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• W2896955363 abstract "•Binucleated S-cells are critical for maintaining the urothelial barrier•S-cells derive from tetraploid I-cells that form via incomplete cytokinesis•Binucleated I- and S-cells increase ploidy via endoreplication•Polyploidy may enable the urothelium to efficiently adjust to environmental changes The urothelium is an epithelia barrier lined by a luminal layer of binucleated, octoploid, superficial cells. Superficial cells are critical for production and transport of uroplakins, a family of proteins that assemble into a waterproof crystalline plaque that helps protect against infection and toxic substances. Adult urothelium is nearly quiescent, but rapidly regenerates in response to injury. Yet the mechanism by which binucleated, polyploid, superficial cells are produced remains unclear. Here, we show that superficial cells are likely to be derived from a population of binucleated intermediate cells, which are produced from mononucleated intermediate cells via incomplete cytokinesis. We show that binucleated intermediate and superficial cells increase DNA content via endoreplication, passing through S phase without entering mitosis. The urothelium can be permanently damaged by repetitive or chronic injury or disease. Identification of the mechanism by which superficial cells are produced may be important for developing strategies for urothelial repair. The urothelium is an epithelia barrier lined by a luminal layer of binucleated, octoploid, superficial cells. Superficial cells are critical for production and transport of uroplakins, a family of proteins that assemble into a waterproof crystalline plaque that helps protect against infection and toxic substances. Adult urothelium is nearly quiescent, but rapidly regenerates in response to injury. Yet the mechanism by which binucleated, polyploid, superficial cells are produced remains unclear. Here, we show that superficial cells are likely to be derived from a population of binucleated intermediate cells, which are produced from mononucleated intermediate cells via incomplete cytokinesis. We show that binucleated intermediate and superficial cells increase DNA content via endoreplication, passing through S phase without entering mitosis. The urothelium can be permanently damaged by repetitive or chronic injury or disease. Identification of the mechanism by which superficial cells are produced may be important for developing strategies for urothelial repair. The urothelium is an epithelial barrier that extends from the renal pelvis to the bladder neck, protects against pathogens and toxins, and controls the passage of water and ions between the mucosa and underlying tissue. The adult urothelium is nearly quiescent but can quickly regenerate after acute injury from urinary tract infection (UTI) or exposure to toxins, indicating that progenitors exist in adults that are able to repair the urothelium. The mouse urothelium contains two sub-populations of basal cells (K5-basal cells and K14-basal cells), intermediate cells (I-cells), and a luminal layer lined with superficial cells (S-cells; Figure 1). S-cells are binucleated, polyploid, and post-mitotic (Hicks, 1975Hicks R.M. The mammalian urinary bladder: an accommodating organ.Biol. Rev. Camb. Philos. Soc. 1975; 50: 215-246Crossref PubMed Scopus (344) Google Scholar). They are cellular machines, specialized for synthesis and transport of Uroplakins, a family of integral membrane proteins that assemble into a crystalline apical plaque that covers most of the urothelial apical surface (Lin et al., 1994Lin J.H. Wu X.R. Kreibich G. Sun T.T. Precursor sequence, processing, and urothelium-specific expression of a major 15-kDa protein subunit of asymmetric unit membrane.J. Biol. Chem. 1994; 269: 1775-1784PubMed Google Scholar, Wu et al., 1990Wu X.R. Manabe M. Yu J. Sun T.T. Large scale purification and immunolocalization of bovine uroplakins I, II, and III. Molecular markers of urothelial differentiation.J. Biol. Chem. 1990; 265: 19170-19179Abstract Full Text PDF PubMed Google Scholar, Wu et al., 1994Wu X.R. Lin J.H. Walz T. Häner M. Yu J. Aebi U. Sun T.T. Mammalian uroplakins. A group of highly conserved urothelial differentiation-related membrane proteins.J. Biol. Chem. 1994; 269: 13716-13724Abstract Full Text PDF PubMed Google Scholar, Wu et al., 2009Wu X.R. Kong X.P. Pellicer A. Kreibich G. Sun T.T. Uroplakins in urothelial biology, function, and disease.Kidney Int. 2009; 75: 1153-1165Abstract Full Text Full Text PDF PubMed Scopus (256) Google Scholar). S-cells connect to one another via high resistance tight junctions, forming a waterproof barrier that prevents leakage during voiding, which occurs under pressure. These cells, which can be as large as 250 μm, are able to respond to environmental cues in a number of ways. For example, during the filling phase of micturition, S-cells increase their apical surface via exocytosis of specialized fusiform vesicles that shuttle Upks to the surface, where they are assembled into uroplaque crystals. During the emptying phase of micturition, S-cells decrease their surface area via endocytosis, shuttling apical membrane/plaque into the cell for degradation (Carattino et al., 2013Carattino M.D. Prakasam H.S. Ruiz W.G. Clayton D.R. McGuire M. Gallo L.I. Apodaca G. Bladder filling and voiding affect umbrella cell tight junction organization and function.Am. J. Physiol. Renal Physiol. 2013; 305: F1158-F1168Crossref PubMed Scopus (34) Google Scholar, Khandelwal et al., 2009Khandelwal P. Abraham S.N. Apodaca G. Cell biology and physiology of the uroepithelium.Am. J. Physiol. Renal Physiol. 2009; 297: F1477-F1501Crossref PubMed Scopus (275) Google Scholar, Wu et al., 2009Wu X.R. Kong X.P. Pellicer A. Kreibich G. Sun T.T. Uroplakins in urothelial biology, function, and disease.Kidney Int. 2009; 75: 1153-1165Abstract Full Text Full Text PDF PubMed Scopus (256) Google Scholar). These specialized features likely depend on the ability of S-cells to maintain a high rate of metabolism, protein synthesis, and intracellular transport. Variation in cell ploidy, or sets of chromosomes generated through duplication(s) of the whole genome, is a natural and important part of development in different organisms and tissues across many taxa (Edgar et al., 2014Edgar B.A. Zielke N. Gutierrez C. Endocycles: a recurrent evolutionary innovation for post-mitotic cell growth.Nat. Rev. Mol. Cell Biol. 2014; 15: 197-210Crossref PubMed Scopus (228) Google Scholar, Fox and Duronio, 2013Fox D.T. Duronio R.J. Endoreplication and polyploidy: insights into development and disease.Development. 2013; 140: 3-12Crossref PubMed Scopus (234) Google Scholar). Polyploidy has been shown to have positive features, for example, enabling cells to better respond to environmental changes or challenges, as well as negative features, including increased potential for aneuploidy, which is commonly associated with cancer and birth defects due to genome instability (Duncan et al., 2010Duncan A.W. Taylor M.H. Hickey R.D. Hanlon Newell A.E. Lenzi M.L. Olson S.B. Finegold M.J. Grompe M. The ploidy conveyor of mature hepatocytes as a source of genetic variation.Nature. 2010; 467: 707-710Crossref PubMed Scopus (365) Google Scholar, Losick et al., 2013Losick V.P. Fox D.T. Spradling A.C. Polyploidization and cell fusion contribute to wound healing in the adult Drosophila epithelium.Curr. Biol. 2013; 23: 2224-2232Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar, Schoenfelder and Fox, 2015Schoenfelder K.P. Fox D.T. The expanding implications of polyploidy.J. Cell Biol. 2015; 209: 485-491Crossref PubMed Scopus (138) Google Scholar, Storchova and Pellman, 2004Storchova Z. Pellman D. From polyploidy to aneuploidy, genome instability and cancer.Nat. Rev. Mol. Cell Biol. 2004; 5: 45-54Crossref PubMed Scopus (620) Google Scholar). Besides cell-cell fusion, polyploidy can be programmed through non-canonical cell cycles, also known as endoreplication or endocycling, in which DNA replication (S phase) proceeds without intervening mitoses (Frawley and Orr-Weaver, 2015Frawley L.E. Orr-Weaver T.L. Polyploidy.Curr. Biol. 2015; 25: R353-R358Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). In this case, genome duplication is uncoupled from daughter cell formation by oscillating G and S phases, as observed in plant trichromes, Drosophila salivary glands, and in many other fly tissues (Edgar and Orr-Weaver, 2001Edgar B.A. Orr-Weaver T.L. Endoreplication cell cycles: more for less.Cell. 2001; 105: 297-306Abstract Full Text Full Text PDF PubMed Scopus (676) Google Scholar, Hammond and Laird, 1985Hammond M.P. Laird C.D. Chromosome structure and DNA replication in nurse and follicle cells of Drosophila melanogaster.Chromosoma. 1985; 91: 267-278Crossref PubMed Scopus (82) Google Scholar, Swanhart et al., 2005Swanhart L. Kupsco J. Duronio R.J. Developmental control of growth and cell cycle progression in Drosophila.Methods Mol. Biol. 2005; 296: 69-94PubMed Google Scholar). Polyploidy can also result from failed cytokinesis in which cells become binucleate (here on referred to as endomitosis), as observed in megakaryocytes, trophoblasts, hepatocytes, and Drosophila male accessory gland cells and larval tissues (Cao et al., 2017Cao J. Wang J. Jackman C.P. Cox A.H. Trembley M.A. Balowski J.J. Cox B.D. De Simone A. Dickson A.L. Di Talia S. et al.Tension creates an endoreplication wavefront that leads regeneration of epicardial tissue.Dev. Cell. 2017; 42: 600-615.e4Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar, Gentric and Desdouets, 2014Gentric G. Desdouets C. Polyploidization in liver tissue.Am. J. Pathol. 2014; 184: 322-331Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar, Nguyen and Ravid, 2010Nguyen H.G. Ravid K. Polyploidy: mechanisms and cancer promotion in hematopoietic and other cells.Adv. Exp. Med. Biol. 2010; 676: 105-122Crossref PubMed Scopus (20) Google Scholar, Sarto et al., 1982Sarto G.E. Stubblefield P.A. Therman E. Endomitosis in human trophoblast.Hum. Genet. 1982; 62: 228-232Crossref PubMed Scopus (42) Google Scholar, Taniguchi et al., 2014Taniguchi K. Kokuryo A. Imano T. Minami R. Nakagoshi H. Adachi-Yamada T. Isoform-specific functions of Mud/NuMA mediate binucleation of Drosophila male accessory gland cells.BMC Dev. Biol. 2014; 14: 46Crossref PubMed Scopus (15) Google Scholar). Importantly, endomitosis and endocycling are not mutually exclusive, and both pathways for increasing genome copy number per cell have been found to occur in polyploid cells, for example, in zebrafish epicardium (Taniguchi et al., 2014Taniguchi K. Kokuryo A. Imano T. Minami R. Nakagoshi H. Adachi-Yamada T. Isoform-specific functions of Mud/NuMA mediate binucleation of Drosophila male accessory gland cells.BMC Dev. Biol. 2014; 14: 46Crossref PubMed Scopus (15) Google Scholar). The cellular mechanism by which S-cells achieve polyploidy is largely unexplored. Lineage studies and pulse-chase analyses suggest that both I-cells and basal cells can produce S-cell daughters depending on the conditions (Colopy et al., 2014Colopy S.A. Bjorling D.E. Mulligan W.A. Bushman W. A population of progenitor cells in the basal and intermediate layers of the murine bladder urothelium contributes to urothelial development and regeneration.Dev. Dyn. 2014; 243: 988-998Crossref PubMed Scopus (44) Google Scholar, Gandhi et al., 2013Gandhi D. Molotkov A. Batourina E. Schneider K. Dan H. Reiley M. Laufer E. Metzger D. Liang F. Liao Y. et al.Retinoid signaling in progenitors controls specification and regeneration of the urothelium.Dev. Cell. 2013; 26: 469-482Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar, Papafotiou et al., 2016Papafotiou G. Paraskevopoulou V. Vasilaki E. Kanaki Z. Paschalidis N. Klinakis A. KRT14 marks a subpopulation of bladder basal cells with pivotal role in regeneration and tumorigenesis.Nat. Commun. 2016; 7: 11914Crossref PubMed Scopus (96) Google Scholar, Schäfer et al., 2017Schäfer F.M. Algarrahi K. Savarino A. Yang X. Seager C. Franck D. Costa K. Liu S. Logvinenko T. Adam R. Mauney J.R. Mode of surgical injury influences the source of urothelial progenitors during bladder defect repair.Stem Cell Reports. 2017; 9: 2005-2017Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar, Shin et al., 2011Shin K. Lee J. Guo N. Kim J. Lim A. Qu L. Mysorekar I.U. Beachy P.A. Hedgehog/Wnt feedback supports regenerative proliferation of epithelial stem cells in bladder.Nature. 2011; 472: 110-114Crossref PubMed Scopus (343) Google Scholar). Yet it remains unclear how binucleated polyploid S-cells are produced from mononucleated diploid populations. Here, we identify a population of binucleated I-cells that reside below the superficial layer of the urothelium that are likely to replace S-cells as they die off during homeostasis and regeneration. Using fate mapping, we show that binucleated I-cells are derived from mononucleated I-cells during urothelial homeostasis and regeneration. Using real-time imaging, we show that binucleated I-cells form via endomitosis in which diploid I-cells enter mitosis but fail to complete cytokinesis. Our studies also suggest that binucleated I- and S-cells can further increase ploidy during differentiation, via endoreplication, passing through S phase without proceeding through mitosis. Interestingly, we found that the pathway to polyploidy is likely to be conserved, regardless of which populations act as progenitors. Binucleated I-cells, are produced from a diploid I-cell population during homeostasis and after acute injury; however, diploid I-cells have a limited capacity for division and are depleted after serial or chronic injury. Under these conditions, basal cells replenish the diploid I-cell population, producing S-cells via endomitosis, thereby preserving the urothelial barrier. The urothelium is stratified, containing layers of basal cells, I-cells, and S-cells critical for producing and maintaining the crystalline plaque that serves as the urothelial barrier. These different populations can be distinguished by combinatorial markers (Figure 1M). Krt20 marks mature S-cells, which are also positive for Upk and negative for P63, K5, and Shh (Figures 1A–1H, 1J, 1L, and 1M, yellow arrows denote S-cells). I-cells co-express Upk, P63, and Shh and display low or undetectable expression of Krt20 and Krt5 (Figures 1b–1F, 1H, and 1K–1M, purple arrows denote I-cells). Analysis of Upk2CreERT2;mTmG mice, in which Gfp is localized to the cell membrane, reveals that I-cells (Gandhi et al., 2013Gandhi D. Molotkov A. Batourina E. Schneider K. Dan H. Reiley M. Laufer E. Metzger D. Liang F. Liao Y. et al.Retinoid signaling in progenitors controls specification and regeneration of the urothelium.Dev. Cell. 2013; 26: 469-482Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar) form very close to one another and display fine cytoplasmic projections that connect with the basement membrane (Figures 1C and 1I) The presence of these connections is likely to be important for maintaining the I-cell population, since epithelial cells require basement membrane connections in order to undergo cell division (Le Bras and Le Borgne, 2014Le Bras S. Le Borgne R. Epithelial cell division - multiplying without losing touch.J. Cell Sci. 2014; 127: 5127-5137Crossref PubMed Scopus (24) Google Scholar, Oliferenko et al., 2009Oliferenko S. Chew T.G. Balasubramanian M.K. Positioning cytokinesis.Genes Dev. 2009; 23: 660-674Crossref PubMed Scopus (91) Google Scholar, Sambandamoorthy et al., 2015Sambandamoorthy S. Mathew-Steiner S. Varney S. Zuidema J.M. Gilbert R.J. Van De Water L. LaFlamme S.E. Matrix compliance and the regulation of cytokinesis.Biol. Open. 2015; 4: 885-892Crossref PubMed Scopus (11) Google Scholar). The basal population contains K5-basal cells that co-express Krt5, P63, and Shh and exhibit low or undetectable expression of K14, Upk, or Krt20 (Figures 1B, 1D, 1E, 1G, 1H, 1J, 1K, and 1M, green arrows denote basal cells), and a second smaller population of basal cells (K14-basal cells) that express K14 as well as K5 and P63 (Figure 1J, K14 basal cells are denoted by white arrows). Lineage studies suggest that both I-cells and Basal cells can divide and produce S-cell daughters (Colopy et al., 2014Colopy S.A. Bjorling D.E. Mulligan W.A. Bushman W. A population of progenitor cells in the basal and intermediate layers of the murine bladder urothelium contributes to urothelial development and regeneration.Dev. Dyn. 2014; 243: 988-998Crossref PubMed Scopus (44) Google Scholar, Gandhi et al., 2013Gandhi D. Molotkov A. Batourina E. Schneider K. Dan H. Reiley M. Laufer E. Metzger D. Liang F. Liao Y. et al.Retinoid signaling in progenitors controls specification and regeneration of the urothelium.Dev. Cell. 2013; 26: 469-482Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar, Papafotiou et al., 2016Papafotiou G. Paraskevopoulou V. Vasilaki E. Kanaki Z. Paschalidis N. Klinakis A. KRT14 marks a subpopulation of bladder basal cells with pivotal role in regeneration and tumorigenesis.Nat. Commun. 2016; 7: 11914Crossref PubMed Scopus (96) Google Scholar, Schäfer et al., 2017Schäfer F.M. Algarrahi K. Savarino A. Yang X. Seager C. Franck D. Costa K. Liu S. Logvinenko T. Adam R. Mauney J.R. Mode of surgical injury influences the source of urothelial progenitors during bladder defect repair.Stem Cell Reports. 2017; 9: 2005-2017Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar, Shin et al., 2011Shin K. Lee J. Guo N. Kim J. Lim A. Qu L. Mysorekar I.U. Beachy P.A. Hedgehog/Wnt feedback supports regenerative proliferation of epithelial stem cells in bladder.Nature. 2011; 472: 110-114Crossref PubMed Scopus (343) Google Scholar). Regardless of which urothelial populations serve as progenitors, it is still unclear how S-cells, which are post-mitotic, binucleated, and polyploid, are formed from basal or I-cells, which are mononucleated and diploid. Because S-cells are post-mitotic, their binucleation is likely to occur in either basal or I-cell progenitors before they begin to differentiate into S-cells. To begin to address this, we analyzed the adult urothelium during homeostasis, when there is little, if any, proliferation, to determine whether we could detect a binucleated population of I-cells or basal cells that could be S-cell precursors. To distinguish mononucleated cells from binucleated cells, we used E-cadherin, which stains adherens junctions to visualize cell boundaries, and DAPI staining to label nuclei. Combinatorial markers were used to distinguish different urothelial sub-populations (Figure 1M). This analysis revealed a previously uncharacterized population of I-cells that are binucleated and reside just below the S-cell layer (Figures 1E and 1K, double purple arrows denote binucleated I-cells). Analysis of whole-mount preps from Upk2CreERT2;mTmG mice revealed that binucleated I-cells are considerably smaller than S-cells, with smaller nuclei compared to S-cells, suggesting that both cell size and DNA content change during S-cell differentiation (Figures 1F and 1L). Consistent with this, DNA content analysis by flow cytometry and Integrated DAPI fluorescence from adult urothelial samples at homeostasis, indicate that binucleated I-cells have a DNA content of about 4n (2n+2n) (Figure S1), half that compared to S-cells, which generally have a DNA content of 8n (4n+4n) (Figure S1). Based on quantification of cell numbers in paraffin-embedded tissue, binucleated I-cells account for about 11% of the total I-cell population during homeostasis, the remainder of which are mononucleated I-cells. The presence of a binucleated I-cell population that resides below the superficial layer of the urothelium suggests that these cells may be progenitors that can quickly replace S-cells upon injury, thereby preserving the urothelial barrier. The existence of a newly identified binucleated I-cell sub-population, raises the possibility that these are direct S-cell progenitors. Recent studies suggest that both I-cells and basal cells can contribute to the S-cell population, depending on the extent of urothelial injury (Schäfer et al., 2017Schäfer F.M. Algarrahi K. Savarino A. Yang X. Seager C. Franck D. Costa K. Liu S. Logvinenko T. Adam R. Mauney J.R. Mode of surgical injury influences the source of urothelial progenitors during bladder defect repair.Stem Cell Reports. 2017; 9: 2005-2017Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar). To determine which populations produce binucleated I-cells, we performed fate-mapping experiments using the Upk3aGCE;mCherry and Krt5CreERT2;mTmG mice to label I-cells and basal cells, respectively, and then we compared their respective contributions to different urothelial sub-populations after regeneration in response to acute injury. We first used UTI, a well-studied model of acute injury and repair (Hung et al., 2009Hung C.S. Dodson K.W. Hultgren S.J. A murine model of urinary tract infection.Nat. Protoc. 2009; 4: 1230-1243Crossref PubMed Scopus (210) Google Scholar), to induce regeneration. For these experiments, females received a transurethral inoculation of UTI 89, a strain of Uropathogenic E. coli (UPEC) isolated from a patient with cystitis (Garofalo et al., 2007Garofalo C.K. Hooton T.M. Martin S.M. Stamm W.E. Palermo J.J. Gordon J.I. Hultgren S.J. Escherichia coli from urine of female patients with urinary tract infections is competent for intracellular bacterial community formation.Infect. Immun. 2007; 75: 52-60Crossref PubMed Scopus (139) Google Scholar). UPEC infects S-cells and establishes intracellular bacterial communities 6- and 12-hr post-infection (p.i.) (Figure S2A, white arrows). UPEC infected S-cells subsequently undergo exfoliation, which occurs between 6- and 12-hr post-inoculation (Figure S2B; arrows denote exfoliating S-cells). Basal and I-cells survive UTI and undergo a burst of cell-cycle activity 24 hr p.i., evidenced by expression of Ki67 and EdU, which is taken up during S phase, markers that are rarely detectable in the homeostatic urothelium, which is virtually quiescent (Jost, 1986Jost S.P. Renewal of normal urothelium in adult mice.Virchows Arch. B Cell Pathol. Incl. Mol. Pathol. 1986; 51: 65-70Crossref PubMed Scopus (23) Google Scholar, Jost and Potten, 1986Jost S.P. Potten C.S. Urothelial proliferation in growing mice.Cell Tissue Kinet. 1986; 19: 155-160PubMed Google Scholar) (Figures 2A–2E, 2H, 2I , S2D, and S2E). A new S-cell layer is established by 72-hr p.i. and regeneration is complete 2 weeks after infection when Krt20 expression is restored in the S-cell layer (Figure S2C). The Upk3aGCE;mCherry line has a labeling efficiency of about 56%, and Cre-dependent recombination will label I-cells and their daughters with mCherry (Gandhi et al., 2013Gandhi D. Molotkov A. Batourina E. Schneider K. Dan H. Reiley M. Laufer E. Metzger D. Liang F. Liao Y. et al.Retinoid signaling in progenitors controls specification and regeneration of the urothelium.Dev. Cell. 2013; 26: 469-482Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar, Van Batavia et al., 2014Van Batavia J. Yamany T. Molotkov A. Dan H. Mansukhani M. Batourina E. Schneider K. Oyon D. Dunlop M. Wu X.R. et al.Bladder cancers arise from distinct urothelial sub-populations.Nat. Cell Biol. 2014; 16: 982-991Crossref PubMed Scopus (148) Google Scholar). Analysis of Upk3aGCE;mCherry mice between 24 hr and 2 weeks p.i. revealed mCherry expression labeling 44% of mononucleated I -cells and 51% of the binucleated I-cell population 24 hr after infection, 43% of S-cells by 72 hr, as well as a small number (5%) of basal cells (Figures 2A–2E, 2N, and S2F–S2I). Importantly, we identified clones containing lineage-marked binucleated I-cells situated just below a lineage-marked binucleated S-cell, as well as clones containing two diploid I-cell and two likely daughters (Figures 2F and 2G), suggesting that I-cells can produce a binucleated population as well as divide, and produce diploid I-cells. Similar findings were observed using the Cyclophosphamide (CPP) model to induce a single round of regeneration. In this case, we used the Up2CreERT2;mTmG line, which has a labeling efficiency of 89% to mark I-cells and their daughters, and we analyzed the distribution of lineage-marked (Gfp+) diploid (mononucleated) I-cells, binucleated I- and S-cells 48 hr after CPP treatment, when proliferation peaks (Figures 2J, 2K, and S2N–S2U). These studies revealed Gfp labeling in 73% of I-cells and binucleated I-cells, and 87% of S-cells (Figure 5A), supporting the idea that I-cells produce binuclear I- and S-cells after acute injury. To examine whether basal cells also contribute to the I- or S-cell populations, we used the Krt5CreERT2;mTmG line, which has a labeling efficiency of about 84%, to follow the fate of basal cells during regeneration. Analysis of the distribution of Gfp+ cells in Krt5CreERT2;mTmG mice 24 hr after UTI revealed extensive labeling (greater than 80%) in the basal population as well as 3% of mononucleated I-cells and 8% of binucleated I-cells, while analysis at 72 hr when newly formed S-cells are present, revealed labeling in 3% of the S-cell population (Figure S2O). These observations indicate that basal cells contribute to the I-cell population. Analysis of regeneration in Krt5CreERT2;mTmG mice using the CPP model of injury and repair revealed a higher rate of contribution to the I- and S-cell lineages. In this case, about 20% of the diploid I-cell population were lineage marked, as well as, 16% of the binucleated I-cell population and 7.5% of the S-cell population after 1X CPP (Figures 2M and 5B). Together, these studies suggest that I-cells are able to divide and to produce binucleated I- and S-cell daughters after acute injury, while K5-basal cells can contribute to the I-cell population after UTI, but make a more significant contribution to the I-cell population after CPP. These findings also raise the possibility that the UTI and CPP injury models affect the behavior of basal and I-cell progenitors differently. Binucleated cells can be produced by cell fusion of mononucleated cells or by endomitosis, in which cells progress partway through mitosis, but fail to complete cytokinesis and daughter cell separation (Duncan et al., 2010Duncan A.W. Taylor M.H. Hickey R.D. Hanlon Newell A.E. Lenzi M.L. Olson S.B. Finegold M.J. Grompe M. The ploidy conveyor of mature hepatocytes as a source of genetic variation.Nature. 2010; 467: 707-710Crossref PubMed Scopus (365) Google Scholar, Fortier et al., 2017Fortier M. Celton-Morizur S. Desdouets C. Incomplete cytokinesis/binucleation in mammals: The powerful system of hepatocytes.Methods Cell Biol. 2017; 137: 119-142Crossref PubMed Scopus (9) Google Scholar, DePamphilis, 2016DePamphilis M.L. Genome duplication: the heartbeat of developing organisms.Curr. Top. Dev. Biol. 2016; 116: 201-229Crossref PubMed Scopus (3) Google Scholar, Rios et al., 2016Rios A.C. Fu N.Y. Jamieson P.R. Pal B. Whitehead L. Nicholas K.R. Lindeman G.J. Visvader J.E. Essential role for a novel population of binucleated mammary epithelial cells in lactation.Nat. Commun. 2016; 7: 11400Crossref PubMed Scopus (58) Google Scholar). If binucleated I-cells are produced by cell fusion, we would expect nuclei to be derived from two different mononucleated I-cells, and hence distributed randomly. Whereas, if binucleated cells arise via endomitosis, we expect that both nuclei would be derived from the same I-cell of origin. To distinguish between these possibilities, we performed fate mapping using Upk2CreERT2;nTnG mice. In this line, cells that undergo Cre-dependent recombination express nuclear Gfp and in cells that have not undergone recombination, nuclei are Tomato positive (Figure 3A). Upk2CreERT2;nTnG mice were treated with tamoxifen to induce recombination, infected with UPEC 2 weeks later, then analyzed 24- or 72-hr p.i. to evaluate the distribution of nuclear Gfp and Tomato. This analysis revealed 522/522 instances in which both nuclei were Gfp+, Tomato+, or negative for both markers and no instances in which marker expression was in one nucleus, but not the other (Figure 3B), strongly suggesting that binucleation occurs via a mechanism other than cell fusion. The failure to observe evidence of mononucleated I-cell fusion after UTI suggests that binucleated I-cells form via endomitosis, entering the cell cycle, and exiting mitosis prior to completing cytokinesis. To begin to investigate how binucleation occurs, we analyzed sections of urothelium from wild-type adult mice 24 hr after infection with UPEC to identify mitotic cells and binucleated cells that are likely products of endomitosis. Urothelial sections were stained for expression of mitotic markers including aurora B kinase, survivin, and anillin. Aurora B and survivin, which are components of the chromosome passenger complex (CPC), localize to the nucleus and centromeres during early mitosis and to the central spindle and cell cortex during cytokinesis (Caldas et al., 2005Caldas H. Jiang Y. Holloway M.P. Fangusaro J. Mahotka C. Conway E.M. Altura R.A. Survivin splice variants regulate the balance between proliferation and cell death.Oncogene. 2005; 24: 1994-2007Crossref PubMed Scopus (177) Google Scholar, Carmena and Earnshaw, 2003Carmena M. Earnshaw W.C. The cellular geography of aurora kinases.Nat. Rev. Mol. Cell Biol. 2003; 4: 842-854Crossref PubMed Scopus (994) Google Scholar). Anillin accumulates at the contractile ring and midbody in late anaphase or telophase during cytokinesis and is localized in the nucleus in late G1, G2, and S phases (Field and Alberts, 1995Field C.M. Alberts B.M. Anillin, a contractile ring protein that cycles from the nucleus to the cell cortex.J. Cell Biol. 1995; 131: 165-178Cro" @default.
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• W2896955363 creator A5091720522 @default.
• W2896955363 date "2018-10-01" @default.
• W2896955363 modified "2023-10-18" @default.
• W2896955363 title "Polyploid Superficial Cells that Maintain the Urothelial Barrier Are Produced via Incomplete Cytokinesis and Endoreplication" @default.
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• W2896955363 doi "https://doi.org/10.1016/j.celrep.2018.09.042" @default.

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