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Lgr5 marks stem/progenitor cells in ovary and tubal epithelia

Abstract

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.

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Figure 1: Lgr5 expression in the developing ovary and ovary-associated structures.
Figure 2: Lgr5 expression in the adult mouse and human ovary and ovary-associated structures.
Figure 3: The adult Lgr5+ OSE transcriptome.
Figure 4: Embryonic and neonate Lgr5+ populations are early stem/progenitor cells of adult OSE lineages in vivo.
Figure 5: Clonal output and characterization of embryonic Lgr5+ cell progeny in vivo.
Figure 6: Adult Lgr5+ OSE populations are capable of epithelial regenerative repair.
Figure 7: Embryonic and neonate Lgr5+ populations are early stem/progenitor cells of the epithelial cell lineage of the oviduct/fimbria and mesovarian ligament in vivo.
Figure 8: Proposed model of Lgr5+ stem/progenitor cell activity during ovary development and adult homeostasis/regenerative repair.

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Acknowledgements

We thank A. Ong, A. Wong and A. Tan for technical assistance, G. Wright and J. Lim for imaging assistance, S. Mustafah for FACS experiments, and D. Tan for technical expertise and manuscript proofreading. We also thank M. Choolani and B. Pang for providing human material and critical discussions before publication. N.B. is supported by the Agency for Science, Technology and Research (A*STAR), Singapore.

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Authors and Affiliations

Authors

Contributions

A.N. designed and performed experiments, analysed data and wrote the manuscript. S.T., P.R. and Y.S. performed microarray experiments and bioinformatics analyses. G.S. performed mouse injections. T.Z.T. and R.Y-J.H. conducted the gene set enrichment analyses. M.L. performed proliferation experiments and provided technical expertise. N.B. supervised the project, analysed the data and wrote the manuscript.

Corresponding author

Correspondence to Nick Barker.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Lgr5 expression in the Lgr5–LacZ reporter knock-in mouse.

P1: Lgr5LacZ expression on the ovary surface (black arrow) and sub-surface (arrowheads), as well as oviduct and fimbria (red arrow). P7: Lgr5LacZ expression in the OSE (arrow). Adult: Lgr5LacZ expression in the OSE (arrow), fimbrial base (black arrowhead), mesovarian ligament (white arrows) and mesovarian-fimbria junction (white arrowhead). WT insets show wild-type littermate ovaries. Neutral red counter-stain. Scale bars: 100 μm (wholemount images); 10 μm (sections).

Supplementary Figure 2 Active Wnt signaling in P7 and adult Lgr5–EGFP+ cells.

(Top) P7 Lgr5-KI/Axin-LacZ ovary section stained for LacZ activity (left) then co-immunostained with EGFP and K8 (right). Arrows point to Axin2+ Lgr5–EGFPhi OSE cells. (Bottom) An adult Lgr5-KI/Axin-LacZ ovary section stained for LacZ activity (left) then co-immunostained with EGFP–HRP (middle). Black and white arrowheads point to Axin2+Lgr5–EGFPhi and Axin2+Lgr5–EGFPlo/neg OSE cells respectively. Neutral red counter-stain. qPCR with Axin2 (right) confirming expression in sorted Lgr5–EGFPhi, Lgr5–EGFPlo and Lgr5–EGFPneg OSE cells, but not in OSE-stripped ovary cells. Histograms represent fold-change relative to Lgr5–EGFPneg cells set as 1. Error bars indicate s.e.m. Gapdh was used as endogenous reference. Scale bars: 10 μm.

Supplementary Figure 3 Spatial distribution of Lgr5–EGFP+ and Aldh1+ cells in the adult ovary.

(Top) EGFP immunostaining of adult Lgr5-KI ovary sections, revealing Lgr5–EGFP+ cells concentrated at cleft regions between growing follicles (a,b) and corpora lutea (c,d). Note OSE cells at surface of corpora lutea were devoid of Lgr5 expression (arrowhead in d) except at ovulating follicles (that is, prospective corpora lutea) comprising stigma-associated OSE/tunica albuginea (arrows in e,f) and rupturing OSE (g,h). F, follicle; CL, corpus luteum; Ov, ovulating follicle; R, rupture site. (Bottom) EGFP/Aldh1 (detecting a1 subunit) co-immunostaining of adult Lgr5-KI ovary sections, revealing Aldh1 expression within OSE cells at the ovary hilum (1) and anterior regions (2,3). Note Aldh1 expression in both Lgr5–EGFPlo (arrows) and Lgr5–EGFPhi (arrowheads) OSE cells. Also note Aldh1 expression in non-OSE cells within the ovary interior. Scale bars: 10 μm.

Supplementary Figure 4 Lgr5 and Aldh1a1/a2 co-FISH in the adult ovary and gene set enrichment analysis (GSEA).

Co-FISH of Lgr5 and Aldh1a1/a2 on adult wild-type ovary sections, confirming Aldh1a1/a2 transcripts within OSE cells at the ovary hilum (1,5) and anterior regions (2,3,6,7). Bright white dots represent positive FISH signals. OSE cells are denoted above yellow dashed lines. Note presence of Aldh1a1/a2 transcripts in Lgr5+ (arrowheads) and Lgr5 (arrows) cells. RNase A-treated control sections revealed specificity of Lgr5 and Aldh1a1/a2 transcripts (4,8). GSEA reveals no significant gene set enrichment of the published Aldh1+ signature (Flesken-Nikitin et al., 2013) in Lgr5–EGFPhi OSE cells (P = 0.24). ES, enrichment score. Scale bars: 10 μm.

Supplementary Figure 5 Lgr5 and Troy co-FISH in the adult ovary.

Co-FISH of Lgr5 and Troy on adult wild-type ovary sections, demonstrating enriched Troy mRNA expression in Lgr5+ OSE cells. Bright white dots represent positive FISH signals. OSE cells are denoted above yellow dashed lines. Note similar expression profile of Troy to that of Lgr5, with abundant transcripts present in OSE cells around a follicle but absent atop a corpus luteum. RNase A-treated sections revealed specificity of Lgr5 and Troy transcripts. F, follicle; CL, corpus luteum. Scale bars: 10 μm.

Supplementary Figure 6 Lgr5–EGFP/Wnt4 and Lgr5–EGFP/Id2 co-IHC.

(Top) EGFP/Wnt4 co-immunostaining of adult Lgr5-KI ovary sections, demonstrating enriched Wnt4 expression in Lgr5–EGFPhi OSE cells (arrowheads). (Bottom) EGFP/Id2 co-immunostaining of adult Lgr5-KI ovary sections, demonstrating down-regulation of Id2 expression in Lgr5–EGFPhi OSE cells. Arrows point to Id2Lgr5–EGFPhi OSE cells. Arrowheads point to Id2+Lgr5–EGFPneg/lo OSE cells. Scale bars: 10 μm.

Supplementary Figure 7 Neonate Lgr5+ populations and their progeny are lifelong contributors of the adult OSE lineage in vivo.

(Top) Wholemount and histological analyses of LacZ activity in ovary at various times post P1-induction. A single limiting dose of 4-OHT induced LacZ activity at clonal density. Sparse isolated LacZ+ cells were first observed 2 days (2d) p.i. Uniform rate of expansion pre-puberty led to similar-sized LacZ+ clones 1 week (1w) and 4 weeks (4w) p.i., whilst variable rate of expansion post-puberty led to heterogenous-sized LacZ+ clones at 3 months (3m) p.i. Frequency of lineage tracing was maintained up to 16 months (16m). Note larger-sized clones adjacent to corpora lutea (red arrowheads) compared to follicles (white arrowheads). Dot plot shows LacZ+ OSE clone size quantification post P1-induction. n = 5 females/time-point, total of 461 clones counted. Each dot represents a disparate LacZ+ OSE clone. Red bars indicate s.d. ranges. Green circles denote mean clone size, which steadily increased over time (green line). F, follicle; CL, corpus luteum. (Bottom) Confocal z-stack images of Lgr5-KI/tdTomato ovary treated with an increased dose of 4-OHT at various times post P1-induction, revealing steady increase in the number of tdTomato+ progeny over time. Scale bars: 100 μm (wholemount images); 10 μm (sections).

Supplementary Figure 8 Adult Lgr5+ cells beyond the hilum are capable of OSE generation.

Confocal z-stack images of near-native Lgr5-KI/Rosa26-tdTomato ovary slices 2 days (2d), 2 weeks (2w) and 4 months (4m) post adult-induction (p.i.). At 2 days p.i., 29.5% of Lgr5–EGFPhi cells have activated the tdTomato reporter (432 tdTomato+ cells out of 1463 Lgr5-GFPhi OSE cells counted). These cells rapidly expanded to form tdTomato+ clones throughout the ovary surface encompassing the ovary hilum and anterior ovary 2w and 4m p.i. Arrowheads point to larger-sized clones at the edges or atop corpora lutea. White dashed line demarcates ovary-mesovarian junction. Yellow dashed lines demarcate follicles. Scale bars: 10 μm.

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Ng, A., Tan, S., Singh, G. et al. Lgr5 marks stem/progenitor cells in ovary and tubal epithelia. Nat Cell Biol 16, 745–757 (2014). https://doi.org/10.1038/ncb3000

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