfection by exogenous plasmids. However recent progress in development of Genetically Encoded Calcium Indicators (GECIs) has produced fluorescent reporters that allow monitoring of calcium transients in living cells and organisms. In the context of the R21 mechanism we propose generation of a multi-cistronic transgenic allele of Sox10 in mice as a novel tool for imaging migration, cell morphology and signaling between glial cells. In Specific Aim 1 we will construct and test multi-cistronic expression vectors in vitro to identify the optimal combination of reporters to monitor calcium signaling, nuclear localization and cell morphology. In Specific Aim 2 we will incorporate a multi-spectral expression cassette into a Sox10 bacterial artificial chromosome backbone and establish transgenic lines that recapitulate endogenous Sox10 expression in vivo. The ability to concurrently track migration, cell morphology and calcium signaling among glial populations will significantly impact the field by enabling analysis of developmental mechanisms that are relevant for directed differentiation of progenitors cells and will enable pharmacologic analyses to identify potential therapeutic agents for treatment of central and peripheral neuropathies.

PROJECT SUMMARY Normal function of the lower urinary tract (LUT) requires coordination of neuronal activities to allow bladder filling for urine storage later followed by bladder contraction to accomplish emptying at an appropriate time and place. Despite the fact that much is known about the neuroanatomy and mature circuitry of the LUT, relatively little is known about the molecular composition, development and postnatal maturation of distinct neuron subtypes that regulate bladder contractility. At present we lack a catalog of receptors expressed by different types of pelvic ganglia neurons that could be used to investigate how these critical neurons reach their targets in different LUT organs or direct regenerative efforts in cases of surgical damage. Even less is known about the molecular makeup of chemosensory brush cells and paraneurons in the urethra that are positioned to modulate LUT contractility. These cells contain high levels of neurotransmitters and thus can act as neuromodulators that stimulate bladder contraction in response to infection or mechanical stretch. Knowledge of the connections between motor neurons in pelvic ganglia and these neuromodulatory cells would enable detailed mechanistic studies of bladder sensitization and contractility in response to infection, inflammation or urine composition. The overall goal of this GUDMAP Atlas project is to fill these gaps in knowledge so that future mechanistic studies can be pursued. Four Specific Aims are proposed: (1) compile an atlas of genes expressed by pelvic ganglia neurons at single cell resolution during development and postnatal maturation; (2) map the first appearance and distribution of urethral neuromodulatory cells during development in the mouse; (3) identify genetic markers for subsets of urethral neuromodulatory cells by RNA-seq ; (4) generate a detailed spatial and temporal map of nerve terminals between neuromodulatory cells and sensory or autonomic pelvic ganglia neurons. These studies will generate novel information that is current lacking from the GUDMAP database. Our results will be summarized in diagrams and tutorial maps on the GUDMAP site and the gene sets identified will expedite comparative studies in human tissues. As a result the research community will be able to investigate the functions of specific cell types and test hypotheses using cell type and gene specific approaches.

PROJECT SUMMARY Gastrointestinal (GI) motility and defecation are absolute prerequisites for nutrient absorption, fecal elimination and overall health. Normal GI motility, vascular perfusion, and intestinal inflammation are coordinated by vast numbers of neurons that reside within ganglia of the enteric nervous system (ENS) intrinsic to the gut wall. While recent work has identified diverse genes that direct the initial development of progenitor cells that give rise to enteric neurons in the wall of the intestine, we know very little about the genes that are expressed in adult enteric neurons. Consequently we are unable to determine whether efforts to generate enteric neurons produce the normal complement of cell types. Moreover we do not fully understand how distinct types of neurons contribute to overall coordination of intestinal motility because the use of common immunohistochemical markers alone does not distinguish functionally distinct subtypes. As a result, our abilities to target and functionally manipulate specific types of neurons in the gut are extremely limited. To surpass these limitations, our application proposes to develop a comprehensive, single cell transcriptome map of adult enteric neurons in normal mice in parallel with deep sequencing of enteric ganglia from distinct regions of human intestine so that a global gene expression atlas of human enteric ganglia is obtained. To capture mouse enteric neurons for single cell RNA-Seq we will use a fluorescent transgenic mouse line that we developed for live-cell imaging of enteric neurons. Human enteric ganglia will be collected by laser capture microdissection from adult surgical remnants. Comparison of enteric neuron expression profiles between mouse and human data sets will identify conserved genes that mark distinct neuronal subtypes. To set the stage for relating specific neuronal subtypes in the mouse to GI motility we will concurrently quantify intestinal transit and motility patterns across inbred strains of mice using novel ex vivo motility imaging methods. The resulting atlas of molecular fingerprints for enteric neurons in combination with high resolution motility patterns will provide essential information needed to begin targeted, functional manipulation of GI motility in distinct regions of the intestine.

PROJECT SUMMARY Normal function of the lower urinary tract (LUT) requires coordination of neuronal activity to allow bladder filling for urine storage followed later by bladder contraction to accomplish urine expulsion at an appropriate time and place. Deficiencies in the development of the neural elements that mediate bladder control can lead to problems in patients such as neurogenic bladder, incontinence, urinary retention, or bladder pain. These LUT diseases result in reduced quality of life for patients, increase healthcare costs, and burden the health care system. While we know that the sacral elements of the peripheral nervous system, which participate in regulation of these bladder processes, include dorsal root ganglion (DRG) sensory neurons and autonomic (motor) pelvic neurons, there are many gaps in our knowledge regarding how these neurons develop and how deficits during development lead to congenital defects in bladder control. By surveying gene expression during development of pelvic ganglia in mice we have identified up-regulation of serotonin receptors that are also expressed in sensory neurons of developing DRG. Mice with loss of function mutations in these serotonin receptors, specifically the serotonin type 3 receptor (5-HT3), have abnormal development of nerves in the bladder wall and later exhibit urinary retention. Our preliminary work in isolated cultures of sacral neural crest stem cells indicates that perturbations of 5-HT3 signaling disrupt neuronal differentiation. We postulate that the bladder deficiencies observed in 5-HT3 mutant mice occur not only as the result of hyper-arborization of developing nerve terminals that lack 5-HT3 signaling, but also as a consequence of altered cell fate specification leading to imbalances among types of developing DRG and pelvic ganglia neurons. Cell fate specification is a novel function that has not previously been ascribed to 5-HT3 and has potential for pronounced impact on the types of neurons formed during development of bladder innervation. Such alterations in neuronal differentiation would predispose to deficiencies in bladder control and add to the potential for increased susceptibility to bladder pain. Three aims are proposed that will elaborate roles for 5-HT3 signaling during development of DRG and pelvic ganglia neurons that innervate the bladder. In Aim 1 we will use genetic and pharmacologic approaches to determine when 5- HT3 signaling is required to develop and maintain normal bladder innervation. In Aim 2 we will use Cre:LoxP lineage tracing studies in Htr3a gain and loss of function mutants to define the developmental processes and time periods that are affected by 5-HT3 signaling. In Aim 3 we will determine whether disruptions of 5-HT3 signaling during development increase vulnerabilty to bladder inflammatory pain. These studies will generate mechanistic knowledge of deficits in neural development that lead to bladder disease and will aid urologists in sculpting personalized therapies for patients.

PROJECT SUMMARY Normal gastrointestinal (GI) motility is an essential prerequisite for nutrient absorption, fecal elimination and overall health. Nearly a quarter of the United States population is affected by intestinal disorders that lead to abnormal GI motility, chronic constipation and other functional bowel disorders. Greater understanding of the mechanisms that regulate differentiation of enteric neural progenitors (ENPs), which form the neurons and glia of the enteric nervous system (ENS), are needed to understand how the normal complement of functional enteric neurons within the intestine is generated. Sox10 is an essential transcription factor that functions in the neural crest derived progenitors that generate the ENS. Defects in Sox10 in patients and mice cause aganglionosis of the distal intestine leading to megacolon. Recent studies of Sox10 mutant mice have identified pronounced alterations of the ratios of different enteric neuron types in proximal innervated bowel of these animals that are accompanied by abnormal intestinal transit and motility. Although Sox10 is expressed in ENPs, these deficiencies among enteric neurons were unexpected because Sox10 is extinguished as neurons begin to differentiate, although its expression is sustained in enteric glia. The results suggest that Sox10 has greater roles in ENS development than simply promoting migration of ENPs during initial phases when the fetal gut is first colonized by progenitors. In the proposed analysis we will test the overarching hypothesis that Sox10 action in ENPs orchestrates transcriptional networks and chromatin accessibility setting in motion a regulatory cascade that orchestrates diversity of enteric neuron subtypes. In Aim 1 we will test the hypothesis that the mutant allels of Sox10 alter enteric neuron ratios by disrupting transcriptional hierarchies in ENPs using single cell RNA sequencing (scRNASeq). In Aim 2 we will examine the hypothesis that Sox10 mutants disrupt chromatin accessibility in developing ENS lineages. In Aim 3 we will test the hypothesis that truncating mutations of Sox10 exert their effects through disrupted binding with other co-factors that are required during ENP differentiation. These studies will distinguish between potential developmental mechanisms that generate the normal repertoire of enteric neuron subtypes and will facilitate efforts to direct differentiation of enteric neurons for treatment of GI disease.