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 proliferation and provide mechanistic insights into Wnt-responsive Results progenitor cells during sensory organ development. Isolated Lgr5+ Cells Behave as Progenitor Cells in Vitro. We previously 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 region 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). These mesenchymal myo7a+ cells residing outside colonies and decreased EdU la- cells do not express hair cell or supporting cell markers (16). beling (Fig. 1O), suggesting that the majority of hair cells were After 10 d in serum-free medium, Lgr5+ cells formed epithelial generated via direct differentiation. In support of their hair cell colonies (consisting of at least five DAPI+ cells), which were identity, myo7a+ cells also expressed other hair cell markers: immunostained with the pan-cytokeratin antibody (Fig. 1 I and calretinin, parvalbumin, and myo6 (Fig. S4 A–C). In addition to − J). When Lgr5+ cells from Lgr5EGFP-CreERT2/+;Actin-DsRed mice myo7a+ cells, many Sox2+ and GFP cells were found both inside were mixed (1:1) with those from Lgr5EGFP-CreERT2/+ animals, and outside colonies (Fig. S4 C and G). Seventy-one percent of 99% of colonies were monochromatic, suggesting that they were myo7a+ cells exhibited polarized protrusions expressing F-actin clonally derived from single cells (Fig. 1 I and J and Fig. S2B). and the actin-bundling protein espin (Fig. S4 D–F) (19). These To determine if Lgr5 serves as a marker for sensory progenitor protrusions occasionally are V-shaped and resemble stereocilia cell enrichment, we compared it with Hes5, which is a Notch of nascent hair cells. These results show that Lgr5+ cells can act as target gene expressed in most cochlear supporting cell types sensory progenitors and led us to investigate their in vivo behavior. (Fig. S3 A and B) (17). We isolated and cultured Hes5+ cells from cochleae of P0–3 Hes5-GFP transgenic mice (18) and found Lgr5+ Cells Behave as Precursors to Hair Cells and Supporting Cells in that the colony counts from Hes5+ and Lgr5+ cells were Vivo. Prior in situ hybridization experiments had shown that Lgr5 − comparable and significantly higher than in Lgr5 cells (Fig. 1K). expression is restricted to supporting cell subtypes (13). Co- Myo7a is a well-established specific marker of hair cells (9, 16). chleae from P3 Lgr5EGFP-CreERT2/+ also showed this expression To investigate Lgr5+ cells’ potential for hair cell differentiation, pattern with no apical-to-basal gradient (Fig. 2A). However, we

8168 | www.pnas.org/cgi/doi/10.1073/pnas.1202774109 Chai et al. Fig. 2. Lineage tracing of Lgr5+ cells in the postnatal cochlea. (A and B)P3Lgr5EGFP-CreERT2/+ cochlea showed GFP signals in supporting cell subtypes. GFP was rarely detected in myo7a+ cells and always was adjacent to a myo7a+,GFP+ cell at the third Deiters’ cell position (arrowheads in B). (B′–B′′) Side views of reconstructed confocal images from B. B′ corresponds to the ′ position and B′′ to the ′′ position in B.(C) Tamoxifen was administered to P3 Lgr5EGFP-CreERT2/+; R26RtdTomato/+ mice, and cochleae were examined 2 and 6 d later. (D and E) Two days later, traced cells included the third Deiters’ cells, inner pillar cells, inner + + +

phalangeal cells, and greater epithelial ridge cells. Traced GFP , myo7a cells were rarely noted, and all were associated with myo7a cells at the the third BIOLOGY Deiters’ cell position (arrow in E′). (E′–E′′′′) Reconstructed z-stack images from E, showing merged labeling (E′), GFP and myo7a (E′′), tdTomato and myo7a (E′′′), and myo7a only (E′′′′). HC, hair cell. (F) Counts of GFP+ cells per cochlea. n is stated in parentheses. (G) At P9, traced cells included Hensen’s cells, DEVELOPMENTAL the second Deiters’ cells (arrowheads), and hair cells (arrows). (H) GFP and myo7a labeling only. H′–H′′′′ are reconstructed z-stack images from G, showing merged labeling (H′), GFP and myo7a (H′′), tdTomato and myo7a (H′′′), and myo7a only (H′′′′). (I) Corn oil controls showed no tdTomato labeling. (J and K) Traced cells among outer and inner hair cells (OHC and IHC). (J′ and J′′) Side view and 3D reconstruction of confocal images from J′′.(K′–K′′′′) Reconstructed z- stack images from K, showing merged labeling (K′), GFP and myo7a (K′′), tdTomato and myo7a (K′′′), and myo7a only (K′′′′). (L) Counts of labeled hair cells and supporting cells from Lgr5EGFP-CreERT2/+; R26RtdTomato/+ mice. n is shown in parentheses. (Scale bars, 25 μm.) noticed rare pairings of GFP+, myo7a+ cells (two pairs in eight oil alone did not alter GFP expression or induce Cre-reporter cochleae), with one located at the outer hair cell and the other at activity (Fig. 2I and Fig. S5E). In addition, traced cells increased the third Deiters’ cell position (Fig. 2B). These pairs of double- significantly from 2 to 6 d after tamoxifen administration (Fig. 2L). positive cells also were rare at P5 (two pairs in four cochleae), The increase in traced cells can represent fully differentiated + and none were found at P9 (Fig. 2 D–F). In light of Lgr5 cells’ cells derived from Lgr5+ cells, but we cannot rule out the pos- ability to differentiate into hair cells in vitro, we postulate that + + sibility of a delayed accumulation of Cre reporter activity. In sum, GFP , myo7a cells may represent differentiating hair cells. To these results support the notion that Lgr5+ cells in the neonatal probe this possibility further, we performed lineage-tracing EGFP-CreERT2/+ tdTomato/+ cochlea behave as precursors to hair cells and supporting cells experiments using Lgr5 ; R26R mice (20). in vivo. Tamoxifen administration at P3 activated tdTomato labeling of + – Lgr5 cells at P5 (Fig. 2 C E and Fig. S5 A and B). In the P5 Active Wnt Signaling Induces Proliferation in Lgr5+ Cells. In self- cochleae, traced cells included rare GFP+, myo7a+ cells but also − renewing organs, overexpression of β-catenin/Wnt signaling occasional GFP hair cells and other supporting cells (Fig. S5 A expands Lgr5+ cell populations and causes tumor formation and B). It is likely that GFP+, myo7a+ cells at both the hair cell (22, 23). To understand how Wnt signaling regulates the mitot- and the third Deiters’ cell positions from the P3 cochlea con- + + + ically quiescent Lgr5 cochlear cells, we characterized the tributed, at least in part, to tdTomato , myo7a cells in the P5 EGFP-CreERT2/+ flox(exon3)/+ and P9 cochleae. Lgr5 ; Catnb mice (24), in which tamox- fi ifen-responsive Cre recombinase initiates overexpression of When the tracing period was extended to P9, signi cantly + + β-catenin in Lgr5 cells. Following tamoxifen injection at P0–1, more traced myo7a hair cells were found. Traced cells were + found in a subset of outer and inner hair cells and supporting we observed the formation of multiple GFP foci at P8 adjacent cells (first and second Deiters’ cells and outer pillar cells; Fig. 2 J to inner hair cells and lateral to outer hair cells (Fig. 3A). Be- and K, Fig. S5 C and D, and Movies S1 and S2). Parallel lineage- cause Lgr5 expression can be an indication of active Wnt sig- tracing experiments using Lgr5EGFP-CreERT2/+; R26RlacZ/+ mice naling in the cochlea (13), these foci represent expanded clusters (21) similarly found traced (lacZ+) cells among hair cells and of Wnt-activated cells that normally become down-regulated and supporting cells (Fig. S5 E–H). In both tracing paradigms, corn are most detectable in the third Deiters’ cells at this age (13).

Chai et al. PNAS | May 22, 2012 | vol. 109 | no. 21 | 8169 Fig. 3. Wnt signaling induces proliferation of Lgr5+ cells. (A–C) Tamoxifen was given to P0–1 Lgr5EGFP-CreERT2/+; Catnbflox(exon3)/+ mice. Foci of GFP+ cells were noted 7 d later, abutting the inner hair cells and laterally in the lesser epithelial ridge. The medial foci (arrowheads) shifted adjacent to inner hair cells and disappeared by P21. (D) Pulse–chase experiments showed BrdU uptake (arrowheads) among formed foci. (D′ and D′′) High magnification and side view of foci. fl (E) Corn oil injection did not induce foci formation. (F) Foci counts per cochlea. (G and H) Tamoxifen was administered to P0–1 Lgr5EGFP-CreERT2/+;Catnb ox(exon3)/+; R26RtdTomato/+ mice. All GFP+ foci expressed tdTomato and Sox2 but not myo7a. G′–G′′′′ Side view of reconstructed images from G.(H′–H′′′′) Individual foci with tdTomato, GFP, Sox2, and Hoescht (H′), Sox2 only (H′′), tdTomato and Hoescht (H′′′), and GFP and Hoescht labeling (H′′′′). (I–O)Purified Lgr5+ cells were cultured (10 d) with Wnt3a or R-spondin1. Treatment cells with Wnt3a or R-spondin1 increased cytokeratin-positive colonies, myo7a+ cells inside colonies, and EdU+,myo7a+ cells, but the total counts of myo7a+ cells were unaffected. *P < 0.05; **P < 0.01. In F and M–O, n is shown in parentheses. (Scale bars, 25 μminA–E, G, H,andJ–L; 10 μminH′–H′′′′.)

Although most foci were adjacent to inner hair cells, a few also these foci underwent cell death. A subset of Lgr5+ cells did not were observed in the region of pillar cells and lateral to outer degenerate, because many tdTomato+ cells remained at P21 hair cells (Fig. S6 G–I). BrdU injected at P4 was incorporated (Fig. S6F). These results support our hypothesis that active Wnt − into these foci, all of which were Sox2+ and myo7a (Fig. 3 D, G, signaling stimulates the proliferation of Lgr5+ cells. To test this and H), suggesting proliferation without further differentiation. theory further, we next examined the effects of Wnt agonists on When Lgr5+ cells were traced simultaneously using the R26R- isolated Lgr5+ cells. tdTomato Cre reporter allele, all GFP+ foci expressed tdTomato, Isolated Lgr5+ cells formed more colonies when cultured with implying that they arose from Lgr5+ cells overexpressing β-catenin. Wnt3a (25) or R-spondin1 (Fig. 3 I–L and N) (23), but colony size Interestingly, we observed a decrease in both the number and was variable and was not affected by either treatment. Myo7a+ size of foci from P8 to P15, and no foci were detected at P21 cell counts were comparable in drug-treated cells and vehicle-only (Fig. 3 A–C and F and Fig. S6 E and F). This result highlights the controls; however, EdU+, myo7a+ cells doubled when treated transient nature of these foci and suggests that cells constituting with either Wnt agonist (P < 0.01 and <0.001, respectively) (Fig. 3 M

8170 | www.pnas.org/cgi/doi/10.1073/pnas.1202774109 Chai et al. Fig. 4. Wnt inhibition reduced proliferation of Lgr5+ cells in vitro. (A) Isolated Lgr5+ cells were cultured (10 d) with IWP-2 or aphidocilin. (B–G)IWP-2or aphidocilin decreased colony formation and EdU+ colonies. Drug treatment also reduced EdU+,myo7a+ cells and myo7a+ cells inside colonies but not outside colonies. In E–G, n isshowninparentheses.**P < 0.01. (Scale bar, 25 μminB–D.) (H) Hypothetical model of Wnt-dependent proliferation and differentiation of Lgr5+ cells. and O). In support of the notion that more hair cells differentiated proliferation of Lgr5+ cochlear supporting cells, a relationship following a mitotic division, Wnt agonists increased the number of observed in other tissue stem/progenitor cells (22, 23). myo7a+ cells residing inside colonies and decreased the number The mitotic quiescence of the mammalian cochlear sensory residing outside colonies (Fig. 3O). epithelium contrasts starkly with that of the nonmammalian vertebrates. However, several lines of evidence show that both + Wnt Inhibition Decreases Proliferation in Lgr5 Cells. IWP-2, a small- mammals and nonmammalian vertebrates are capable of regen- + molecule Wnt inhibitor (26), significantly decreased Lgr5 cells’ erating hair cells by direct differentiation (5, 28). In cultures of < + + colony-forming capacity and EdU uptake (P 0.001 for both) Lgr5 cells, most myo7a hair cells were outside epithelial col- (Fig. 4 B–F). In comparison with vehicle-treated controls, IWP-2, − + onies, were EdU , and formed despite aphidocilin treatment. reduced EdU, myo7a double-labeled cells, myo7a cells inside These results raise the possibility that Lgr5+ cells can differenti- colonies, and EdU-labeled colonies. As a positive control, we BIOLOGY + ate into hair cells via two routes: mitotic or direct differentiation, treated Lgr5 cells with the DNA polymerase inhibitor aphido- in which active Wnt signaling promotes proliferation before hair DEVELOPMENTAL cilin and found similar results. The number of myo7a+ cells fi cell differentiation (Fig. 4H). In the mitotically quiescent cochlear outside colonies did not change signi cantly with aphidocilin but epithelium, hair cell formation by direct differentiation may ex- increased slightly with IWP-2 treatment (Fig. 4G). These results plain the results of lineage-tracing experiments in which labeling show that Wnt inhibition diminishes the proliferative capacity of + + of Lgr5 cells subsequently yielded labeled hair cells. The low Lgr5 cells, whose ability to differentiate into hair cells does not level of labeled hair cells may be related to residual Cre recom- require proliferation. Similarly, IWP-2 and aphidocilin decreased − binase activity, compounded by a delayed clearance of tamoxifen the colony-forming capacity of and EdU uptake in isolated Lgr5 or its active metabolites. However, we cannot rule out the pos- cells (Fig. S7 C and D). sibility that the increase in traced hair cells also may be attributed Discussion to a delayed accumulation of Cre reporter activity. Although our Multiple studies have demonstrated that the neonatal cochlea control experiments indicate tight regulation of Cre recombinase harbors cells capable of proliferating and regenerating hair cells activity, we acknowledge these limitations when interpreting the lineage-tracing results. in vitro. Here we show that Lgr5-marked supporting cells exhibit fi similar progenitor cell behavior in vitro. When cochlear sensory During development, sensory hair cells rst arise in the pros- epithelial cells were isolated and cultured, others have observed ensory region in the basal turn of the cochlea at E13.5, and hair a transition from mitotic quiescence to proliferation (7–9). Al- cell formation was presumed complete by E16.5 (29). Our data though their colony-forming capacity is comparable to that of raise the possibility of a low level of differentiating hair cells in the Hes5-expressing supporting cells, Lgr5+ cells are enriched hair neonatal cochlea. Postnatal addition of hair cells has been observed cell precursors. On average, 7.8% of cultured Lgr5+ cells versus inothermammalianspecies(30,31);whetherthistimecourse + + is similar in the mouse cochlea will be of interest in future studies. 3.9% of Hes5 cells generated myo7a cells. EGFP-CreERT2/+ In vivo, overactive Wnt signaling was sufficient for Lgr5+ cells It is important to point out that although Lgr5 to overcome cycle arrest and proliferate, albeit temporarily. cochleae have normal morphology and function (13), it is pos- Proliferation foci were observed more commonly in the medial sible that Lgr5 haploinsufficiency can affect progenitor cell region and could have resulted from heterogeneity of Lgr5+ cells behavior. Recent studies show that Lgr5, like Lgr4 and Lgr6, and/or their corresponding microenvironments. Although the functions as a receptor for R-spondins and may play a role in precise mechanism underlying the disappearance of these pro- fine-tuning Wnt signals (32). It would be of interest to explore liferation foci currently is unknown, it likely involves activation whether Lgr5 functions similarly with complementary homologs of the cell-death pathway, a phenomenon occurring when sup- in the cochlea. porting cells are induced to proliferate via retinoblastoma de- In summary, we report that Lgr5-marked cochlear supporting letion (27). In vitro, Wnt agonists promoted and Wnt antagonist cells behave as sensory precursor cells in vitro and in vivo. Wnt suppressed proliferation in Lgr5+ cells. Together, these data led signals function to regulate proliferation of these cells, which us to conclude that the degree of Wnt signaling dictates have an intrinsic ability to generate new hair cells. We therefore

Chai et al. PNAS | May 22, 2012 | vol. 109 | no. 21 | 8171 propose Lgr5+ cells and Wnt signaling as candidate targets in Flow Cytometry. Cochleae from P0–3 Lgr5EGFP-CreERT2/+ or Hes5-GFP mice were future hair cell regeneration efforts. isolated under sterile conditions as described previously (13). Materials and Methods ACKNOWLEDGMENTS. We thank members of our laboratories for fruitful discussions; V. Nookala, Z. Sayyid, J. Kim, and K. Harnish for excellent tech- Further details are given in SI Materials and Methods. nical assistance; and V. Taylor for providing the Hes5-GFP mouse line. This work was supported by a Stanford University Dean’s Fellowship (to R.C.), Mice. Actin-DsRed (33), Rosa26R-lacZ (21), Rosa26R-tdTomato (20), and National Institute on Deafness and Other Communication Disorders/National Lgr5EGFP-CreERT2/+ (11) (all from Jackson Laboratory), Hes5-GFP (provided by Institutes of Health Grant P30DC010363; American Lebanese Syrian Associated Charities Grant CA21765; Office of Naval Research Grants N000140911014 and Verdon Taylor, University of Sheffield, Sheffield, United Kingdom) (18), and R01DC06471; the Hartwell Foundation (to J.Z.), the American Otological Soci- fl Catnb1- ox(exon3) (24) in C57BL/6 background were used. Primers used for ety, Triological Society Grant K08DC011043, and the Akiko Yamazaki and Jerry genotyping are shown in Table S2. All protocols were approved by the in- Yang Faculty Scholar Fund (to A.G.C.). R.N. is a Howard Hughes Medical stitutional Animal Care and Use Committees. Institute investigator.

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8172 | www.pnas.org/cgi/doi/10.1073/pnas.1202774109 Chai et al.