ARTICLES Lgr5 marks stem/progenitor cells in ovary and tubal epithelia Annie Ng 1 , Shawna Tan 1 , Gurmit Singh 1 , Pamela Rizk 1 , Yada Swathi 1 , Tuan Zea Tan 2 , Ruby Yun-Ju Huang 2,3 , Marc Leushacke 1 and Nick Barker 1,4,5,6 The ovary surface epithelium (OSE) undergoes ovulatory tear and remodelling throughout life. Resident stem cells drive such tissue homeostasis in many adult epithelia, but their existence in the ovary has not been definitively proven. Lgr5 marks stem cells in multiple epithelia. Here we use reporter mice and single-molecule fluorescent in situ hybridization to document candidate Lgr5 + stem cells in the mouse ovary and associated structures. Lgr5 is broadly expressed during ovary organogenesis, but becomes limited to the OSE in neonate life. In adults, Lgr5 expression is predominantly restricted to proliferative regions of the OSE and mesovarian–fimbria junctional epithelia. Using in vivo lineage tracing, we identify embryonic and neonate Lgr5 + populations as stem/progenitor cells contributing to the development of the OSE cell lineage, as well as epithelia of the mesovarian ligament and oviduct/fimbria. Adult Lgr5 + populations maintain OSE homeostasis and ovulatory regenerative repair in vivo. Thus, Lgr5 marks stem/progenitor cells of the ovary and tubal epithelia. Little is known about the underlying mechanisms governing epithelial homeostasis in the ovary and oviduct 1 . In many adult epithelia, resident stem cells are critical effectors of tissue renewal 2–5 and cancer initiation following genetic mutation 2,6 . In the ovary and oviduct, efforts to establish the existence of stem cells have long been frustrated by the lack of available stem cell markers. Several studies have identified a subset of epithelia, juxtaposed at the edges of ovulatory follicles and fimbrial fringes that exhibit surrogate features of stemness 7–10 . The stem cell marker lymphocyte antigen 6 complex locus A Ly6a (Sca-1) also highlights a side-population- enriched OSE subpopulation 9 . However, a formal demonstration of stem cell function for these epithelial subpopulations is lacking. More recently, a pool of aldehyde dehydrogenase 1 (Aldh1)-expressing OSE cells restricted to the adult ovary hilum, enriched for stem cell markers including Lgr5, was shown to be capable of replenishing ovary epithelia in vivo 11 , providing the most compelling evidence of a stem cell niche in the ovary hilum. We have previously identified the Wnt target gene Lgr5 as a marker of stem cells in various epithelia including the small intestine, colon, stomach, hair follicle and kidney 2–5 . Here, we employ reporter mice and sensitive single-molecule fluorescent in situ hybridization (FISH) analyses to document the existence of Lgr5-expressing cells in the ovary and associated structures, and evaluate their endogenous stem/progenitor cell identity using in vivo lineage tracing. RESULTS Lgr5 expression in developing ovary and oviduct/fimbria Quantitative PCR (qPCR) identified Lgr5 transcripts in the adult ovary (Fig. 1a). Using Lgr5–EGFP–ires–CreER T 2 (Lgr5-KI ) reporter mice, Lgr5 + cells were first observed at embryonic day 13.5 (E13.5), scat- tered throughout the ovary surface and subsurface (Fig. 1b). At postna- tal day 1 (P1), Lgr5 + surface cells were readily detectable, whereas the number of Lgr5 + subsurface cells was diminished (Fig. 1b). By P7, Lgr5 expression was restricted to the ovary surface (Fig. 1b). Independent FISH analyses 12 confirmed the presence of Lgr5 transcripts in surface and subsurface cells at P1, and in surface cells at P7 (Fig. 1c). Co-immunofluorescence of EGFP and cytokeratin 8 (K8) revealed the epithelial identity of Lgr5 + surface cells in P1 ovaries (Fig. 1d). Lgr5 + subsurface cells did not express K8, but co-labelled with the granulosa marker Foxl2 (arrowheads, Fig. 1d). Most of the Foxl2 + granulosa precursors situated within the ovary interior were, however, Lgr5 − (Fig. 1d). Oocytes, marked by Ddx4, were also Lgr5 − (Fig. 1d). qPCR analysis of Lgr5 expression in fluorescence-activated cell sorted (FACS) EGFP hi cells confirmed their identity as the ovary-resident 1 A-STAR Institute of Medical Biology, 8A Biomedical Grove, 06-06 Immunos, 138648, Singapore. 2 Cancer Science Institute of Singapore, National University of Singapore, 117599, Singapore. 3 Department of Obstetrics & Gynaecology, National University Hospital, 119228, Singapore. 4 Centre for Regenerative Medicine, 47 Little France Crescent, University of Edinburgh, EH16 4TJ, UK. 5 Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore, 117596, Singapore. 6 Correspondence should be addressed to N.B. (e-mail: nicholas.barker@imb.a-star.edu.sg) Received 29 November 2013; accepted 29 May 2014; published online 6 July 2014; DOI: 10.1038/ncb3000 NATURE CELL BIOLOGY VOLUME 16 | NUMBER 8 | AUGUST 2014 745 © ƐƎƏ4 Macmillan Publishers Limited. All rights reserved.