Michelle Southard-Smith (PI)
Vanderbilt University Medical Center
Knowledge of the developmental cues that promote growth, survival, and appropriate synaptic targeting during formation of pelvic ganglia is essential for regenerative strategies to repair pelvic innervation in adults. Presently, we do not know when distinct pelvic ganglia neuron subtypes appear during fetal organogenesis or what genes regulate their development and postnatal maturation – nor do we know the full complement of genes expressed in pelvic ganglia neurons at maturity. The goal of this Atlas proposal is to produce a unified, consistently annotated dataset that will provide researchers ready access to a portfolio of genes expressed in LUT autonomic neurons and neuromodulatory cells.
Wegner, KA; Abler, LL; Oakes, SR; Mehta, GS; Ritter, KE; Hill, WG; Zwaans, BMM; Lamb, LE; Wang, Z; Bjorling, DE; Ricke, WA; Macoska, J; Marker, PC; Southard-Smith, EM; Eliceiri, KW; Vezina, CM.. Am J Physiol Renal Physiol. July 2018.
Mouse urinary behavior is quantifiable and used to pinpoint mechanisms of voiding dysfunction and evaluate potential human therapies. Approaches to evaluate mouse urinary function vary widely among laboratories, however, complicating cross-study comparisons. Here, we describe development and multi-institutional validation of a new tool for objective, consistent and rapid analysis of mouse void spot assay (VSA) data. Void Whizzard is a freely available software plugin for FIJI (a distribution of ImageJ) that facilitates VSA image batch processing and data extraction. We describe its features, demonstrate them by evaluating how specific VSA method parameters influence voiding behavior, and establish Void Whizzard as an expedited method for VSA analysis. This study includes control and obese diabetic mice as models of urinary dysfunction to increase rigor and ensure relevance across distinct voiding patterns. In particular, we show that Void Whizzard is an effective tool for quantifying non-concentric overlapping void spots, which commonly confound analyses. We also show that mouse genetics are consistently more influential than assay design parameters when it comes to VSA outcomes. None of the following procedural modifications to reduce overlapping spots masked these genetic-related differences: reduction of VSA testing duration, water access during the assay period, placement of a wire mesh cage bottom on top of or elevated over the filter paper, treatment of mesh with a hydrophobic spray, and size of wire mesh opening. The Void Whizzard software and rigorous validation of VSA methodological parameters described here advance the goal of standardizing mouse urinary phenotyping for comprehensive urinary phenome analyses.
Wiese, CB; Deal, KK; Ireland, SJ; Cantrell, VA; Southard-Smith, EM. Dev Biol. vol. 429(1), 356–369. September 2017.
Ritter, KE; Southard-Smith, EM. Front Neurosci. vol. 10(592) June 2017.
Georgas, KM; Armstrong, J; Keast, JR; Larkins, CE; McHugh, KM; Southard-Smith, EM; Cohn, MJ; Batourina, E; Dan, H; Schneider, K; Buehler, DP; Wiese, CB; Brennan, J; Davies, JA; Harding, SD; Baldock, RA; Little, MH; Vezina, CM; Mendelsohn, C. Development. vol. 142(10), 1893–908. May 2015.
Malformation of the urogenital tract represents a considerable paediatric burden, with many defects affecting the lower urinary tract (LUT), genital tubercle and associated structures. Understanding the molecular basis of such defects frequently draws on murine models. However, human anatomical terms do not always superimpose on the mouse, and the lack of accurate and standardised nomenclature is hampering the utility of such animal models. We previously developed an anatomical ontology for the murine urogenital system. Here, we present a comprehensive update of this ontology pertaining to mouse LUT, genital tubercle and associated reproductive structures (E10.5 to adult). Ontology changes were based on recently published insights into the cellular and gross anatomy of these structures, and on new analyses of epithelial cell types present in the pelvic urethra and regions of the bladder. Ontology changes include new structures, tissue layers and cell types within the LUT, external genitalia and lower reproductive structures. Representative illustrations, detailed text descriptions and molecular markers that selectively label muscle, nerves/ganglia and epithelia of the lower urogenital system are also presented. The revised ontology will be an important tool for researchers studying urogenital development/malformation in mouse models and will improve our capacity to appropriately interpret these with respect to the human situation.
Wiese, CB; Ireland, S; Fleming, NL; Yu, J; Valerius, MT; Georgas, K; Chiu, HS; Brennan, J; Armstrong, J; Little, MH; McMahon, AP; Southard-Smith, EM. Front Neurosci. vol. 6, 130. September 2012.
Relative positions of neurons within mature murine pelvic ganglia based on expression of neurotransmitters have been described. However the spatial organization of developing innervation in the murine urogenital tract (UGT) and the gene networks that regulate specification and maturation of neurons within the pelvic ganglia of the lower urinary tract (LUT) are unknown. We used whole-mount immunohistochemistry and histochemical stains to localize neural elements in 15.5 days post coitus (dpc) fetal mice. To identify potential regulatory factors expressed in pelvic ganglia, we surveyed expression patterns for known or probable transcription factors (TF) annotated in the mouse genome by screening a whole-mount in situ hybridization library of fetal UGTs. Of the 155 genes detected in pelvic ganglia, 88 encode TFs based on the presence of predicted DNA-binding domains. Neural crest (NC)-derived progenitors within the LUT were labeled by Sox10, a well-known regulator of NC development. Genes identified were categorized based on patterns of restricted expression in pelvic ganglia, pelvic ganglia and urethral epithelium, or pelvic ganglia and urethral mesenchyme. Gene expression patterns and the distribution of Sox10+, Phox2b+, Hu+, and PGP9.5+ cells within developing ganglia suggest previously unrecognized regional segregation of Sox10+ progenitors and differentiating neurons in early development of pelvic ganglia. Reverse transcription-PCR of pelvic ganglia RNA from fetal and post-natal stages demonstrated that multiple TFs maintain post-natal expression, although Pax3 is extinguished before weaning. Our analysis identifies multiple potential regulatory genes including TFs that may participate in segregation of discrete lineages within pelvic ganglia. The genes identified here are attractive candidate disease genes that may now be further investigated for their roles in malformation syndromes or in LUT dysfunction.
Buehler, DP; Wiese, CB; Skelton, SB; Southard-Smith, EM. J Vis Exp. vol. 66 August 2012.
During development neural crest (NC)-derived neuronal progenitors migrate away from the neural tube to form autonomic ganglia in visceral organs like the intestine and lower urinary tract. Both during development and in mature tissues these cells are often widely dispersed throughout tissues so that isolation of discrete populations using methods like laser capture micro-dissection is difficult. They can however be directly visualized by expression of fluorescent reporters driven from regulatory regions of neuron-specific genes like Tyrosine hydroxylase (TH). We describe a method optimized for high yields of viable TH+ neuronal progenitors from fetal mouse visceral tissues, including intestine and lower urogenital tract (LUT), based on dissociation and fluorescence-activated cell sorting (FACS). The Th gene encodes the rate-limiting enzyme for production of catecholamines. Enteric neuronal progenitors begin to express TH during their migration in the fetal intestine and TH is also present in a subset of adult pelvic ganglia neurons . The first appearance of this lineage and the distribution of these neurons in other aspects of the LUT, and their isolation has not been described. Neuronal progenitors expressing TH can be readily visualized by expression of EGFP in mice carrying the transgene construct Tg(Th-EGFP)DJ76Gsat/Mmnc. We imaged expression of this transgene in fetal mice to document the distribution of TH+ cells in the developing LUT at 15.5 days post coitus (dpc), designating the morning of plug detection as 0.5 dpc, and observed that a subset of neuronal progenitors in the coalescing pelvic ganglia express EGFP. To isolate LUT TH+ neuronal progenitors, we optimized methods that were initially used to purify neural crest stem cells from fetal mouse intestine. Prior efforts to isolate NC-derived populations relied upon digestion with a cocktail of collagenase and trypsin to obtain cell suspensions for flow cytometry. In our hands these methods produced cell suspensions from the LUT with relatively low viability. Given the already low incidence of neuronal progenitors in fetal LUT tissues, we set out to optimize dissociation methods such that cell survival in the final dissociates would be increased. We determined that gentle dissociation in Accumax (Innovative Cell Technologies, Inc), manual filtering, and flow sorting at low pressures allowed us to achieve consistently greater survival (\textgreater70% of total cells) with subsequent yields of neuronal progenitors sufficient for downstream analysis. The method we describe can be broadly applied to isolate a variety of neuronal populations from either fetal or adult murine tissues.
Harding, SD; Armit, C; Armstrong, J; Brennan, J; Cheng, Y; Haggarty, B; Houghton, D; Lloyd-MacGilp, S; Pi, X; Roochun, Y; Sharghi, M; Tindal, C; McMahon, AP; Gottesman, B; Little, MH; Georgas, K; Aronow, B; Potter, SS; Brunskill, EW; Southard-Smith, EM; Mendelsohn, C; Baldock, RA; Davies, JA; Davidson, D. Development. vol. 138(13), 2845–53. July 2011.
The GenitoUrinary Development Molecular Anatomy Project (GUDMAP) is an international consortium working to generate gene expression data and transgenic mice. GUDMAP includes data from large-scale in situ hybridisation screens (wholemount and section) and microarray gene expression data of microdissected, laser-captured and FACS-sorted components of the developing mouse genitourinary (GU) system. These expression data are annotated using a high-resolution anatomy ontology specific to the developing murine GU system. GUDMAP data are freely accessible at www.gudmap.org via easy-to-use interfaces. This curated, high-resolution dataset serves as a powerful resource for biologists, clinicians and bioinformaticians interested in the developing urogenital system. This paper gives examples of how the data have been used to address problems in developmental biology and provides a primer for those wishing to use the database in their own research.