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Wnt Signaling Induces Proliferation of Sensory Precursors in the Postnatal Mouse Cochlea

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Wnt Signaling Induces Proliferation of Sensory Precursors in the Postnatal Mouse Cochlea Wnt signaling induces proliferation of sensory precursors in the postnatal mouse cochlea Renjie Chaia,1, Bryan Kuob,1, Tian Wanga, Eric J. Liawa, Anping Xiaa, Taha A. Jana,c, Zhiyong Liub, Makoto M. Taketod, John S. Oghalaia, Roeland Nussec,e,2, Jian Zuob, and Alan G. Chenga,2 Departments of aOtolaryngology-Head and Neck Surgery and eDevelopmental Biology and cHoward Hughes Medical Institute, Stanford University School of Medicine, Stanford, CA 94305; bDepartment of Developmental Neurobiology, St. Jude Children’s Research Hospital, Memphis, TN, 38103; and dDepartment of Pharmacology, Graduate School of Medicine, Kyoto University, Kyoto 606-8501, Japan Contributed by Roeland Nusse, April 9, 2012 (sent for review February 20, 2012) Inner ear hair cells are specialized sensory cells essential for found that these cells exhibit such progenitor behavior. These auditory function. Previous studies have shown that the sensory results corroborated with those of White et al. (7), who isolated epithelium is postmitotic, but it harbors cells that can behave as supporting cells using the p27(Kip1)-GFP mouse line. When progenitor cells in vitro, including the ability to form new hair p75-nerve growth factor receptor was used as an enrichment cells. Lgr5, a Wnt target gene, marks distinct supporting cell types marker, hair cell formation increased, raising the possibility that in the neonatal cochlea. Here, we tested the hypothesis that distinct supporting cell types are more capable of differentiating Lgr5+ cells are Wnt-responsive sensory precursor cells. In contrast into hair cells in vitro. To date, the in vivo behavior of cochlear to their quiescent in vivo behavior, Lgr5+ cells isolated by flow supporting cells in the postnatal period and the role of Wnt cytometry from neonatal Lgr5EGFP-CreERT2/+ mice proliferated and signaling in regulating their behavior remain unclear. formed clonal colonies. After 10 d in culture, new sensory cells Wnt signaling plays a critical role in regulating tissue ho- formed and displayed specific hair cell markers (myo7a, calretinin, meostasis, including the maintenance of somatic stem cells (10). parvalbumin, myo6) and stereocilia-like structures expressing F-ac- Leucine-rich repeat-containing G-protein coupled receptor 5 tin and espin. In comparison with other supporting cells, Lgr5+ cells (Lgr5), a Wnt target gene, has been shown to mark endogenous were enriched precursors to myo7a+ cells, most of which formed stem cells in rapidly proliferating organs (11, 12). In the postnatal without mitotic division. Treatment with Wnt agonists increased cochlea we have shown that Lgr5 expression is Wnt dependent proliferation and colony-formation capacity. Conversely, small- and limited to supporting cell subtypes (13). The current study molecule inhibitors of Wnt signaling suppressed proliferation demonstrates that Lgr5+ cells behave as hair cell precursors as + without compromising the myo7a cells formed by direct differen- supported by fate-mapping studies. In culture, they exhibited + tiation. In vivo lineage tracing supported the idea that Lgr5 cells progenitor cell ability and formed clonal colonies and new hair + EGFP-CreERT2/+ give rise to myo7a hair cells in the neonatal Lgr5 co- cells. Moreover, both in vitro and in vivo, Wnt signaling enhanced chlea. In addition, overexpression of β-catenin initiated prolifera- proliferation of Lgr5+ cells. Together, these data indicate that + tion and led to transient expansion of Lgr5 cells within the Lgr5 marks Wnt-regulated sensory precursor cells in the postnatal BIOLOGY cochlear sensory epithelium. These results suggest that Lgr5 marks cochlea. DEVELOPMENTAL sensory precursors and that Wnt signaling can promote their pro- liferation and provide mechanistic insights into Wnt-responsive Results progenitor cells during sensory organ development. Isolated Lgr5+ Cells Behave as Progenitor Cells in Vitro. We pre- viously characterized the Lgr5EGFP-CreERT2/+ mouse and found it to regeneration | hearing | transdifferentiation | stem cells have normal cochlear morphology and hearing thresholds (11, 13). In the cochleae of neonatal Lgr5EGFP-CreERT2/+ mice, GFP is egeneration of sensory hair cells causes hearing loss and coexpressed with Sox2 in the third Deiters’ cells, inner pillar Ddeafness, a sensory disorder affecting more than 48 million cells, inner phalangeal cells, and lateral greater epithelial ridge Americans (1). In the mature mammalian cochlea, hair cell loss cells (Fig. 1 A–C). When the thymidine analog 5-ethynyl-2′- is considered irreversible because no hair cell regeneration has deoxyuridine (EdU) was injected on postnatal day (P) 0–2 been observed (2). Conversely, in nonmammalian vertebrates (50 mg/kg two times/d), no labeling was noted in the sensory ep- including birds and amphibians, supporting cells are capable of ithelium (Fig. 1D), confirming previous findings that supporting regenerating lost hair cells to restore hearing function (3, 4). cells are mitotically quiescent (6, 14). Next, we dissociated P0–3 Pulse–chase experiments found that supporting cells proliferated cochleae from Lgr5EGFP-CreERT2/+ miceandisolatedGFP+ cells before differentiating into new hair cells in the avian cochlea, but via flow cytometry; GFP+ cells constituted ∼2.1% of viable cells they also could acquire a hair cell fate directly without dividing, (Fig. 1E). Immunostained Lgr5+ cells were 94% GFP+,96% − a process termed “direct differentiation” (5). Sox2+,and0%myo7a+ (Fig. 1 F and G), whereas Lgr5 cells The developing mammalian cochlear epithelium becomes were 0% GFP+,3%Sox2+, and 1% myo7a+ (Fig. S1 A and B). mitotically quiescent between embryonic day (E) 12.5 and 14.5 Quantitative RT-PCR showed that sorted Lgr5+ cells, in com- − and expresses the cell-cycle inhibitor p27(Kip1), which marks parisonwithLgr5 cells, expressed higher levels of Lgr5 and Sox2 the prosensory region (6). Sensory hair cells emerge in this re- gion and become organized in a checkerboard pattern and are interspersed with supporting cells. Although the postnatal sen- Author contributions: R.C., B.K., J.Z., and A.G.C. designed research; R.C., B.K., T.W., E.J.L., sory epithelium remains mitotically quiescent, several recent A.X., T.A.J., Z.L., and A.G.C. performed research; M.M.T., R.N., J.Z., and A.G.C. contributed new reagents/analytic tools; R.C., B.K., E.J.L., T.A.J., Z.L., J.S.O., R.N., J.Z., and A.G.C. ana- studies have reported isolation of cells with proliferative capacity lyzed data; and R.C., B.K., T.A.J., Z.L., J.S.O., J.Z., and A.G.C. wrote the paper. from the neonatal cochlea. When isolated cochlear cells were The authors declare no conflict of interest. cultured in growth factor or serum-supplemented conditions, 1R.C. and B.K. contributed equally to this work. they proliferated, demonstrated colony-formation capacity, and 2To whom correspondence may be addressed. E-mail: [email protected] or aglcheng@ also generated new hair cell-like cells (7, 8). To begin identifying stanford.edu. cells with progenitor cell potentials, Sinkkonen and colleagues This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. + low low (9) isolated CD326 /CD146 /CD271 supporting cells and 1073/pnas.1202774109/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1202774109 PNAS | May 22, 2012 | vol. 109 | no. 21 | 8167–8172 Fig. 1. Lgr5+ cochlear supporting cells act as progenitor cells in vitro. (A) Cryosection of P3 Lgr5EGFP-CreERT2/+ cochlea showed GFP expression in the third Deiters’ cells (DC), inner pillar cells (PC), inner phalangeal cells (IPC), and the lateral greater epithelial ridge (GER). IHC, inner hair cells; LER, lesser epithelial ridge; OHC, outer hair cells. (B) Schematic depicting cell types in the P0–3 cochlea. BC, Boettcher cells; CC, Claudius cells; HEC, Hensen’s cells; SG, spiral ganglia; TBC, tympanic border cells. (C) Sox2 is expressed in Lgr5+ cells and other supporting cell types. (D) Cochlea from P3 mouse injected with EdU (P0–2) dem- − onstrated no EdU uptake in the sensory epithelium. (E) Lgr5EGFP-CreERT2/+ cochleae were dissociated, and GFP+ and GFP cells were isolated using flow cytometry. (F–H) Immunostaining and quantitative PCR showed that isolated GFP+ cells did not contain myo7a+ cells and robustly expressed Lgr5 and Sox2 but not brn3.1.(I and J)Lgr5+ cells isolated from Lgr5EGFP-CreERT2/+; Actin-DsRed mice were mixed (1:1) with those from Lgr5EGFP-CreERT2/+ mice and cultured. After 10 d, the majority of cytokeratin-positive colonies formed were monochromatic. I and J show DsRed and DAPI labeling only. I′ and J′ show DsRed, cytokeratin − (CK), and DAPI labeling. (K)Lgr5+ and Hes5+ cells formed more colonies than Lgr5 cells. Forty percent of colonies from Lgr5+ cells contained myo7a+ cells. − (L–N)Lgr5+ cells generated more myo7a+ cells than Lgr5 or Hes5+ cells, most of which were outside (O/S) colonies. I/S, inside colonies. (O) In the presence of EdU, only a minority of myo7a+ cells were EdU+; double-positive cells were noted more commonly inside colonies. Data are represented as mean ± SD. **P < 0.01. In F, G, I–K, N, and O, n is shown in parentheses. (Scale bars, 25 μm.) and lower levels of the hair cell marker Brn3.1 (Fig. 1H and Table we immunostained and characterized two classes of myo7a+ cells: S1) (15). These data indicate that sorted Lgr5+ supporting cells those residing inside or outside epithelial colonies. On average, were highly pure. the majority of myo7a+ cells (308 of 389) resided outside colo- − To study the behavior of Lgr5+ cells, we cocultured 5,000 nies (Fig. 1 L–N). In comparison with Hes5+ and Lgr5 cells, Lgr5+ cells with mitomycin-inactivated feeder cells derived from Lgr5+ cells generated significantly more myo7a+ cells. With embryonic chicken utricle mesenchyme (Fig. S2). Inner ear-de- EdU supplementation, 11.0% of myo7a+ cells within epithelial rived mesenchymal tissues have been shown to foster differen- colonies were labeled. We observed a correlation between tiation of cochlear supporting cells (7, 9).
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