Characterization of the Transcriptomes of Lgr5+ Hair Cell Progenitors and Lgr5- Supporting Cells in the Mouse Cochlea

Cochlear supporting cells (SCs) have been shown to be a promising resource for hair cell (HC) regeneration in the neonatal mouse cochlea. Previous studies have reported that Lgr5+ SCs can regenerate HCs both in vitro and in vivo and thus are considered to be inner ear progenitor cells. Lgr5+ progenitors are able to regenerate more HCs than Lgr5- SCs, and it is important to understand the mechanism behind the proliferation and HC regeneration of these progenitors. Here, we isolated Lgr5+ progenitors and Lgr5- SCs from Lgr5-EGFP-CreERT2/Sox2-CreERT2/Rosa26-tdTomato mice via flow cytometry. As expected, we found that Lgr5+ progenitors had significantly higher proliferation and HC regeneration ability than Lgr5- SCs. Next, we performed RNA-Seq to determine the gene expression profiles of Lgr5+ progenitors and Lgr5- SCs. We analyzed the genes that were enriched and differentially expressed in Lgr5+ progenitors and Lgr5- SCs, and we found 8 cell cycle genes, 9 transcription factors, and 24 cell signaling pathway genes that were uniquely expressed in one population but not the other. Last, we made a protein–protein interaction network to further analyze the role of these differentially expressed genes. In conclusion, we present a set of genes that might regulate the proliferation and HC regeneration ability of Lgr5+ progenitors, and these might serve as potential new therapeutic targets for HC regeneration.


INTRODUCTION
Sensorineural hearing loss is a common sensory disorder caused by the loss of hair cells (HCs). HCs are responsible for converting vibrational sound waves into the electrical impulses that are transmitted to the brain. Regeneration of damaged HCs could possibly yield a cure for sensorineural hearing loss, which still has no treatment other than prosthetic devices. Although the mature mammalian cochlea lacks the ability to regenerate HCs, new HCs are spontaneously regenerated in non-mammalian vertebrates from the resident supporting cells (SCs) that surround the HCs (Corwin and Cotanche, 1988;Balak et al., 1990;Roberson et al., 1992;Stone and Cotanche, 2007;Bermingham-McDonogh and Reh, 2011;Warchol, 2011). Previous studies have shown that some cochlear SCs in neonatal mammals are HC progenitors that possess a limited capacity to regenerate HCs (Chai et al., 2012;Shi et al., 2013;Bramhall et al., 2014;Cox et al., 2014;Wang et al., 2015). However, these SCs lose their intrinsic regenerative potential as the animal ages (Oesterle et al., 2008;Bermingham-McDonogh and Reh, 2011;Warchol, 2011;Cox et al., 2014), and as a result hearing loss tends to be permanent and incurable in mature mammals.
Supporting cells in the mouse inner ear are known to be a potential resource for HC regeneration after damage (White et al., 2006;Sinkkonen et al., 2011), and when isolated by flow cytometry neonatal SCs have the ability to proliferate and to differentiate into HCs in vitro. Upon HC injury, cochlear SCs also display a limited capacity to proliferate and to regenerate HCs (White et al., 2006;Sinkkonen et al., 2011). Leucine-rich repeat-containing G-protein coupled receptor 5 (Lgr5), a Wnt target gene, is a marker of endogenous stem cells in rapidly proliferating organs (Barker et al., 2007;Jaks et al., 2008). Lgr5 is widely expressed in the cochlear duct prosensory region during embryonic development, but it becomes restricted to a subset of SCs after birth . Recently, Lgr5+ cells in newborn mice have been shown to be a population of HC progenitors that can regenerate HCs through both direct differentiation and through mitotic regeneration (Chai et al., 2012;Shi et al., 2013;Bramhall et al., 2014;Cox et al., 2014;Hegarty et al., 2015;Wang et al., 2015;Lu et al., 2016). However, the detailed gene expression profile differences between the Lgr5+ progenitors and the Lgr5− SCs have not yet been investigated.
The current study focused on identifying the molecular mechanism behind the increased proliferation and HC regeneration ability of Lgr5+ progenitors in the neonatal mouse cochlea. We performed RNA-Seq profiling of the Lgr5+ progenitors and the Lgr5− SCs from Lgr5-EGFP-CreERT2/Sox2-CreERT2/Rosa26-tdTomato transgenic mice and identified the differentially expressed genes that might be involved in regulating proliferation, differentiation, or signaling pathways in these two cell populations. To further analyze the role of differentially expressed genes between Lgr5+ progenitors and Lgr5− SCs, we created a protein-protein interaction network using STRING (Search Tool for the Retrieval of Interacting Genes/Proteins). These datasets are expected to serve as a resource for future work in determining the detailed regulatory mechanisms behind HC regeneration.

Mice and Genotyping
Lgr5 EGFP-Ires-CreERT2 (stock no. 008875) (Barker et al., 2007), Sox2-CreER (stock no. 008875), and Rosa26-tdTomato (stock no. 007914) (Madisen et al., 2010) mice were obtained from the Jackson Laboratory. Transgenic mice were genotyped using genomic DNA from tail tips by adding 180 µl 50 mM NaOH, incubating at 98 • C for 1 h, and adding 20 µl 1M Tris-HCl. The genotyping primers were as follows: Lgr5: (F) CTG CTC TCT GCT CCC AGT CT; wild-type (R) ATA CCC CAT CCC TTT TGA GC; mutant (R) GAA CTT CAG GGT CAG CTT GC; tdTomato: wild-type (F) AAG GGA GCT GCA GTG GAG T; (R) CCG AAA ATC TGT GGG AAG TC; mutant (F) GGC ATT AAA GCA GCG TAT C; (R) CTG TTC CTG TAC GGC ATG G. Sox2-CreER mutant (F) GCG GTC TGG CAG TAA AAA CTA TC; Sox2-CreER mutant (R) GTG AAA CAG CAT TGC TGT CAC TT; Sox2-CreER wild-type (F) CTA GGC CAC AGA ATT GAA AGA TCT; Sox2-CreER wild-type (R) GTA GGT GGA AAT TCT AGC ATC ATC C. Tamoxifen (Sigma, diluted in corn oil) was injected i.p. at post-natal day (P) 2 at 0.1 mg/g bodyweight to induce the Cre recombinase activity, and the cochleae were harvested at P4. All animal procedures were performed according to protocols approved by the Animal Care and Use Committee of Southeast University and were consistent with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. All efforts were made to minimize the number of animals used and to prevent their suffering.

RNA Extraction for RNA-Seq Analysis
Approximately 5,000 Lgr5+ HC progenitors and 5,000 Lgr5− SCs were isolated by FACS and split into three fractions for separate replicates. RNA-Seq libraries of FACS-purified cells were generated using the SMART-Seq v4 Ultra Low Input RNA Kit for Sequencing and the Illumina mRNA-Seq Sample Prep Kit. FACSpurified cells were suspended in 10 × lysis buffer. First strand and second strand cDNA synthesis, adaptor ligation, and PCR amplification were performed using the Illumina mRNA-Seq Sample Prep Kit. SPRI beads (Ampure XP, Beckman) were used in each purification step after RNA fragmentation for size selection. All libraries were analyzed for quality and concentration using an Agilent Bioanalyzer. Sequencing was performed using the Illumina HiSeq2500 150-bp Paired-End Platform, and FASTQ files of paired-end read files were generated.

Data Analysis
RNA-Seq reads in the FASTQ files were trimmed using Trimmomatic. Clean reads were mapped to the mouse reference genome (mm9) using TopHat (Trapnell and Schatz, 2009) followed by transcript assembly and differential gene expression analysis using Cufflinks. Genes and transcripts were annotated using the RefGene database (NCBI). Genes with p-values < 0.05 were marked as significant. To assess the extent of functional enrichment, we performed gene ontology (GO) analysis with the functional annotation tool DAVID 6.7 (Huang da et al., 2009), which determines whether biological processes are enriched within a list of genes.

Statistical Analysis
All of the data are shown as the mean ± SD, and statistical analyses were conducted using GraphPad Prism6 software. For all experiments, n represents the number of replicates, and at least three individual experiments were conducted. Two-tailed, unpaired Student's t-tests were used to determine statistical significance when comparing two groups. A value of p < 0.05 was considered to be statistically significant.

Lgr5+ HC Progenitors Generate More HCs Compared to the Lgr5− SCs In Vitro
Lgr5-EGFP-CreERT2 transgenic mice were used to report Lgr5 expression with EGFP (Hume et al., 2007). Lgr5-EGFP is expressed in a subset of SCs, including third-row Deiters' cells, inner pillar cells, and inner phalangeal cells . To separate the Lgr5+ HC progenitors from the other Lgr5− SCs, we crossed Lgr5-EGFP-CreERT2/Rosa26-tdTomato mice with Sox2-CreERT2/Rosa26-tdTomato mice to generate Lgr5-EGFP-CreERT2/Sox2-CreERT2/Rosa26-tdTomato triple-positive mice among the offspring. Tamoxifen was injected at P2, and the cochleae were harvested at P4 (Figure 1A). Several previous studies have noted that the apical turn of the cochlea has greater HC regeneration ability than the basal turn (Chai et al., 2012;Cox et al., 2014), so to avoid the gene expression differences between the apical and basal turns (Waqas et al., 2016a) we used the middle turn of the cochlea for all of the experiments and analyses. For flow cytometry, we first used the tdTomato channel to sort out all the tdTomato+ cells (Red), which represent all of the Sox2+ SCs. From these tdTomato+ cells, we used the FITC channel to isolate the EGFP+ cells, which were the Lgr5+ progenitors, and the rest of the tdTomato+ but EGFP− cells were the Lgr5− SCs ( Figure 1A). The Lgr5+ progenitors expressed both tdTomato and EGFP and thus were labeled in yellow in Figure 1. Using this strategy, we could separate the purified Lgr5+ progenitors from the other Lgr5− SCs ( Figure 1B).
To determine the HC regeneration capability of Lgr5+ progenitors and Lgr5− SCs, we cultured 5,000 cells in laminincoated 4-well dishes at a density of 50 cells/µl for 10 days in serum-free medium. We added 10 µM EdU to the culture medium from day 4 to 7 during culture to label the mitotically regenerated HCs ( Figure 1C). After 10 days of culture, the cells were immunostained with the HC marker Myo7a. We found that the Lgr5+ progenitors generated significantly more Myo7a+ colonies and total colonies than the Lgr5− SCs ( * * p < 0.01, * * * p < 0.001, n = 3) (Figures 1D-H and Supplementary  Figures S1A,B), while the number of Myo7a-colonies was significantly greater for the Lgr5− SCs ( * * p < 0.01, n = 3) ( Figure 1H). Isolated Lgr5+ progenitor SCs generated HCs through both direct differentiation and mitotic regeneration. In the differentiation assay, the HCs inside of the colonies represent the mitotically regenerated HCs, and the HCs outside of the colonies represent the directly differentiated HCs. Next, we characterized and counted the Myo7a+ cells inside and outside of the epithelial colonies and found that Lgr5+ progenitors generated significantly more Myo7a+ HCs both inside and outside of the colony than the Lgr5− SCs ( * p < 0.05, * * p < 0.01, n = 3) ( Figure 1I). When we counted the Myo7a+/EdU+ cells, we found that the majority of the Myo7a+/EdU+ cells were inside of the colonies and only a few of the Myo7a+/EdU+ cells were outside of the colonies (Figures 1D-G,J) and that Lgr5+ progenitors generated significantly more Myo7a+/EdU+ HCs both inside and outside of the colonies than the Lgr5− SCs ( * p < 0.05, * * p < 0.01, * * * p < 0.001, n = 3) ( Figure 1J).
To further verify our findings, we used multiple HC markers, including Myo6 and parvalbumin (PV), to label the newly regenerated HCs. We found that all of the Myo7a+ cells were also Myo6+ and PV+ in both the population of HCs regenerated from Lgr5+ progenitors and the population generated from Lgr5− SCs (Supplementary Figures S1C-F). To further investigate the bundle morphology of newly regenerated HCs, we used the common hair bundle markers phalloidin and espin1. We found that 68.7 and 66.3% of newly regenerated HCs had hair bundles from Lgr5+ progenitors and Lgr5− SCs, respectively (Supplementary Figures S1E,F' ,G,H). To test whether the newly regenerated HCs have mechanosensory transduction function, we performed additional FM1-43 dye experiments. Almost all of the newly regenerated HCs from both Lgr5+ progenitors and Lgr5− SCs could take up FM1-43 dye, suggesting that the majority of the newly regenerated HCs had the mechanosensory transduction function (Supplementary  Figures S1I,J). Taken together, these results suggest that Lgr5+ progenitors generate significantly more HCs than the Lgr5− SCs in vitro.

Lgr5+ Progenitors have Higher Sphere-Forming Ability than Lgr5− SCs
Sphere-forming assays have been used to evaluate cell proliferation in many studies (Li et al., 2003;Sinkkonen et al., 2011;Chai et al., 2012;Shi et al., 2012;Jan et al., 2013; FIGURE 1 | Lgr5+ HC progenitors generate more HCs compared to Lgr5− SCs in vitro. (A) We crossed the Lgr5-EGFP-CreERT2/Rosa26-tdTomato mice with Sox2-CreERT2/Rosa26-tdTomato mice to get the Lgr5-EGFP-CreERT2/Sox2-CreERT2/Rosa26-tdTomato triple-positive mice. We used the tdTomato channel to sort out all of the Sox2+ SCs, and then we used the FITC channel to separate the tdTomato/EGFP double-positive Lgr5+ progenitors from the other tdTomato+ but EGFP− SCs. Because Lgr5+ progenitors expressed both EGFP and tdTomato, the yellow dots represent the Lgr5+ progenitor cells. In Lgr5+ progenitors, some of the Myo7a+ cells were co-labeled with EdU inside of the colony and outside of the colony ( * p < 0.05, * * p < 0.01, * * * p < 0.001, n = 3. Scale bars are 20 µm in D-G). Lu et al., 2016). To determine the sphere-forming ability of Lgr5+ progenitors and Lgr5− SCs, we purified the two cell populations by flow cytometry, cultured 200 sorted cells at a density of 2 cells/µl in a Costar ultra-low attachment plate for 5 days, and counted the number of spheres before passage to the next generation (Figure 2A). We found that Lgr5+ progenitors formed significantly more spheres than the Lgr5− SCs (p < 0.05, n = 3), but there was no difference in the size of the spheres (Figures 2B,C). To further evaluate the HC regeneration ability of these spheres, we isolated the spheres derived from Lgr5+ progenitors and the Lgr5− SCs from the first generation and differentiated the spheres for 10 days. EdU was added from day 4 to 7 during the culture, and the cells were stained for Myo7a after culture ( Figure 2D). We counted the Myo7a+ HCs in each differentiated sphere as well as the total number of Myo7a+ HCs. We found that each sphere that originated from Lgr5+ progenitors generated almost 8 times as many Myo7a+ HCs than spheres from the Lgr5− SCs (Figures 2E-H), and the total spheres originating from Lgr5+ progenitors gave rise to significantly more total Myo7a+ HCs than those originating from the Lgr5− SCs (Figures 2I,J). In a similar manner, we used Myo6 and PV to label the newly regenerated HCs, and we found that all of the Myo7a+ cells were also Myo6+ and PV+ in both groups (Supplementary Figures  S2A-D). We also used phalloidin and espin1 to show the bundle morphology of newly regenerated HCs, and we found that 61.4 and 58.3% of newly regenerated HCs had hair bundles from Lgr5+ progenitors and Lgr5− SCs, respectively (Supplementary Figures S2A' ,B' ,G,H,G' ,H'). We also performed the FM1-43 dye experiments to test whether the newly regenerated HCs had the mechanosensory transduction function. We found that almost all of the newly regenerated HCs from both groups could take up FM1-43 dye, suggesting that the majority of the newly regenerated HCs had the mechanosensory transduction function (Supplementary Figures S2E,F). These results showed that Lgr5+ progenitors had a greater ability to proliferate and to generate HCs than the other Lgr5− SCs.

Analysis of RNA-Seq Results
We performed RNA-Seq analysis to identify differences in gene expression between Lgr5+ progenitors and Lgr5− SCs. Between 30.8 and 47.7 million paired-end reads were obtained for each sample, with 58.1-75.5% of the read pairs mapping correctly to the reference genome (mouse mm9). The expression of every gene was measured by FPKM (Fragments Per Kilobase of transcript per Million fragments mapped), and we filtered out genes with low expression (FPKM < 1). Three replicates of each population showed high reproducibility (Pearson's r = 0.923-0.957 for the Lgr5− SC populations and 0.941 for the Lgr5+ progenitor populations) ( Figure 3A). After excluding genes with FPKM below the baseline, 13,997 and 12,392 genes were expressed in the Lgr5+ progenitors and the Lgr5− SCs, respectively, and 11,225 of these genes were expressed in both cell populations ( Figure 3B).

Genes Enriched in Lgr5+ Progenitors or Lgr5− SCs
In order to characterize the gene-expression profiles in Lgr5+ progenitors and Lgr5− SCs, we explored the most abundantly expressed genes in both populations. Figure 3C shows the expression levels for the top 200 most abundant genes in Lgr5− SCs. For comparison, expression levels for the same transcripts in the Lgr5+ progenitors and abundance rankings for these transcripts are also illustrated. Figure 3D similarly shows the 200 most abundant transcripts in Lgr5+ progenitors compared to the same transcripts in Lgr5− SCs. As shown in both figures, the majority of the transcripts that are richly expressed in one population are also abundantly expressed in the other. However, among the most abundantly expressed genes, Mbp, Pmp22, and H1f0 were significantly highly expressed in Lgr5− SCs, and Pcp4, Acbd7, 4930170H14Rik, and Gm6537 were significantly highly expressed in Lgr5+ progenitors. None of these genes have been previously reported to be expressed in the inner ear.

Differentially Expressed Genes in Lgr5+ Progenitors and Lgr5− SCs
To determine which genes are differentially expressed in Lgr5+ progenitors and Lgr5− SCs, we compared the expression levels of all of the transcripts in Lgr5+ progenitors with those of Lgr5− SCs and selected the top differentially expressed genes ( Figure 4A). Differentially expressed genes were categorized as those whose expression levels were above background and at least 2-fold different between the Lgr5+ progenitors and Lgr5− SCs (p < 0.05). We found 1,826 genes that were differentially highly expressed in Lgr5− SCs and 986 genes that were differentially highly expressed in Lgr5+ progenitors.    Deng et al., 2006), and this supports our notion that Lgr5+ progenitors have a much greater potential to generate more sensory HCs in the neonatal cochlea. However, a significant number of the differentially expressed genes have not been characterized before and need to be further studied in the future.

Cell Cycle Analysis
Lgr5+ progenitors had significantly greater proliferation and mitotic HC regeneration ability than the other Lgr5− SCs; however, the detailed mechanism behind this difference remains unknown. To identify the possible genes regulating the cell cycling of Lgr5+ progenitors, we used RNA-Seq analysis to compare the expression of genes regulating the cell cycle and cell proliferation in Lgr5+ progenitors and Lgr5− SCs. It has been reported that over 1,000 cell cycle genes might exist in the mammalian cell (Forrest et al., 2003), and we examined the expression of 80 genes known to be involved in the cell cycle and that are commonly assayed in cell cycle PCR arrays. We found that Cdkn1b, Cdkn2a, E2f1, Rad51, and Shc1 were significantly highly expressed in Lgr5+ progenitors and that Aurka, Bcl2, Ccnd1, Ccnd2, Ccnd3, Ccnf, Cdkn3, Itgb1, Mki67, Mre11a, Msh2, Pmp22, and Trp63 were significantly highly expressed in Lgr5− SCs ( Figure 5A). However, most of the differentially expressed cell cycle-regulating genes that we identified in Lgr5+ progenitors and Lgr5− SCs have not been characterized before in the inner ear and need to be further studied. We performed q-PCR to confirm the RNA-Seq data, and the results were consistent with the microarray analysis data (Figure 5C). We did not detect the expression of Aurka or Mre11a, possibly because of their low gene expression.

Transcription Factors Analysis
Transcription factors (TFs) are proteins that bind to either enhancer or promoter regions of genes thereby controlling the expression level of these target genes. TFs are involved in various processes, including inner ear development and HC regeneration.
To determine which TFs might be involved in regulating HC regeneration, we examined the expression of 1,324 TFs in the mouse genome in Lgr5+ progenitors and Lgr5− SCs. Figure 5B shows the 83 significantly differentially expressed TFs in Lgr5+ progenitors and Lgr5− SCs (p < 0.05, fold change > 2). We found that the genes for six TFs (Barhl1, Lbx2, Nhlh1, Insm1, Egr4, and Sp5) were richly expressed in Lgr5+ progenitors but not detected in the Lgr5− SCs at all. Some of the TF genes that were highly expressed in Lgr5+ progenitors have been reported to play roles in promoting HC fate and patterning regulation during inner ear development, including Atoh1, Barhl1, Hmga2, Pou3f3, and Sox11 (Mutai et al., 2009;Chonko et al., 2013;Smeti et al., 2014;Gnedevaa and Hudspeth, 2015), and some of the TF genes that are highly expressed in Lgr5− SCs have been reported to play critical roles in regulating cell survival and apoptosis in the inner ear, including Gata2 (Haugas et al., 2010), Hif1a (Chung et al., 2011), Thrb (Ng et al., 2001), Jun (Sanz et al., 1999), Smad4 (Yang et al., 2009), andHes1 (Kelley, 2006). We performed q-PCR to confirm the RNA-Seq data, and the results were consistent with the microarray analysis data ( Figure 5D). We did not detect the expression of Sp5, possibly as a result of the low gene expression. We have identified many TFs that have not been characterized before, and their involvement in regulating the HC regeneration capacity of Lgr5+ progenitors and Lgr5− SCs should be investigated in the future.
We performed q-PCR to confirm the RNA-Seq data, and the result was consistent with the microarray analysis data ( Figure 6F). We did not detect the expression of Wnt8, Ptch1, Wif1, Krt1, or Fgf4, possibly as a result of their low gene expression. The different expression of these genes might be involved in regulating the different proliferation and regeneration ability of Lgr5+ progenitors compared to the Lgr5− SCs.

Gene Ontology Analysis of the Differentially Expressed Genes in Lgr5+ Progenitors and Lgr5− SCs
In order to obtain a comprehensive view of the gene network involved in inner ear HC regeneration, we performed a STRING protein-protein interaction analysis (Franceschini et al., 2013), which assembles the predicted networks of the differentially expressed genes (fold change > 2.0, p < 0.01) with the functional categories highlighted by GO analysis (DAVID) (Figure 7C). This integrated GO analysis suggests a complex network of genes that are involved in inner ear HC development and are predicted to participate in regulating inner ear development, cell proliferation, and Wnt signaling. GO analysis was also applied to the genes that are upregulated in Lgr5+ progenitors and Lgr5− SCs (fold change > 2.0, p < 0.01) (Figures 7A,B). As shown in Figure 7A, genes upregulated in Lgr5+ progenitors were highly enriched in functional categories such as hearing, mechanoreceptor differentiation, and inner ear development, while the genes upregulated in Lgr5− SCs were slightly enriched in functional categories such as signaling and the extracellular matrix.

DISCUSSION
In the mouse inner ear, SCs can divide and transdifferentiate into HCs (White et al., 2006;Cox et al., 2014;Lu et al., 2016). Lgr5 is only expressed in a subset of SCs, and it is enriched in the population of HC progenitors Shi et al., 2012). The Lgr5+ progenitors have a greater capacity to regenerate HCs both in vitro and in vivo than Lgr5− SCs, and Lgr5+ progenitors can be regulated by Wnt and Notch signaling to regenerate HCs via both direct differentiation and mitotic regeneration Li et al., 2016;Lu et al., 2016;Ni et al., 2016;Waqas et al., 2016b). When isolated by FACS, the Lgr5+ progenitors can be passaged for at least five generations (Chai et al., 2012;Shi et al., 2012Shi et al., , 2013Bramhall et al., 2014;Li et al., 2016;Waqas et al., 2016a;Wu et al., 2016). In this study, we isolated the Lgr5+ progenitors and the other Lgr5− SCs from transgenic mice by flow cytometry. The Lgr5+ progenitors differentiated to form more Myo7a+ HCs, and they formed more spheres than Lgr5− SCs. To understand the mechanism behind the different proliferation and HC regeneration ability of Lgr5+ progenitors compared to the other Lgr5− SCs, we determined the genomewide transcriptional profiles of these two cell populations via RNA-Seq profiling.

Differentially Expressed Genes in Lgr5+ Progenitors and Lgr5− SCs
Among the top 150 differentially expressed genes, most of them have not been reported in the inner ear, and only a few of them have been described before. The genes that are highly expressed in Lgr5+ progenitors include Cib2, Epha4, Espn, Atoh1, Lhfpl5, Smpx, and Lmo1. Epha4 is expressed in outer HCs and spiral ganglion neurons (SGNs), and it mediates afferent signaling to HCs (Defourny et al., 2013). Lhfpl5 mutation affects tip-link assembly (Zhao et al., 2014), and Smpx is strongly expressed in the sensory epithelium and plays a role in HC formation (Huebner et al., 2011;Schraders et al., 2011). Lmo1 is suggested to play an important role in HC differentiation and is specifically expressed in cochlear HCs and vestibular HCs during the development of the inner ear (Deng et al., 2006). The genes Cib2 and Espn are involved in the formation of stereocilia in the inner ear, and their disruption can lead to hearing impairment (Sekerkova et al., 2006;Ahmed et al., 2013). Our analysis showed that some of the genes that are highly expressed in Lgr5+ progenitors are crucial for HC formation during inner ear development, and expression of these genes might be the source of the high HC regeneration capabilities of Lgr5+ progenitors.
The set of reported genes that are highly expressed in Lgr5− SCs includes Ednrb, S1pr1, and Tekt2. Ednrb mutation causes syndromic hearing loss due to congenital defects in the melanocytes in the stria vascularis of the inner ear (Matsushima et al., 2014). S1pr1 is expressed in both the organ of Corti and the SGN, and it plays a role in maintaining the function of vestibular and cochlear HCs (Nakayama et al., 2014). Tekt2 is also expressed in HCs and participates in the transient appearance of the microtubule-based kinocilium in the cochlear HCs (Yoon et al., 2011). The genes that are highly expressed in Lgr5− SCs are mainly involved in the function of the cochlea.
Furthermore, we analyzed the cell cycle genes, TF genes, and signaling pathway factor genes that might regulate proliferation and HC regeneration ability, and we found 8 cell cycle genes, 9 TF genes, and 24 signaling pathway factor genes that are uniquely expressed in either Lgr5+ progenitors or Lgr5− SCs.

Cell Cycle Analysis
The highly expressed genes in Lgr5+ progenitors include E2f1, Cdkn1b, and Rad51. E2f1 is expressed in the SGN, and mitochondrial reactive oxygen species-mediated E2f1 activation induces apoptosis in the SGN (Raimundo et al., 2012). The Cdkn1b and Rad51 genes in the auditory sensory epithelium promote the proliferation and formation of supernumerary HCs in the post-natal and adult cochlea (Chen and Segil, 1999;Lowenheim et al., 1999;Oesterle et al., 2014;Walters et al., 2014).
The genes that are highly expressed in Lgr5− SCs include Bcl2, Birc5, Pkd1, Trp63, and Itgb1. Bcl2 knockout mice have high-frequency hearing loss due to a developmental defect in the stapes (Carpinelli et al., 2012;Liu et al., 2014). Birc5 is broadly expressed in the cochlea, and it can protect HCs against damage (Habtemichael et al., 2010). Pkd1 is localized to the HC stereocilia, and it plays an essential role in stereocilia structure and maintenance (Steigelman et al., 2011). Trp63 is important for normal development of the cochlea by activating the Notch signaling pathway (Terrinoni et al., 2013). Itgb1 is expressed throughout the otic area -including the sensory epithelium and the periodic mesenchyme -during inner ear development (Matilainen et al., 2007). We also found other cell cycle-promoting genes (including Ran, Stmn1, and Smc1a) and cell cycle-inhibiting genes (including E2f4, Rbl1, and Mdm2) that are abundantly expressed in both cell populations. In addition to these, the other newly identified cell cycle regulatory genes in Lgr5+ progenitors and Lgr5− SCs need to be further characterized.

Transcription Factor Analysis
The highly expressed TF genes in Lgr5+ progenitors include Barhl1, Atoh1, Hmga2, Pou3f3, and Sox11. Atoh1 promotes cochlear HC survival and differentiation, and Barhl1 is a downstream gene of Atoh1 that is essential for HC maintenance (Chonko et al., 2013). Hmga2 is broadly expressed during inner ear development, which suggests its potential dual role in early differentiation and in the maintenance of both HC and SC phenotypes (Smeti et al., 2014). Pou3f3 is specifically expressed in SCs and mesenchymal cells, and it is important for the maintenance and functional development of the post-natal cochlea (Mutai et al., 2009). Sox11 promotes the differentiation of SCs into HCs (Gnedevaa and Hudspeth, 2015).
The TF genes that were highly expressed in Lgr5− SCs include Gata2, Hif1a, Thrb, Jun, Smad4, and Hes1. Gata2 is required for vestibular morphogenesis (Haugas et al., 2010). Hif1a and Thrb are expressed in HCs, and high expression of Hif1a prevents noise-induced hearing loss (Chung et al., 2011), while a lack of Thrb leads to the developmentally delayed establishment of potassium currents (Ng et al., 2001). The Jun gene plays a critical role during inner ear development by mediating apoptosis through the JNK pathway (Sanz et al., 1999). Smad4 is required for inner ear development (Yang et al., 2009), and Hes1 inhibits SC differentiation by decreasing the expression of Atoh1 (Kelley, 2006). Our results suggest that the higher expression of these negative transcriptional regulators might be involved in the reduced proliferation capacity of Lgr5− SCs compared to Lgr5+ progenitors

Signaling Pathway Analysis
The signaling factor genes that are highly expressed in Lgr5+ progenitors include Axin2, Wnt4, Tjp2, Dll1, and Dll3. Axin2 acts as a Wnt target gene, and its expression in tympanic border cells allows them to behave as HC progenitors (Jan et al., 2013;Jansson et al., 2015). Combined with other growth factors, Egf can protect HCs from ototoxic damage (Lou et al., 2015). Wnt4 is detected in the inner and outer spiral sulcus cells, as well as in the Claudius and Hensen's cells, and downregulation of Wnt4 expression significantly reduces the proliferation of SCs (Alvarado et al., 2011). The Tjp2 gene is mainly expressed in the membrane between the HCs and SCs, and mutation of the Tjp2 gene causes hearing loss (Op de Beeck et al., 2011;Kim et al., 2014). Both Dll1 and Dll3 can repress HC formation and can promote HC differentiation (Kiernan et al., 2005;Hartman et al., 2007;Chrysostomou et al., 2012;Petrovic et al., 2014).
The signaling factor genes that are highly expressed in Lgr5− SCs include Dusp6, Egf, Rhoa,Fgf9,Frzb,Fzd1,Fzd4,Fzd9,Fkbp8,Hey2,Src,Smo,Vangl2,Wnt5a,Wnt6,and Wif1. Dusp6 is expressed in the otic region during embryonic development and acts as a negative feedback regulator of FGF signaling (Urness et al., 2008). Rhoa, Wnt5a, Src, Vangl2, and Wif1 mediate planar cell polarity in the inner ear (Dabdoub et al., 2003;Qian et al., 2007;Copley et al., 2013;Andreeva et al., 2014;Sai et al., 2014), and Src inhibitors protect HCs from noise-induced damage (Bielefeld, 2015). Fgf9 participates in regulating the number of cochlear progenitors and the length of the cochlea through mesenchymal FGFR signaling (Pirvola et al., 2004;Huh et al., 2015). Smo is an effector of Hedgehog signaling that inhibits prosensory cell differentiation into HCs or SCs and delays differentiation in the apical region (Tateya et al., 2013). Fzd1, Fzd4, and Fzd9 are expressed in adult mouse SGNs, and Fzd9 guides neurite regeneration in SGNs (Shah et al., 2009). Frzb is expressed in the lateral region of the developing organ of Corti and regulates stereociliary orientation and cochlear extension (Qian et al., 2007). Fkbp8 is localized in the SGN and is important for the onset of hearing processes in rodents (Zak et al., 2011). Hey2 is highly expressed in HCs and SCs, and it is critical for maintaining prosensory cells in an undifferentiated state (Benito-Gonzalez and Doetzlhofer, 2014). Wnt6 is the first Wnt gene expressed in the otic epithelium at embryonic day 8.5, and its expression is confined to the dorsal portion of the otic placode (Lillevali et al., 2006). Our analysis shows that the genes that are highly expressed in Lgr5− SCs are mainly involved in the function of the cochlea but not in cell proliferation or HC regeneration.

STRING Prediction of Inner Ear HC Development
In this protein-protein interaction network, most of the genes in the GO categories of sensory organ development were highly expressed in Lgr5+ progenitors, such as Gfi1, Pou4f3, and Pax2, although several genes such as Eya1 and Bcl2 were richly expressed in Lgr5− SCs. It would be interesting to further investigate the involvement of these genes in regulating the progenitor cells.
All of the data we provided in this paper were from the neonatal mouse cochlea. The situation might be completely different in the adult mouse cochlea or in the damaged mouse cochlea, and the approaches to promote HC regeneration in the neonatal cochlea might not have the same effects in the adult cochlea. Thus, further investigations in the adult cochlea or damaged cochlea need to be performed in the future.

CONCLUSION
We found that Lgr5+ progenitors have significantly greater proliferation and HC regeneration ability than Lgr5− SCs. We investigated the transcriptome differences between Lgr5+ progenitors and Lgr5− SCs and found significantly differentially expressed genes that might regulate the Lgr5+ progenitors' proliferation and HC regeneration capacity. The most interesting of these are the genes that are uniquely expressed in Lgr5+ progenitors but not in Lgr5− SCs. To further analyze the role of differentially expressed genes in HC regeneration and proliferation, we constructed a STRING prediction map. The transcriptomes of Lgr5+ progenitors and Lgr5− SCs reported here establish a framework for future characterization of the genes that regulate the proliferation and HC regeneration ability of Lgr5+ progenitors, and these genes might represent new therapeutic targets for HC regeneration.

AUTHOR CONTRIBUTIONS
CC, LG, LL, HS, HL, and RC designed the study. CC, LG, HS, XX, and SZ performed the laboratory experiments. RC, CC, LG, HS, CX, HL, MW, YC, FC, XZ, XG, and MT contributed to critical discussion and data analysis. CC, MW, HS, RC, HL, JG, and LG wrote the paper. All authors read and approved the final manuscript.

SUPPLEMENTARY MATERIAL
The Supplementary Material for this article can be found online at: http://journal.frontiersin.org/article/10.3389/fnmol. 2017.00122/full#supplementary-material