Recruitment of endogenous progenitors is critical during tissue repair. The inner ear utricle requires mechanosensory hair cells (HCs) to detect linear acceleration. After damage, non-mammalian utricles regenerate HCs via both proliferation and direct transdifferentiation. In adult mammals, limited transdifferentiation from unidentified progenitors occurs to regenerate extrastriolar Type II HCs. Here we show that HC damage in neonatal mouse utricle activates the Wnt target gene Lgr5 in striolar supporting cells. Lineage tracing and time-lapse microscopy reveal that Lgr5+ cells transdifferentiate into HC-like cells in vitro. In contrast to adults, HC ablation in neonatal utricles in vivo recruits Lgr5+ cells to regenerate striolar HCs through mitotic and transdifferentiation pathways. Both Type I and II HCs are regenerated, and regenerated HCs display stereocilia and synapses. Lastly, stabilized ß-catenin in Lgr5+ cells enhances mitotic activity and HC regeneration. Thus Lgr5 marks Wnt-regulated, damage-activated HC progenitors and may help uncover factors driving mammalian HC regeneration.
Tissue injury activates endogenous stem/progenitor cells to regenerate lost cells, and disproportionate responses can result in disease states. The integumentary and intestinal systems are capable of self-renewing at baseline and after damage1,2,3,4, whereas the mature mammalian cochlea, which relies on sensory hair cells (HC) to detect auditory information, lacks the ability to regenerate new mechanoreceptive HCs. Within this spectrum of regenerative capacity is the mammalian utricle, a vestibular organ that requires HCs to detect linear acceleration and has a limited capacity to regenerate.
In birds, both the basilar papilla (cochlea) and utricle robustly regenerate lost HCs5,6,7,8,9. This regenerative process consists of: (1) renewed/upregulated supporting cell (SC) proliferation and subsequent differentiation into HCs; and (2) phenotypic conversion, a mechanism termed direct transdifferentiation. In this mechanism, a SC converts into a HC without undergoing a mitotic division. After damage, these two processes (mitotic and non-mitotic regeneration) occur in distinct spatiotemporal patterns10,11.
The utricle consists of two types of HCs (Type I and II or HC-I and HC-II), which are distinguished by morphology, ion channel composition, expression of calcium-binding proteins and synaptic innervation patterns12,13,14. HC-I’s reside predominantly in the J-shaped striolar region and HC-II’s in the extrastriolar region (Supplementary Fig. 1). During regeneration of the avian utricle, both HC-I’s and HC-II’s are robustly replenished to restore vestibular function7,15. In contrast, the mammalian utricle regenerates only a small percentage of lost HCs in vitro and in vivo16,17,18,19,20,21,22,23,24, with little to no proliferation observed after HC loss16,18,19,21,23,25. Therefore, the primary mode of regeneration is assumed to be direct transdifferentiation. These replacement HCs appear to be exclusively of the Type II phenotype, and spontaneous regeneration of HC-I’s has not been reported in mammals23,26. Finally, regeneration occurs mainly in the extrastriolar region with the extent of recovery reaching only 18% at 6–8 months after damage23,26.
Within the sensory epithelium, HCs are interdigitated by SCs. Several lines of evidence suggest that SCs are the sources of regenerated HCs. First, cells isolated from the sensory epithelium can behave as HC progenitors27,28. Second, a subset of SCs express the HC transcription factor Atoh1 after damage18,23. Third, SCs are occasionally labelled with mitotic tracers after damage16,19,20,25. However, a lineage marker of HC progenitors in the mammalian utricle has not been identified.
Wnt signalling plays essential roles in the regulation of tissue homoeostasis by directing self-renewal of somatic stem cells29. Lgr5 is a Wnt target gene that marks somatic stem cells in self-regenerating organs1,2 as well as progenitor cells recruited in the damaged liver and pancreas30,31. In some tissues, injury upregulates Wnt signalling, which in turn promotes repair32,33. However, overactive Wnt signalling also induces pathologic states by perturbing cellular differentiation and causing uncontrolled proliferation34,35. Such conflicting effects have also been found in the developing cochlea, where in vivo activation of Wnt signalling in Lgr5+ SCs induces proliferation and a failure of differentiation into HCs36,37. In contrast, the identical manipulation in Sox2+ SCs induces proliferation and limited ectopic HC formation37,38. At present, the roles of Wnt-responsive cells after damage and whether Wnt/ß-catenin signalling promotes HC regeneration in the utricle are unknown.
Here we report the emergence of Lgr5+ SCs in the striolar region of the utricle after HC loss in vitro and in vivo. In both models of HC loss, lineage tracing demonstrated that Lgr5+ SCs can act as HC precursors with both HC-Is and HC-IIs regenerated in vivo. Furthermore, ß-catenin stabilization enhanced mitotic HC regeneration by Lgr5+ cells. Based on these data, we propose Lgr5+ SCs as damage-recruited Wnt-regulated HC progenitors in the mammalian utricle.
Damage activates Lgr5 expression in supporting cells in vitro
We examined utricles from neonatal Lgr5EGFP-CreERT2/+ mice1, which report active Wnt signalling and Lgr5 expression in the cochlea36,39. In the cochlea, Lgr5-enhanced green fluorescent protein (Lgr5–EGFP) expression is regulated by Wnt signals, mirrors Lgr5 messenger RNA (mRNA) expression and persists into early adulthood39,40. In contrast, utricles from postnatal day 3 (P3) Lgr5EGFP-CreERT2/+ mice demonstrated no detectable EGFP signal (Fig. 1c,h) but had comparable HC densities and sensory epithelium dimensions to those of wild-type littermates (Supplementary Table 1). Because mechanical damage to the sensory epithelium led to robust upregulation of Lgr5–EGFP (Supplementary Fig. 1b), we examined whether HC damage induces Lgr5 expression using a well-characterized paradigm of aminoglycoside-induced HC death (1.0 mM neomycin × 24 h, Fig. 1a)14,16,18. This paradigm resulted in HC death preferentially in the striolar region (Fig. 1b), which is defined by oncomodulin expression in undamaged tissues (Supplementary Fig. 1a)13.
Two days after neomycin treatment, many Lgr5–EGFP+ cells occupied the SC layer in the striolar region, whereas only a few were found in the extrastriolar region (Fig. 1d, Supplementary Fig. 1j,u). We measured the dimension and location of the oncomodulin+ striolar region before damage and of Lgr5–EGFP+ domains after neomycin damage and found no significant differences (Supplementary Table 2); we thus refer the Lgr5–EGFP+ domain as striolar. In situ hybridization showed Lgr5 mRNA expression in the striolar region 2 days after neomycin treatment but not in undamaged control tissues (Fig. 1f,g), thus corroborating spatiotemporal expression pattern of the Lgr5–EGFP reporter mice. Longitudinal analyses revealed that Lgr5+ cells first appeared after 4 h of neomycin treatment, subsequently increased in number and remained predominantly in the striolar region (Fig. 1p, Supplementary Fig. 1c–m). In the extrastriolar region where damage was less severe, rare Lgr5+ cells were transiently detected (Fig. 1p, Supplementary Fig. 1n–x). Twenty-four hours after damage, occasional Myo7a+ and Lgr5+ cells were stained by terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL; Fig. 1m), indicating degenerating cells. While dying Myo7a+ and Lgr5+ cells significantly decreased 48 h post damage (Fig. 1l,m); the number of Lgr5+ cells increased and reached a plateau between 48 and 72 h and remained robust for at least 6 days post damage (Fig. 1p, Supplementary Fig. 1c–m). At all time points examined, Lgr5+ cells resided in the SC layer (Fig. 1i–k). Jag1 and Sox2 are both expressed in SCs with the latter also marking Type II HCs. Immunostaining 48 h after neomycin treatment showed that all Lgr5+ cells expressed Jag1 (n=4), but only a subset of them expressed Sox2 (26.9±38.2%, n=9, Fig. 1i–k). Lgr5+ cells occasionally expressed the HC markers Gfi1 (3.2±1.9%) and Myo7a (2.5±1.3%), but never the proliferation marker Ki67 (Fig. 1i–k, Supplementary Fig. 6a–c, Supplementary Table 6).
To validate and quantify Lgr5 expression after damage, we measured Lgr5 mRNA levels in utricles from P3 wild-type mice identically damaged by neomycin in vitro. In comparison to non-damaged, cultured controls, Lgr5 mRNA expression tripled in neomycin-treated cultures (P<0.01, Student’s t-test), whereas Pou4f3 decreased significantly (P<0.05, Student’s t-test) and Sox2 expression did not significantly change (Fig. 1e). The increase in Lgr5 transcripts in damaged wild-type organs is consistent with Lgr5 mRNA detected by in situ hybridization and also with the increased EGFP signal in damaged utricles from neonatal Lgr5–EGFP reporter mice.
Damage-activated Lgr5+ SCs generate HC-like cells in vitro
Prior studies on adult utricles have demonstrated regenerated HCs after aminoglycoside-induced HC loss in vitro; however, almost no proliferation has been observed16,18,20. Here we allowed neomycin-damaged neonatal utricles from P3 Lgr5EGFP-CreERT2/+ mice to recover in aminoglycoside-free media for 5 or 12 days and did not detect any significant increase in the number of Myo7a+ HCs (Fig. 2e). Addition of the thymidine analogue EdU (1 μM × 5 days) failed to label Lgr5+ or Myo7a+ cells (Supplementary Table 6), indicating that Lgr5+ cells were not proliferative in vitro. We then used lineage tracing to determine whether damage-activated Lgr5+ cells can act as precursors for regenerated HCs observed in prior studies. After utricles from P3 Lgr5EGFP-CreERT2/+; Rosa26RtdTomato/+ mice41 were damaged with neomycin, 4OH-tamoxifen (500 nM × 5 days) was added to aminoglycoside-free media to prospectively follow damage-induced Lgr5+ cells (Fig. 2a). In this model, tamoxifen-induced activation of Cre recombinase results in permanent labelling (tdTomato) of Lgr5+ cells and their progeny. Two days after neomycin-induced damage (48 h of 4OH-tamoxifen), few tdTomato-positive, Lgr5 lineage-traced cells were observed, none of which expressed Myo7a (Supplementary Fig. 2a). By 5 days after neomycin-induced damage, few cells expressed tdTomato and Myo7a. One week later (12 days after damage), the number of tdTomato+/Myo7a+ cells increased by 2.6-fold (P<0.05, Student’s t-test, 7.3±2.5 cells and 19.3±5.0 cells at 7DIV and 14DIV, respectively, Fig. 2c,d,g). These findings suggest that Lgr5+ SCs recruited after HC loss can contribute to the regeneration of HC-like cells in vitro. Addition of EdU (1 μM × 5 days) during the post-damage period did not label any tdTomato+/Myo7a+ cells (Supplementary Table 6), suggesting that Lgr5+ SCs acquired a HC fate directly without undergoing mitosis.
To further examine whether damage-recruited Lgr5+ cells undergo direct transdifferentiation, we performed time-lapse imaging concurrent with lineage tracing of damage-recruited Lgr5+ cells. As before, utricles from P3–5 Lgr5EGFP-CreERT2/+; Rosa26RtdTomato/+ mice were first cultured and damaged with neomycin, then exposed to 4OH-tamoxifen to initiate labelling of Lgr5+ cells. Imaging began 2–4 days after damage and continued for 45–97 h (Fig. 2h, Supplementary Fig. 2b). Utricular HCs and SCs are morphologically distinct with HCs being shorter, round-bottomed and crowned apically with prominent stereocilia, while SCs are taller and narrower than HCs. Using these morphologic criteria, we analyzed time-lapse videos of 34 tdTomato+ cells (fate mapped from the Lgr5+ lineage) from four utricles. We observed 2 tdTomato-labelled lineage-traced cells that appeared to convert into HC-like cells (Fig. 2i, Supplementary Fig. 2c–d, Supplementary Movies 1–3). After fixation, we also detected Myo7a expression in a subset of tdTomato+ cells (Supplementary Fig. 2e). Together these data indicate that damage-activated Lgr5+ SCs can act as HC precursors in vitro.
Embryonic Lgr5+ cells contribute to reactivated Lgr5+ cells
To determine the source of damage-activated Lgr5+ cells, we examined the embryonic utricle. Utricle development in mice begins around embryonic day 11.5 (E11.5) and extends into the first two postnatal weeks42,43,44. Prior to birth, 49% of HCs are present, and the sensory epithelium reaches 81% of its adult size42. Utricles from E15.5 Lgr5EGFP-CreERT2/+ mice showed robust Lgr5–EGFP expression in the central region of the sensory epithelium and the EGFP+ cells more commonly expressed Sox2 than Myo7a (84.1±24.7% and 15.2±5.5%, respectively, n=428 cells from four organs, Fig. 3a). Between E15.5 and P3, both Lgr5–EGFP+ cell counts and mRNA levels significantly decreased (P<0.01, Student’s t-test, Fig. 3a–f). At P3 and later ages, Lgr5–EGFP+ cells were undetectable (n=3, Fig. 3d,e).
To determine the relationship between embryonic Lgr5+ cells and those re-expressing Lgr5 in response to HC loss, we performed lineage tracing in Lgr5EGFP-CreERT2/+; Rosa26RtdTomato/+ mice. When tamoxifen was administered at E15.5, and the utricles were viewed at E19.5, a small number of both HCs and SCs in the striolar region expressed tdTomato (4.6±1.4% Myo7a+ and 6.0±1.1% Sox2+ cells, n=1,532–3,184 cells from 3 organs). Next we harvested utricles at E19.5 and cultured them for 4 days (equivalent to P3) before damaging them with neomycin (Fig. 3g). In control undamaged organs, lineage-traced cells (tdTomato+) but no Lgr5–EGFP+ cells were detected (Fig. 3i). Neomycin-treated utricles contained Lgr5–EGFP+ cells in the striolar region at 7DIV, a subset of which (29.4±35.7%, n=479 cells from 8 utricles) were tdTomato+ (Fig. 3j,k), indicating that they derived from embryonic Lgr5+ cells.
In vivo HC ablation in Pou4f3DTR/+ mice
To investigate whether Lgr5+ cells can act as HC precursors after damage in vivo, we utilized Pou4f3-DTR mice23. In this mouse strain, the HC promoter Pou4f3 drives expression of the human diphtheria toxin receptor (DTR), thereby allowing selective HC ablation that can be temporally controlled by DT administration23,45,46. In P1 mice, a single DT injection (6.25 ng g−1, i.p.) results in HC loss 2 days later (Fig. 4a). In contrast to the rapid HC loss centred in the striolar region caused by neomycin treatment in vitro, DT administration produced gradual HC loss encompassing the entire sensory epithelium. In comparison to age-matched control animals (wild-type littermates injected with DT), utricles from P3 Pou4f3DTR/+ mice showed comparable reductions in HC densities in the striolar and extrastriolar regions (Fig. 4b–k, Supplementary Fig. 3f,j, Supplementary Table 3). This degeneration was compounded by an overall decrease in the sensory epithelium (Myo7a+) area (Fig. 4j, Supplementary Table 3). Over the next 12 days, both the HC number and the total area of the sensory epithelium decreased and reached their lowest points between P7 and P15 (Fig. 4j,k, Supplementary Fig. 3n, Supplementary Table 3). The nadir of HC number of all time points (P3-30) examined was P15, at which point the striolar and extrastriolar HC densities were 29.1±20.5% and 24.5±17.4% of age-matched controls, respectively (Fig. 4k, Supplementary Fig. 3n).
In comparison to P15, both the number of HCs and the area of the sensory epithelium area had significantly (but incompletely) recovered by P30 (Fig. 4j,k, Supplementary Fig. 3c–d,h–i,l–n, Supplementary Table 3). In undamaged utricles from P30 wild-type mice, 42.6±3.5% of striolar Myo7a+ HCs (n=2,121 cells from 12 organs, Fig. 4l, Supplementary Fig. 3e) expressed Sox2 and were classified as Type II (refs 23, 47). However, more HCs in damaged utricles from P30 Pou4f3DTR/+ mice expressed Sox2 (80.7±7.6%, P<0.0001, Student’s t-test, n=1,678 cells from 12 organs), suggesting that regenerated HCs were predominantly Type II. Our data using the Pou4f3DTR/+ mice indicate that the neonatal mouse utricle spontaneously regenerates HCs in vivo.
To investigate whether in vivo HC ablation also activates Lgr5 expression in SCs, we examined Lgr5EGFP-CreERT2/+; Pou4f3DTR/+ mice. After DT treatment at P1, Lgr5+ cells appeared 1 day later and increased in the subsequent 2 days (Fig. 4p, Supplementary Fig. 3o–q). At P4, Lgr5+ cells resided in the SC layer distributed in a striolar pattern and primarily expressed the SC marker Sox2 (Fig. 4n′). The dimensions and location of Lgr5–EGFP domain after DT-mediated damage in vivo was not different from that of oncomodulin+ striolar region in age-matched, undamaged control utricles. At P4, the Lgr5–EGFP domain surrounded the remaining oncomodulin+ striolar HCs (Fig. 4o). Lgr5+ cells were rare at P7 and undetectable at P15 (Fig. 4p, Supplementary Fig. 3r–s). Thus similar to our findings in the in vitro preparation, these data indicate that in vivo HC ablation also reactivates Lgr5 in striolar SCs and led us to next determine their cell fate via lineage tracing.
Damage-activated Lgr5+ SCs regenerate HC-like cells in vivo
While most damage-recruited Lgr5+ cells did not express Myo7a (97.5±1.1%, P4), a significant portion expressed the early HC marker Gfi1 (21.2±5.7%, Supplementary Fig. 6e, Supplementary Table 6). Most Lgr5+ cells that were Gfi1+/Myo7a− were found in the supporting cell layer (Supplementary Fig. 6e). To determine the significance of this, we fate mapped Lgr5+ cells in Pou4f3DTR/+; Lgr5EGFP-CreERT2/+; Rosa26RtdTomato/+ mice. After DT was injected at P1 to initiate HC degeneration, tamoxifen was administered at P3 to lineage-trace Lgr5+ cells (Fig. 5a). At P4, 18.9±4.7% of Sox2+ SCs expressed Lgr5 after damage (n=1,866 cells from 4 organs, Supplementary Fig. 3q). Two days after tamoxifen treatment, occasional tdTomato-expressing SCs and few Myo7a+ HCs were traced (Fig. 5c,f,g, Supplementary Table 4). In parallel controls (Pou4f3+/+; Lgr5EGFP-CreERT2/+; Rosa26RtdTomato/+ identically administered with DT at P1 and tamoxifen at P3), no Lgr5–EGFP+ cells and only rare tdTomato+ cells were found (Fig. 5b,f,g, Supplementary Fig. 4b–c, Supplementary Table 4).
Because we had seen a partial recovery of HC number at P30 using this in vivo damage paradigm, we extended the tracing period to this age and found a significant increase in tdTomato+/Myo7a+ cells in the striolar region (P<0.0001, Student’s t-test, Fig. 5d–f, Supplementary Table 4), suggesting that some Lgr5+ cells gave rise to Myo7a+ HCs. At P5, Myo7a+ cells were rarely tdTomato positive (0.2±0.3%, n=850 cells from 5 organs), whereas 10.7±2.5% expressed tdTomato at P30 (n=3,523 cells from 28 organs), indicating that a significant proportion of newly generated HCs derived from Lgr5+ cells (Fig. 5g). Among all tdTomato+ cells, the proportion that was also Myo7a+ increased from 3.1±4.5% at P5 (n=53 cells from 5 organs) to 60.6±9.1% at P30 (P<0.0001, Student’s t-tests, n=635 cells from 28 organs, Fig. 5f, Supplementary Table 4), suggesting that many lineage-traced cells had converted to HCs in the striola. In undamaged control utricles, there was no significant increase in lineage-traced Myo7a+ cells at P30 (P=0.32, Student’s t-test; Fig. 5d,f, Supplementary Fig. 4e,f, Supplementary Table 4). We also performed lineage tracing using Pou4f3DTR/+; Plp1CreERT/+; Rosa26RtdTomato/+ mice and found a similar increase in lineage-traced Myo7a+ cells in the striolar region between P3 and P30 (Supplementary Fig. 4j–l,n, Supplementary Table 4). These data further indicate that Lgr5+ cells gave rise to new HCs after damage to the postnatal mouse utricle.
We next examined whether lineage-traced Myo7a+ cells possess apical features of utricle HCs, including the actin-rich stereocilia bundle and expression of the actin-bundling protein espin48. Using these markers, we detected three major morphologic patterns in regenerated HCs lineage-traced from the Lgr5+ lineage: long stereocilia (12.9±7.8%), short stereocilia (72.4±11.5%) or no stereocilia (14.7±10.4%; n=69 HCs from 7 organs; Fig. 5i,j).
To relay sensory information centrally, HCs form synaptic connections with afferent projections from the vestibular ganglia. These synapses contain pre and postsynaptic components comprised of Ctbp2 and Shank1, respectively49,50. At P30, all Lgr5 lineage-traced Myo7a+ HCs expressed Ctbp2 and Shank1 on their basolateral surfaces (n=33–35 HCs from 3 organs; Fig. 5k,l). Immunostaining for Tuj1 also revealed neural elements juxtaposed to most lineage-traced Myo7a+ HCs (92.8±6.7%; n=37 HCs from 3 organs, Fig. 5m), suggesting that regenerated HCs are neurally integrated.
Utricular HCs are subdivided into Type I and II with the former expressing calbindin and encased by afferent calyces on their basolateral surface; HC-IIs lack these elements yet express Sox2 (refs 12, 14, 51). As a marker of HC-Is, calbindin was expressed in 48.4±6.5% of Myo7a+ HCs traced from the Lgr5+ lineage (n=47 cells from 3 organs, Fig. 5n,p), and 45.6±5.1% of lineage-traced HCs exhibited Tuj1+ calyx-like innervation (n=37 cells from 3 organs, Fig. 5m,p). Conversely, 53.7±3.2% lineage-traced HCs were immuno-positive for Sox2 (n=28 cells from 3 organs, Fig. 5o,p). In sum, these results show that damage-recruited Lgr5+ cells can give rise to bundle-bearing, neurally connected HCs of both Type I and II.
Distinct SC populations contribute to HC regeneration
We observed an increase in HC number in both the striolar and extrastriolar regions in the utricles of DT-treated Pou4f3-DTR mice at P30. However, Lgr5+ SCs almost exclusively contributed to regeneration of striolar HCs. Therefore, we hypothesized that Lgr5-negative SCs may act as HC precursors in the extrastriolar domain. Since prior studies have shown that tamoxifen preferentially activates Cre recombinase in extrastriolar SCs in Plp1-CreERT mice42,52, we employed Plp1CreERT/+; Rosa26RtdTomato/+ mice to test this hypothesis. Tamoxifen (0.75 mg g−1 gavage) administration at P1 induced tdTomato labelling of Sox2+, Myo7a-negative SCs in the striolar and extrastriolar regions 2 days later (Fig. 6b, Supplementary Fig. 4i). Using Pou4f3+/+; Plp1CreERT/+; Rosa26RtdTomato/+ littermates as undamaged controls, we examined whether Plp1-Cre+ SCs also contribute to extrastriolar HC regeneration using Pou4f3DTR/+; Plp1CreERT/+; Rosa26RtdTomato/+ mice. After tamoxifen administration at P1, DT treatment (8 h later) similarly led to HC loss and sensory epithelium shrinkage at P7 and subsequent HC regeneration at P30 (Fig. 6a, Supplementary Fig. 4j–l). In comparison to undamaged controls, significantly more Myo7a+ HCs were tdTomato+ in the extrastriolar region at P30 (81.1±1.8% and 22.7±4.3%, respectively, P<0.0001, Student’s t-test, Fig. 6b–g, Supplementary Table 4). These data show that Plp1+ SCs contribute to HC regeneration in the extrastriolar region after in vivo HC ablation.
To determine if Lgr5+ cells are more likely to regenerate HCs than Plp1+ cells in the striolar region, we analyzed utricles from P30 transgenic mice used to trace both lineages. Despite the reported low Cre activity in the striola of Plp1-CreERT mice42, we observed many Plp1 lineage-traced SCs using the Rosa26R-tdTomato Cre reporter mice (Supplementary Fig. 4i). Because our tracing schemes led to significantly more tdTomato+ cells in the Plp1+ than in Lgr5+ lineages, we measured regenerative capacity as the percentage of tdTomato+ lineage-traced cells that were Myo7a+. In the striolar region where Lgr5 expression was robust (Fig. 4n), significantly more tdTomato+ cells of the Lgr5+ lineage than those of the Plp1+ lineage expressed Myo7a at P30 (60.6±9.1% versus 25.7±2.7%, P<0.0001, Student’s t-test, Fig. 6h, Supplementary Table 4). In contrast, the extrastriolar region lacked Lgr5 expression and harboured traced Myo7a+ cells from the Plp1+ lineage and not the Lgr5+ lineage (Fig. 6e, Supplementary Fig. 4g). These results suggest a spatial segregation of HC progenitors in which a higher proportion of Lgr5+ SCs, compared with Plp1+ SCs, converted to HCs in the striola.
Lgr5+ supporting cells proliferate and differentiate into HCs
We next asked whether Lgr5+ SCs are more proliferative than Lgr5-negative SCs in response to HC loss. Prior studies demonstrated some proliferative regeneration of HCs in the neonatal mouse utricle16. We first marked proliferative cells by administering the mitotic marker EdU (50 mg kg−1 once daily at P4–6) after DT-mediated HC ablation in P1 Pou4f3DTR/+ mice (Supplementary Fig. 5a). At P7 and P30, sensory epithelium in damaged organs had robust EdU labelling, which was rare in undamaged, age-matched controls (wild-type littermates identically injected with DT and EdU, Supplementary Fig. 5b–e). In undamaged controls, most EdU-labelled cells resided in the stromal layer below the sensory epithelium (Supplementary Fig. 5b,d). In damaged organs, significantly more EdU+/Sox2+ SCs resided in the striolar than extrastriolar regions at P7 and P30 (P<0.0001, Student’s t-tests, for both, Fig. 6n, Supplementary Fig. 5c,e). Similarly, the striolar region contained more EdU+/Myo7a+ HCs than the extrastriolar region at P7 and P30 (Supplementary Fig. 5c,e). Thus, the Lgr5-containing striolar region is significantly more proliferative than the extrastriolar region. To determine the identity of these proliferative cells, we immunostained utricles from P4 Pou4f3DTR/+; Lgr5EGFP-CreERT2/+ mice and found that 8.1±3.5% Lgr5+ cells expressed the proliferative marker Ki67 (Supplementary Fig. 6f and Supplementary Table 6).
Next, we investigated the fate of these damage-activated proliferative cells by combining lineage and mitotic tracing in Pou4f3DTR/+; Lgr5EGFP-CreERT2/+; Rosa26RtdTomato/+ mice (DT at P1, tamoxifen at P3, EdU at P4–6 with Pou4f3+/+; Lgr5EGFP-CreERT2/+; Rosa26RtdTomato/+ mice as controls (Fig. 6i). EdU labelled a significantly higher percentage of SCs of the Lgr5 lineage (tdTomato+) than of Sox2+ SCs in the striolar and extrastriolar regions at P7 and P30 (Fig. 6k,m,n, Supplementary Fig. 5c,e). At P7, 5.9±2.6% of Myo7a+ HCs were tdTomato labelled, but no EdU+/tdTomato+/Myo7a+ HCs were observed (n=602 cells from 6 organs, Fig. 6k). At P30, 22.8±9.8% of tdTomato+/Myo7a+ HCs were EdU labelled (n=60 cells from 6 organs, Fig. 6m,n). These results indicate that Lgr5+ cells represent spatially specified HC progenitors that are capable of mitotic HC regeneration in vivo.
ß-catenin stabilization promotes proliferation of damage-activated Lgr5+ supporting cells in vivo
As the central mediator of canonical Wnt signalling, ß-catenin overexpression has been shown to exert contrasting effects on SCs in the developing cochlea36,37. We examined the roles of Wnt/ß-catenin signalling on damaged-recruited Lgr5+ cells by generating the Pou4f3DTR/+; Lgr5EGFP-CreERT2/+; Catnbflox(exon3)/+ mice53. In this set of experiments, DT was administered at P1 to ablate HCs, tamoxifen (0.075 mg g−1) was given at P3 to activate Cre recombinase in Lgr5+ cells and EdU (25 mg kg−1 daily at P4–6) was used to label proliferating cells. Utricles were examined at P30 (Fig. 7a). In comparison to DT-damaged utricles without stabilized ß-catenin, damaged organs with stabilized ß-catenin contained larger sensory epithelia (46.2±15.6% and 64.7±4.9% of undamaged controls, respectively, Fig. 7d–f). In the striolar region where most EdU+ HCs and SCs resided, ß-catenin stabilization caused a modest but significant increase in HCs (P<0.005, Student’s t-test, Fig. 7d′–e′) but not SCs. In addition, activating Wnt signalling in Lgr5+ cells significantly increased both the numbers of EdU+/Myo7a+ cells and EdU+ SCs in the striola (Fig. 7d′–e″) while those in the extrastriola were comparable to DT-damaged organs (Supplementary Table 5). Without DT-mediated damage, tamoxifen did not increase EdU uptake in Lgr5EGFP-CreERT2/+; Catnbflox(exon3)/+ utricles (Fig. 7b,c,h,i), suggesting that Lgr5 was not expressed and Cre recombinase activity was absent. Together, these results implicate that ß-catenin stabilization stimulates proliferation and mitotic HC regeneration by damage-activated Lgr5+ SCs.
Hearing and balance functions require mechanosensory HCs, and diseases that cause HC degeneration manifest as hearing or balance disorders. In the mature mammalian cochlea, the absence of HC regeneration underlies the permanence of hearing loss54,55. The mammalian vestibular epithelium, however, exhibits a limited regenerative capacity and therefore serves a useful preparation to examine mammalian HC regeneration. Here we have identified Lgr5+ cells as region-specific HC progenitors capable of both mitotic and non-mitotic HC regeneration at early postnatal stages. Moreover, we show that Wnt/ß-catenin signalling can modestly enhance mitotic regeneration by Lgr5+ cells in the damaged neonatal utricle.
While utricular HCs are functional in the neonatal period56,57, prior studies comparing neonatal to adult utricles found differences in cellular composition12,42, responsiveness to Notch inhibition18,58 and proliferative responses to damage16,23. Specifically, the neonatal utricle can proliferate and mitotically regenerate HCs, whereas the primary mode of HC regeneration in the adult organ is direct transdifferentiation. Because both modes of HC regeneration operate in non-mammalian vertebrates to restore auditory and vestibular functions, they are likely important in restoring the proper cytoarchitecture and functions of HCs and SCs7,59. Our data indicate that Lgr5+ cells in the neonatal utricle are capable of regenerating HCs via both direct transdifferentiation and mitotic regeneration. Moreover, in the adult utricle, HC regeneration is mainly in the extrastriolar region in which regenerated HCs are primarily Type II (refs 17, 23). While both HC-Is and HC-IIs function as mechanoreceptors, the former are concentrated in the striolar region, and they are morphologically and functionally distinct from HC-IIs12. Our study shows that Lgr5+ cells in the neonatal utricle can regenerate both HC-Is and HC-IIs, behaviour that is reminiscent of SCs from avian utricles in which both types of HCs are spontaneously regenerated7. Therefore, future studies defining the molecular signatures of Lgr5+ cells may help reveal factors regulating the regeneration of distinct HC subtypes and also those dictating the two modes of HC regeneration. As HCs are still being born with committed HCs maturing, SCs in the neonatal utricle may be less mature and more plastic than those in the adult organ, possibly accounting for their differential responses to damage. In addition, it is important to note that while Lgr5 expression was induced by damage both in vitro and in vivo, behaviour of Lgr5+ cells in the two systems differ in that direct transdifferentiation predominated and mitotic regeneration was absent in vitro, and the degree of HC regeneration was much less robust than that observed in vivo (Supplementary Fig. 6 and Supplementary Table 6).
After neomycin damage in vitro (14DIV), 19.3±5.0 striolar HCs per organ were fate-mapped from the Lgr5 lineage. One month after HC ablation in vivo, 38.7±6.2 Lgr5 lineage-traced HCs were noted in the striola of each organ. We postulate that the different damage models used (neomycin in vitro versus diphtheria toxin in vivo) may have, at least in part, caused these differences by inflicting more damage in vitro as degenerating Lgr5+ SCs (which appeared smaller, TUNEL positive and Sox2 negative) were more frequently observed in vitro than in vivo. As a result, the persistence of Lgr5 expression observed in vitro (and not in vivo) may reflect an undifferentiated state1,30, in which damaged-recruited Lgr5+ cells lack additional cues that are required for them to proliferate and/or differentiate. To add to this complexity, the culture system may also lack secreted mitogenic/prosensory factors normally present in vivo, with candidates including TGFα, EGF and possibly other family members60,61. Thus future studies comparing the in vitro and in vivo systems may shed light on factors limiting or driving spontaneous HC regeneration in mammals.
Canonical Wnt signalling becomes activated after damage and mediates repair in multiple organ systems4,30,32. In the current study, embryonic Lgr5+ cells contributed to otherwise quiescent resident cells that became recruited in response to tissue injury. Similarly in the mammary gland, Wnt-responsive cells contribute to organ development as well as to the resident progenitor cells that are recruited for glandular outgrowth during pregnancy62. In less regenerative organs such as the liver and pancreas, tissue damage similarly elicits the emergence of Lgr5+ progenitors cells, which can be expanded in vitro in a Wnt-dependent manner to form organoids consisting of mature cell types30,31.
Overactive Wnt signalling can also induce pathologic states as a result of uncontrolled proliferation and a lack of cellular differentiation34,35. In the developing cochlea, conflicting results have been observed as a result of ß-catenin stabilization36,37. Our data indicate that ß-catenin stabilization resulted in a modest increase in HC density and more remarkably the number of EdU-labelled HCs and SCs, suggesting that canonical Wnt signalling primarily serves as a proliferative signal to Lgr5+ cells but does not limit their HC fate (Fig. 8). These results lead us to propose that other instructive signals, possibly including increased Atoh1 expression and decreased Notch signalling18,63,64,65,66,67, can be complementary for driving HC regeneration. While active Wnt signalling may be used to increase the population of competent cells, more work is necessary to test the feasibility and success of such combinatorial approaches.
In summary, we report that HC loss results in induction of Lgr5 in a subset of striolar SCs, which can behave as facultative HC progenitors in the neonatal utricle in vitro and in vivo. Therefore, Lgr5+ cells may be useful for further dissecting the mechanisms governing mammalian HC regeneration.
Lgr5–EGFP-CreERT2 (Jackson Laboratory, #8875)1, Plp1-CreERT (Jackson Laboratory, #5975)68, Rosa26R-tdTomato (Jackson Laboratory, #7908)41, Pou4f3-DTR23 and Catnb-flox(exon3)53 mice of both genders were used. For Cre activation, tamoxifen (dissolved in corn oil; Sigma) was given via gavage to neonatal mice (0.075 mg g−1 for Lgr5–EGFP-CreERT2 and 0.75 mg g−1 Plp1-CreERT strains) and dams (0.225 mg g−1, i.p.). Diphtheria toxin (6.25 ng g−1 i.p., List Biological Laboratories) and EdU (25–50 mg kg−1 i.p., Invitrogen) were used. All protocols were approved by Animal Care and Use Committee of the Stanford University School of Medicine, St. Jude Children’s Research Hospital and NIH.
Cell quantification and statistics
Cells were quantified from z-stack images of 20,000 μm2 using Volocity software (v6.1.0; Improvision) unless otherwise stated. Images were taken from 1–2 representative areas from the striolar (each area is ∼20,000 μm2 and represent 35–48% of the striola, which we defined as oncomodulin+, Lgr5+ or Lgr5 lineage-traced domains, see Fig. 4f′, Supplementary Fig. 1a, Supplementary Table 2 for details) or extrastriolar regions for analyses. In damaged tissues where oncomodulin-marked HC-I had degenerated, Lgr5 or Lgr5 lineage-traced expression was used to define the striolar region. For all experiments, n values represent the number of cells or organs examined unless otherwise stated. Statistical analyses were conducted using Microsoft Excel (Microsoft) and GraphPad Prism 5.0 software (GraphPad). Two-tailed, unpaired Student’s t-tests were used to determine statistical significance. P<0.05 was considered as significant. Data are shown as mean±s.d.
Whole organ cultures
Utricles were harvested from E19.5-P5 mice of both genders and otoconia removed in sterile conditions, and then attached to coverslips (10 mm, Marienfeld) pre-coated with CellTaK (BD Biosciences) and placed in four-well Petri dishes (CellStar). Whole organs were cultured in growth factor-enriched, serum-free media, consisting of DMEM/F12 (1:1; Cellgro), N2 (1:100), B27 (1:50, both from Invitrogen), bFGF (1 ng ml−1), IGF-1 (50 ng ml−1), EGF (20 ng ml−1), heparin sulfate (50 ng ml−1) and ampicillin (50 ng ml−1; all from Sigma). The following agents were added to a subset of cultures: neomycin (1.0 mM, Sigma), EdU (1.0 μM), 4OH-tamoxifen (500 nM, Sigma).
Genotyping and real time quantitative PCR
Standard PCR was performed to genotype transgenic mice using genomic DNA. DNA was isolated by adding 200 μl of 50 mM NaOH to cut tail tips, incubating at 98 °C for 1 h, and then adding 20 μl of 1 M Tris-HCl.
For quantitative PCR (qPCR), total RNA was isolated with RNeasy mini extraction kits (Qiagen), then complementary DNA was synthesized using SuperScript III First-Strand Synthesis System kits (Invitrogen). SYBR Green PCR Mix kit (Applied Biosystems) was used to perform qPCR reactions on a 7900HT-Fast Real time PCR system (Applied Biosystems). All qPCR reactions were performed in triplicate and relative quantification of gene expression was analyzed using the ΔΔCT method with GAPDH as the endogenous reference69. Primers used are listed in Supplementary Table 7.
Utricles were fixed for 1 h in 4% paraformaldehyde (in PBS, pH 7.4; Electron Microscopy Services) at room temperature.70 Tissues were blocked with 5% goat or donkey serum, 0.1% Triton-X100, 1% bovine serum albumin (BSA), and 0.02% sodium azide (NaN3) in PBS at pH 7.4 for 1–2 h at room temperature, followed by incubation with primary antibodies diluted in the same blocking solution overnight at 4 °C in a humidified chamber. The next day, after washing with PBS, tissues were incubated with secondary antibodies diluted in 0.1% Triton-X100, 0.1% BSA and 0.02% NaN3 solution in PBS for 1 h at room temperature. After PBS washing, tissues were then mounted in antifade Fluorescence Mounting Medium (DAKO) and coverslipped. We used antibodies against the following markers: Myosin7a (1:1,000; Proteus Bioscience or Labome), Tuj1 (1:1,000; Neuromics), Ki67 (1:1,000; Abcam), Gfi1 (1:1,000; gift from H. Bellen), Calbindin (1:1,000; Millipore), Espin (1:1,000, gift from S. Heller), Ctbp2 (1:100; BD Transduction Laboratories), Shank1 (1:100; Neuromics), Sox2 (1:400), Jag1 (1:200), and Oncomodulin (1:250, all from Santa Cruz Biotechnology). The secondary antibodies were conjugated with FITC, TRITC or Cy5 (1:500, Invitrogen). Fluorescent-conjugated phalloidin (1:1,000; Sigma), DAPI (1:10,000; Invitrogen) and Alexa Fluor 488, 555 or 647 EdU detection kit (Invitrogen) were used. TUNEL Alexa Fluor 594 imaging Assay kit (Invitrogen) was used in accordance to kit instructions. Images were acquired using epifluorescent or confocal microscopy (Axioplan 2, Zeiss Pascal or LSM700) and analyzed with Image J64 (NIH) and Photoshop CS4 (Adobe Systems). Three-dimensional reconstruction of z-stack images was performed with Volocity software.
In situ hybridization
In situ hybridization was performed on cultured P3 wild-type utricles using RNAscope probes for Lgr5 according to manufacturer’s instructions (Advanced Cell Diagnostics). Probes for DapB were used as negative controls. Utricles were fixed for 1.5 h in 4% paraformaldehyde and washed in PBS before being hybridized with probes for 2 h at 40 °C. Signal was amplified and detected with 2.0 HD reagent Kit-RED and imaged on the EVOS XL Core Imaging system (Life Technologies).
Utricles harvested from P3–P5 Lgr5EGFP-CreERT2/+; Rosa26RtdTomato/+ mice were attached to 35 mm glass bottom dishes (MatTek) pre-coated with CellTaK. Whole organs were cultured overnight, then treated with neomycin (1.0 mM × 24 h, Sigma) as above. 4OH-tamoxifen (500 nM, Sigma) in growth factor-enriched, serum-free media was present for 2–4 days first. Then organs were imaged in DMEF/F12 without phenol red media using a spinning disc confocal imaging system (Zeiss Axio Observer Z1 or OlympusIX-81 coupled with a Yokogawa spinning disc system CSU-X1A 5000) connected to an incubating chamber (37 °C, 5% CO2). One to two EGFP+ regions with tdTomato+ cells were selected from each utricle, and z-stack images spanning the sensory epithelium were taken at 0.5–1 h intervals. Collected videos were processed and analyzed with Image J64, MetaMorph (NX 2.0; Olympus) and Volocity software.
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We thank E. Rubel, D. Wu, S. Heller, M. Drummond, B. Cox for critical reading; R. Nusse, O. Bermingham-McDonogh, J. Corwin, E. Oesterle, J. Stone, A. Ricci and our laboratory members for fruitful discussions; V. Nookala, J. Luo, G. Huang, M. Drummond, L. Tong and Z. Sayyid for excellent technical assistance; M. Taketo for sharing the Catnb-flox(exon3) mouse, E. Rubel for sharing the Pou4f3-DTR mouse, H. Bellen for sharing antibodies; and E. Monzack for figure illustration (5 h). This work was supported by NIH-NCATS-CTSA (UL1 TR001085), the Lucile Packard Foundation for Children's Health, the Child Health Research Institute, the National Basic Research Program of China 2012CB967904, 2012CB967900 (T.W.), the National Basic Research Program of China 2015CB965000, the National Natural Science Foundation of China 81470692, the Natural Science Foundation from Jiangsu Province BK20140620 (R.C.), the Stanford University Medical Scholars Program, the Howard Hughes Medical Institute Medical Scholars Fellowship (G.S.K.), the Swedish Research Council C0657401 (L.J.), R01DC006471, P30CA21765, the American Lebanese Syrian Associated Charities, the Office of Naval Research (J.Z.), NIDCD Division of Intramural Research 1ZIADC000079 (L.L.C.), NIDCD/NIH P30DC010363, K08DC011043, RO1DC013910, Department of Defense W81XWH-14-1-0517 and the Akiko Yamazaki and Jerry Yang Faculty Scholar Fund, the American Hearing Research Foundation and the California Initiative in Regenerative Medicine RN3-06529 (A.G.C.).
The authors declare no competing financial interests.
Supplementary Figures 1-6, Supplementary Tables 1-7 (PDF 2053 kb)
Time-lapse movie of traced hair cell-like cells from facultative Lgr5+ cells (related to Figure 2). Time-lapse imaging of traced Lgr5+ cells from P5 Lgr5EGFP-CreERT2/+; Rosa26RtdTomato/+ utricle (Fig. 2h-i). After hair cell ablation with neomycin treatment, 4OHtamoxifen was added to initiate tracing. EGFP+ areas with tdTomato+ (red) cells were selected, and 67-80 μm thick z-stack images (1 μm intervals) spanning the sensory epithelium were taken every 0.5-1 hr from 84 hr to 178 hr. Collected stacks were compiled as brightest point projection images, from which this movie was generated. (MOV 431 kb)
Three-dimensional reconstruction of traced hair cell-like cells from facultative Lgr5+ cells (related to Figure 2). 3D reconstruction of z-stack images of traced Lgr5+ cells from P5 Lgr5EGFPCreERT2/+; Rosa26RtdTomato/+ utricle at 178 hr (Fig. 2i). One of the traced cells appears flaskshaped with apical projections. (MOV 1492 kb)
Time-lapse movie of traced supporting cell-like cells from facultative Lgr5+ supporting cells (related to Figure 2 and Supplementary Figure 2). Time-lapse imaging of traced Lgr5+ cells from P3 Lgr5EGFP-CreERT2/+; Rosa26RtdTomato/+ utricle (Supplementary Fig. 2a-b). 80-90 μm thick z-stack images (1 μm intervals) spanning the sensory epithelium were taken hourly from 135 hr to 180 hr. Collected stacks were compiled, reconstructed and analyzed in an orthogonal view, from which this movie was generated. (MOV 1792 kb)
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Wang, T., Chai, R., Kim, G. et al. Lgr5+ cells regenerate hair cells via proliferation and direct transdifferentiation in damaged neonatal mouse utricle. Nat Commun 6, 6613 (2015). https://doi.org/10.1038/ncomms7613
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