Chad Vezina (PI)
University of Wisconsin-Madison
- Mark Cadena
University of Wisconsin-Madison
- Anne Turco
University of Wisconsin-Madison
Our goals are to identify mouse cell lineages giving rise to key prostatic stromal subtypes, map RNAs/proteins that mediate stromal cell behaviors in developing human prostate, and generate searchable resources and tutorials to enable researchers to leverage this data. We are ideally positioned to meet these objectives. Our research team has the expertise (>500 publications in clinical urology and mechanism based prostatic research), experience (previous contributions of >1300 mouse prostatic RNA expression images to the GUDMAP database) and research tools (deep tissue and multispectral imaging, extensive experience with mouse genetics, 28 human fetal prostates ready for analysis, and access to ~50 fresh human fetal prostatic specimens per year) to achieve these goals.
Turco, Anne E.; Cadena, Mark T.; Zhang, Helen L.; Sandhu, Jaskiran K.; Oakes, Steven R.; Chathurvedula, Thrishna; Peterson, Richard E.; Keast, Janet R.; Vezina, Chad M.. Histochem Cell Biol. vol. 152(1), 35–45. July 2019.
Prostate autonomic and sensory axons control glandular growth, fluid secretion, and smooth muscle contraction and are remodeled during cancer and inflammation. Morphogenetic signaling pathways reawakened during disease progression may drive this axon remodeling. These pathways are linked to proliferative activities in prostate cancer and benign prostate hyperplasia. However, little is known about which developmental signaling pathways guide axon investment into prostate. The first step in defining these pathways is pinpointing when axon subtypes first appear in prostate. We accomplished this by immunohistochemically mapping three axon subtypes (noradrenergic, cholinergic, and peptidergic) during fetal, neonatal, and adult stages of mouse prostate development. We devised a method for peri-prostatic axon density quantification and tested whether innervation is uniform across the proximo-distal axis of dorsal and ventral adult mouse prostate. Many axons directly interact with or innervate neuroendocrine cells in other organs, so we examined whether sensory or autonomic axons innervate neuroendocrine cells in prostate. We first detected noradrenergic, cholinergic, and peptidergic axons in prostate at embryonic day (E) 14.5. Noradrenergic and cholinergic axon densities are uniform across the proximal-distal axis of adult mouse prostate while peptidergic axons are denser in the periurethral and proximal regions. Peptidergic and cholinergic axons are closely associated with prostate neuroendocrine cells whereas noradrenergic axons are not. These results provide a foundation for understanding mouse prostatic axon development and organization and, provide strategies for quantifying axons during progression of prostate disease.
Wegner, Kyle A.; Mehta, Vatsal; Johansson, Jeanette A.; Mueller, Brett R.; Keil, Kimberly P.; Abler, Lisa L.; Marker, Paul C.; Taketo, M. Mark; Headon, Denis J.; Vezina, Chad M.. Biology Open. vol. 8(3), bio037945. March 2019.
Beta-catenin (CTNNB1) directs ectodermal appendage spacing by activating ectodysplasin A receptor (EDAR) transcription, but whether CTNNB1 acts by a similar mechanism in the prostate, an endoderm-derived tissue, is unclear. Here we examined the expression, function, and CTNNB1 dependence of the EDAR pathway during prostate development. In situ hybridization studies reveal EDAR pathway components including Wnt10b in the developing prostate and localize these factors to prostatic bud epithelium where CTNNB1 target genes are co-expressed. We used a genetic approach to ectopically activate CTNNB1 in developing mouse prostate and observed focal increases in Edar and Wnt10b mRNAs. We also used a genetic approach to test the prostatic consequences of activating or inhibiting Edar expression. Edar overexpression does not visibly alter prostatic bud formation or branching morphogenesis, and Edar expression is not necessary for either of these events. However, Edar overexpression is associated with an abnormally thick and collagen-rich stroma in adult mouse prostates. These results support CTNNB1 as a transcriptional activator of Edar and Wnt10b in the developing prostate and demonstrate Edar is not only important for ectodermal appendage patterning but also influences collagen organization in adult prostates.This article has an associated First Person interview with the first author of the paper.
Henry, GH; Malewska, A; Joseph, DB; Malladi, VS; Lee, J; Torrealba, J; Mauck, RJ; Gahan, JC; Raj, GV; Roehrborn, CG; Hon, GC; Macconmara, MP; Reese, JC; Hutchinson, RC; Vezina, CM; Strand, DW. Cell Rep. vol. 25(12), 3530-3542. December 2018.
A cellular anatomy of normal human organs is essential for solving the cellular origins of disease. We report the first comprehensive cellular atlas of the young adult human prostate and prostatic urethra using an iterative process of single cell RNA sequencing and flow cytometry on ~98,000 cells taken from different anatomical regions. Two previously unrecognized epithelial cell types were identified by KRT13 and SCGB1A1 expression and found to be highly similar to hillock and club cells of the proximal lung. It was demonstrated by immunohistochemistry that prostate club and hillock cells are similarly concentrated in the proximal prostate. We also optimized a new flow cytometry antibody panel to improve cell type-specific purification based on newly established cellular markers. The molecular classification, anatomical distribution, and purification methods for each cell type in the human prostate create a powerful new resource for experimental design in human prostate disease.
Joseph, DB; Chandrashekar, AS; Abler, LL; Chu, LF; Thomson, JA; Mendelsohn, C; Vezina, CM. Proc Natl Acad Sci U S A. vol. 115(33), 8394–8399. August 2018.
The bladder’s remarkable regenerative capacity had been thought to derive exclusively from its own progenitors. While examining consequences of DNA methyltransferase 1 (Dnmt1) inactivation in mouse embryonic bladder epithelium, we made the surprising discovery that Wolffian duct epithelial cells can support bladder regeneration. Conditional Dnmt1 inactivation in mouse urethral and bladder epithelium triggers widespread apoptosis, depletes basal and intermediate bladder cells, and disrupts uroplakin protein expression. These events coincide with Wolffian duct epithelial cell recruitment into Dnmt1 mutant urethra and bladder where they are reprogrammed to express bladder markers, including FOXA1, keratin 5, P63, and uroplakin. This is evidence that Wolffian duct epithelial cells are summoned in vivo to replace damaged bladder epithelium and function as a reservoir of cells for bladder regeneration.
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.
Wegner, KA; Cadena, MT; Trevena, R; Turco, AE; Gottschalk, A; Halberg, RB; Guo, J; McMahon, JA; McMahon, AP; Vezina, CM. PLoS ONE. vol. 12(11) November 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.
Keil, KP; Altmann, HM; Mehta, V; Abler, LL; Elton, EA; Vezina, CM. Gene Expr Patterns. vol. 13(8), 413–24. December 2013.
The mouse prostate develops from a component of the lower urinary tract (LUT) known as the urogenital sinus (UGS). This process requires androgens and signaling between mesenchyme and epithelium. Little is known about DNA methylation during prostate development, including which factors are expressed, whether their expression changes over time, and if DNA methylation contributes to androgen signaling or influences signaling between mesenchyme and epithelium. We used in situ hybridization to evaluate the spatial and temporal expression pattern of mRNAs which encode proteins responsible for establishing, maintaining or remodeling DNA methylation. These include DNA methyltransferases, DNA deaminases, DNA glycosylases, base excision repair and mismatch repair pathway members. The mRNA expression patterns were compared between male and female LUT prior to prostatic bud formation (14.5 days post coitus (dpc)), during prostatic bud formation (17.5 dpc) and during prostatic branching morphogenesis (postnatal day (P) 5). We found dramatic changes in the patterns of these mRNAs over the course of prostate development and identified examples of sexually dimorphic mRNA expression. Future investigation into how DNA methylation patterns are established, maintained and remodeled during the course of embryonic prostatic bud formation may provide insight into prostate morphogenesis and disease.
Keil, KP; Mehta, V; Branam, AM; Abler, LL; Bresh-Stiemke, RA; Joshi, PS; Schmitz, CT; Marker, PC; Vezina, CM. Endocrinology. vol. 153(12), 6091–103. December 2012.
Fetal prostate development from urogenital sinus (UGS) epithelium requires androgen receptor (AR) activation in UGS mesenchyme (UGM). Despite growing awareness of sexually dimorphic gene expression in the UGS, we are still limited in our knowledge of androgen-responsive genes in UGM that initiate prostate ductal development. We found that WNT inhibitory factor 1 (Wif1) mRNA is more abundant in male vs. female mouse UGM in which its expression temporally and spatially overlaps androgen-responsive steroid 5α-reductase 2 (Srd5a2). Wif1 mRNA is also present in prostatic buds during their elongation and branching morphogenesis. Androgens are necessary and sufficient for Wif1 expression in mouse UGS explant mesenchyme, and testicular androgens remain necessary for normal Wif1 expression in adult mouse prostate stroma. WIF1 contributes functionally to prostatic bud formation. In the presence of androgens, exogenous WIF1 protein increases prostatic bud number and UGS basal epithelial cell proliferation without noticeably altering the pattern of WNT/β-catenin-responsive Axin2 or lymphoid enhancer binding factor 1 (Lef1) mRNA. Wif1 mutant male UGSs exhibit increased (Sfrp)2 and (Sfrp)3 expression and form the same number of prostatic buds as the wild-type control males. Collectively our results reveal Wif1 as one of the few known androgen-responsive genes in the fetal mouse UGM and support the hypothesis that androgen-dependent Wif1 expression is linked to the mechanism of androgen-induced prostatic bud formation.
Keil, KP; Mehta, V; Abler, LL; Joshi, PS; Schmitz, CT; Vezina, CM. Differentiation. vol. 84(3), 232–9. October 2012.
The purpose of this study was to validate a combined in situ hybridization (ISH)/immunohistochemistry (IHC) staining method for visualizing and quantifying mouse prostatic buds. To refine animal usage in prostate development studies, we also determined whether a comparable number of prostatic buds were formed in male and female mouse urogenital sinus (UGS) explants grown in vitro in the presence of androgen. We used IHC to label UGS epithelium and ISH to label prostatic buds with one of three different prostatic bud marking riboprobes: a previously identified prostatic bud marker, NK-3 transcription factor, locus 1 (Nkx3-1), and two newly identified prostatic bud markers, wingless-related MMTV integration site 10b (Wnt10b) and ectodysplasin-A receptor (Edar). We calculated total buds formed per UGS and the proportion marked by each mRNA after male UGS development in vivo and male and female UGS development in vitro. Nkx3-1 was first to mark the prostate field during UGS development in vivo but all three mRNAs marked prostatic buds during later developmental stages. The mRNAs localized to different domains: Nkx3-1 was present along about half the prostatic bud length while Edar and Wnt10b were restricted to distal bud tips. None of the mRNAs marked all buds formed in vitro and the proportion marked was developmental stage- and gender-dependent. Nkx3-1 marked the highest proportion of prostatic buds during in vitro UGS development. Together, our results reveal that ISH staining of mouse UGS can be used to quantify prostatic bud number, Nkx3-1 is currently the best suited riboprobe for this method, and female UGSs cannot be used interchangeably with male UGSs when conducting prostate development studies in vitro. We also found that Nkx3-1, Edar, and Wnt10b mark different prostatic bud regions and are likely to be useful in future studies of regional differences in prostatic bud gene expression.
Mehta, V; Abler, LL; Keil, KP; Schmitz, CT; Joshi, PS; Vezina, CM. Dev Dyn. vol. 240(11), 2548–60. November 2011.
Prostate development is influenced by β-catenin signaling, but it is unclear which β-catenin activators are involved, where they are synthesized, and whether their mRNA abundance is influenced by androgens. We identified WNT/β-catenin-responsive β-galactosidase activity in the lower urogenital tract (LUT) of transgenic reporter mice, but β-galactosidase activity differed among the four mouse strains we examined. We used in situ hybridization to compare patterns of Wnts, r-spondins (Rspos, co-activators of β-catenin signaling), β-catenin-responsive mRNAs, and an androgen receptor-responsive mRNA in wild type fetal male, fetal female, and neonatal male LUT. Most Wnt and Rspo mRNAs were present in LUT during prostate development. Sexually dimorphic expression patterns were observed for WNT/β-catenin-responsive genes, and for Wnt2b, Wnt4, Wnt7a, Wnt9b, Wnt10b, Wnt11, Wnt16, and Rspo3 mRNAs. These results reveal sexual differences in WNT/β-catenin signaling in fetal LUT, supporting the idea that this pathway may be directly or indirectly responsive to androgens during prostate ductal development.
Abler, LL; Keil, KP; Mehta, V; Joshi, PS; Schmitz, CT; Vezina, CM. Dev Dyn. vol. 240(10), 2364–77. October 2011.
Epithelial-stromal interactions in the lower urogenital tract (LUT) are integral to prostatic and seminal vesicle development in males, vaginal and uterine development in females, and urethral development in both sexes. Gene expression profiling of isolated LUT stroma and epithelium has unraveled mechanisms of LUT development, but such studies are confounded by heterogeneous and ill-defined cell sub-populations contained within each tissue compartment. We used in situ hybridization to synthesize a high-resolution molecular atlas of 17-day post-coitus fetal mouse LUT. We identified mRNAs that mark selective cell populations of the seminal vesicle, ejaculatory duct, prostate, urethra, and vagina, subdividing these tissues into 16 stromal and 8 epithelial sub-compartments. These results provide a powerful tool for mapping LUT gene expression patterns and also reveal previously uncharacterized sub-compartments that may play mechanistic roles in LUT development of which we were previously unaware.
Abler, LL; Mehta, V; Keil, KP; Joshi, PS; Flucus, CL; Hardin, HA; Schmitz, CT; Vezina, CM. J Vis Exp. vol. 54 August 2011.
Development of the lower urogenital tract (LUT) is an intricate process. This complexity is evidenced during formation of the prostate from the fetal male urethra, which relies on androgenic signals and epithelial-mesenchymal interactions(1,2). Understanding the molecular mechanisms responsible for prostate development may reveal growth mechanisms that are inappropriately reawakened later in life to give rise to prostate diseases such as benign prostatic hyperplasia and prostate cancer. The developing LUT is anatomically complex. By the time prostatic budding begins on 16.5 days post conception (dpc), numerous cell types are present. Vasculature, nerves and smooth muscle reside within the mesenchymal stroma(3). This stroma surrounds a multilayered epithelium and gives rise to the fetal prostate through androgen receptor-dependent paracrine signals(4). The identity of the stromal androgen receptor-responsive genes required for prostate development and the mechanism by which prostate ductal epithelium forms in response to these genes is not fully understood. The ability to precisely identify cell types and localize expression of specific factors within them is imperative to further understand prostate development. In situ hybridization (ISH) allows for localization of mRNAs within a tissue. Thus, this method can be used to identify pattern and timing of expression of signaling molecules and their receptors, thereby elucidating potential prostate developmental regulators. Here, we describe a high throughput ISH technique to identify mRNA expression patterns in the fetal mouse LUT using vibrating microtome-cut sections. This method offers several advantages over other ISH protocols. Performing ISH on thin sections adhered to a slide is technically difficult; cryosections frequently have poor structural quality while both cryosections and paraffin sections often result in weak signal resolution. Performing ISH on whole mount tissues can result in probe trapping. In contrast, our high throughput technique utilizes thick-cut sections that reveal detailed tissue architecture. Modified microfuge tubes allow easy handling of sections during the ISH procedure. A maximum of 4 mRNA transcripts can be screened from a single 17.5dpc LUT with up to 24 mRNA transcripts detected in a single run, thereby reducing cost and maximizing efficiency. This method allows multiple treatment groups to be processed identically and as a single unit, thereby removing any bias for interpreting data. Most pertinently for prostate researchers, this method provides a spatial and temporal location of low and high abundance mRNA transcripts in the fetal mouse urethra that gives rise to the prostate ductal network.