β-Catenin transcriptional activity is required for establishment of inner pillar cell identity during cochlear development

The mammalian cochlea is composed of sensory hair cells as well as multiple different types of non-sensory supporting cells. Pillar cells are one type of supporting cell that form the tunnel of Corti and include two morphologically and functionally distinct subtypes: inner pillar cells (IPCs) and outer pillar cells (OPCs). The processes of specification and differentiation of inner versus outer pillar cells are still unclear. Here, we show that β-Catenin is required for establishing IPC identity in the mammalian cochlea. To differentiate the transcriptional and adhesion roles of β-Catenin in establishing IPC identity, we examined two different models of β-Catenin deletion; one that deletes both transcriptional and structural functions and one which retains cell adhesion function but lacks transcriptional function. Here, we show that cochleae lacking β-Catenin transcriptional function lost IPCs and displayed extranumerary OPCs, indicating its requirement for establishing IPC identity. Overexpression of β-Catenin induced proliferation within IPCs but not ectopic IPCs. Single-cell transcriptomes of supporting cells lacking β-Catenin transcriptional function show a loss of the IPC and gain of OPC signatures. Finally, targeted deletion of β-Catenin in IPCs also led to the loss of IPC identity, indicating a cell autonomous role of β-Catenin in establishing IPC identity. As IPCs have the capacity to regenerate sensory hair cells in the postnatal cochlea, our results will aid in future IPC-based hair cell regeneration strategies.


Introduction
The sensory epithelium of the mammalian cochlea (the organ of Corti) consists of multiple unique cell types arranged in a highly ordered pattern that is necessary for proper auditory function.Cells in this organ are categorized into sensory hair cells and non-sensory supporting cells.Hair cells are arranged into 4 rows: three rows of outer hair cells and one row of inner hair cells.Supporting cells consist of multiple cell types including (from lateral to medial): several rows of Hensen's cells, 3 rows of Deiters' cells (DCs) that underlie the outer hair cells, one row of outer pillar cells (OPCs), one row of inner pillar cells (IPCs) and few rows of inner phalangeal cells that support inner hair cells (reviewed in [1] and displayed in Fig 1A).In the mature organ, IPCs and OPCs form the boundaries of a triangular space called the tunnel of Corti [2].
Supporting cells play crucial roles in cochlear development, maturation, and repair after injury.During development, supporting cells regulate patterning of the epithelium and synaptogenesis [3][4][5][6].Supporting cells provides structural support during sound stimulation, maintain extracellular ion concentrations essential for hair cell function, maintain the epithelial reticular lamina, and support the survival of hair cells and neurons [7][8][9][10].After damage to the auditory epithelium, supporting cells regulate scar formation and clearance of debris [11].Additionally, it has been shown that supporting cells can replace hair cells after damage in the neonatal mouse cochlea, yet this ability decreases significantly during the postnatal period [12][13][14].
During mammalian cochlear development, both hair cells and supporting cells are derived from the prosensory cells that are specified in a narrow domain in the floor of the cochlear duct [15].Around embryonic day (E)13, prosensory cells begin to exit mitosis and upregulate the cell cycle inhibitor CDKN1B (p27) [16].Once hair cells are specified, Notch signaling mediates lateral inhibitory interactions to prevent prosensory cells from adopting a hair cell fate, which creates the mosaic hair cell/supporting cell pattern in the mature cochlea [17][18][19].As hair cells differentiate, surrounding supporting cells differentiate [20,21].There is emerging evidence that distinct mechanisms regulate specification of different subtypes of supporting cells.For instance, multiple studies have shown that pillar cell fate is induced by the FGF8 secreted from inner hair cells around E16.5.FGF8 binds to and activates FGFR3 in adjacent precursors of supporting cells, leading to the formation of pillar cells [22][23][24][25][26][27].However, how inner versus outer pillar cells are individually specified is still unknown.
Canonical Wnt/β-Catenin signaling regulates a wide range of developmental processes including cell proliferation, migration, epithelial-mesenchymal transition, and differentiation [28,29].In the absence of Wnt, cytoplasmic β-Catenin protein is constantly degraded by the action of the Axin complex, which prevents β-Catenin from reaching the nucleus.Once a Wnt ligand binds to a Frizzled (Fz) receptor and its co-receptor, a complex is formed that eventually leads to the inhibition of Axin-mediated β-Catenin degradation and to the stabilization of β-Catenin, which in turn accumulates and translocates to the nucleus to form complexes with transcription factors such as Tcf and Lef and thereby activates Wnt target gene expression (reviewed in [28,30]).In addition to its role in canonical Wnt signaling, β-Catenin connects Ecadherin and α-Catenin to generate an important component in the cell-to-cell adhesion system which is required for the structural integrity of epithelial cells [31].
There is growing evidence that Wnt/β-Catenin signaling pathway is essential for mammalian cochlear development.In the developing cochlea, canonical Wnt/β-Catenin signaling reporter activity becomes restricted to medial supporting cells including pillar cells and inner phalangeal cells [32].In two independent studies, deletion of β-Catenin during differentiation of prosensory cells (E13.5)resulted in loss of NGFR (p75) expression in the IPCs [33,34].Additionally, it is shown that β-Catenin deletion, via its cell adhesion role, disrupts the boundaries between medial and lateral compartments of the developing cochlea [34].To differentiate the canonical Wnt signaling from the structural roles of β-Catenin in supporting cell development, we have examined two different β-Catenin deletion alleles in this study: (1) the β-Catenin fl allele that has a deletion from exon 2 (which contains the ATG translation start site) to exon 6, generating a β-Catenin null allele that deletes both transcriptional and structural functions [35], and (2) the β-Catenin dm allele which retains cell adhesion function but deletes transcriptional function by point mutating Aspartic Acid of 164 to Alanine (D164A) and deleting the C-terminus [36].In this study, we analyzed the differences between these two β-Catenin deletion models, to determine the role of β-Catenin in pillar cell fate determination.Through genetic modification of β-Catenin gene, lineage tracing, and single cell RNA sequencing, we showed that that β-Catenin transcriptional activity within supporting cells is required to establish IPC identity during cochlear development.

β-Catenin transcriptional activity regulates pillar cell development
In our previous study [34], we utilized β-Catenin fl allele to eliminate both the transcriptional and cell adhesion functions of β-Catenin, and showed that Sox2-Cre-and Fgf20-Cre-mediated deletions of β-Catenin caused hair cell and supporting cell disorganization throughout the cochlear duct.Additionally, p75 expression (IPC marker) was downregulated or absent.To further investigate the role of β-Catenin in the development of cochlear supporting cells, we utilized an additional Cre driver, Emx2-Cre, which is active in cochlear epithelial cells by E12.5.Emx2-Cre has a broader activity domain and higher efficiency compared to the Sox2-Cre and Fgf20-Cre models, as well as higher animal survival rates [34,37,38].Analysis of cochleae from β-Catenin conditional mutants (β-Catenin fl/fl ; Emx2 Cre/+ ) and littermate controls (β-Catenin fl/+ ; Emx2 Cre/+ , β-Catenin fl/+ ; Emx2 +/+ , or β-Catenin fl/fl ; Emx2 +/+ ) at E18.5 showed hair cell disorganization in all cochlear turns, disrupted boundaries between inner and number of CD44+ cells in β-Catenin cKO.White dashed lines indicate the boundary between the first row of Deiters' cells and the OPCs (D) Quantification of HCs, Prox1+, p75+, and CD44+ cells at E18.5 in β-Catenin cKO versus control (apical, middle, and basal turns averaged).(E-F) Immunostaining of whole mount E18.5 cochlear epithelium (basal turn) from β-Catenin fl/fl ; Fgf20 Cre/+ (Complete deletion; cKO) and β-Catenin dm/fl ; Fgf20 Cre/+ (Transcriptional deletion; dm-cKO) for phalloidin, p75, Prox1 and CD44 showing loss of p75 staining, increased CD44 staining and increased density of Prox1+ cells in both deletion models versus controls.(G-H) Immunostaining of cochlear sections for p75 (green), CD44 (gray), and Sox2 (red) at E18.5 from both deletion models versus control showing loss of the IPC maker and gain of the OPC marker.White dashed lines indicate the boundary between the 1 st row of Deiters' cells and the OPCs (I-J) Quantification of the density of HCs, Prox1+, p75+, and CD44+ cells at E18.5 in both deletion models versus controls showing a statistically significant loss of p75+ cells and gain of CD44+ cells.Scale bar = 100μm.n = 3-5 per group, mean±SE, Student's t-test p-value indicated.https://doi.org/10.1371/journal.pgen.1010925.g001outer hair cells (as indicated by E-cadherin staining), without a significant change in hair cell density (Fig 1A and 1B), a phenotype consistent with our previous results [34].On the other hand, β-Catenin mutants revealed an increase in Prox1+ supporting cell density, loss of IPCs (p75+ cells), and gain of OPCs (CD44+ cells) across all cochlear turns (n = 4, Student's t-test p<0.05) (Fig 1C and 1D).Most ectopic CD44+ cells were located medial to the first row of Prox1+ Deiters' cells (Fig 1C).To investigate the underlying cause of increased Prox1+ cell density, we immunostained for Ki67 proliferation marker and Sox2, and found no significant changes in Sox2/Ki67 double positive cells in the prosensory domain in β-Catenin conditional mutants.This indicates that the increase in Prox1+ cell density is not due to increased cell proliferation (S1A and S1B Fig) .Altogether, our data suggest that β-Catenin is required for IPC identity within the developing cochlea.
To test whether the phenotype observed in β-Catenin conditional mutant cochleae is due to the deletion of β-Catenin cell adhesion or transcriptional function, we generated and compared 2 different mouse models: β-Catenin fl/fl ; Fgf20 Cre/+ (complete deletion; cKO) to delete both β-Catenin cell adhesion and transcriptional functionalities, and β-Catenin dm/fl ; Fgf20 Cre/+ (transcriptional deletion; dm-cKO) to delete β-Catenin transcriptional activity while retaining the cell-cell adhesion function [36].Since homozygous β-Catenin dm/dm mice exhibit early embryonic lethality [36], we utilized Fgf20 Cre mice to delete one copy of β-Catenin fl allele, while the other allele is β-Catenin dm (β-Catenin dm/fl ; Fgf20 Cre/+ ).β-Catenin fl/+ ; Fgf20 Cre/+ was used as the control for both cKO and dm-cKO models.Both models have demonstrated high efficiency of β-Catenin deletion from the cochlear supporting cells [34].Postnatal day (P)0 cochleae from both models (cKO and dm-cKO) showed loss of p75+ IPCs and gain of CD44 + OPCs along with an overall increase in density of Prox1+ supporting cells (Fig 1E -1J) (n = 5, Student's t-test p<0.05).Hair cells and supporting cells appeared less disorganized in the dm-cKO model compared to the cKO model, likely because the dm-cKO cochleae retain the cell adhesion function of β-Catenin.Immunostaining of cochlear sections from both models confirmed that supporting cells in the region of IPCs have lost expression of p75 and gained CD44.Of note, we observed fewer supernumerary CD44+ cells in dm-cKO compared to cKO cochleae (Fig 1I and 1J), which may indicate a less severe phenotype in the former model.It is notable that these supporting cells retained Sox2 expression, indicating that they maintained their supporting cell signature (Fig 1G and 1H).Since both cKO and dm-cKO models displayed the loss of IPC staining and gain of OPC staining, β-Catenin transcriptional activity is likely required for establishing IPC fate.

Lineage tracing of β-Catenin-deficient cells shows that β-Catenin transcriptional activity is required for IPC fate
To concurrently trace and delete β-Catenin in prosensory cells around the time of pillar cell specification, we utilized an inducible Cre allele (Lgr5 CreERT2/+ ) that is active in the Sox2+ supporting cells of the cochlear duct [39], along with Rosa tdTomato/+ allele that expresses tdTomato upon Cre recombinase activity to trace Cre activity [40].This setup allows spatial and temporal control for β-Catenin deletion at later time points compared to Fgf20-Cre line.We generated pregnant female mice carrying β-Catenin fl/fl ; Lgr5 CreERT2/+ ; Rosa tdTomato/+ embryos (lacking both β-Catenin functions) and β-Catenin fl/dm ; Lgr5 CreERT2/+ ; Rosa tdTomato/+ embryos (lacking β-Catenin transcription but retaining cell adhesion function).Pregnant females were induced with tamoxifen (250 mg/kg) once at E14.5, before pillar cell differentiation.Analysis of the cochleae from control mice at E18.5 for the recombination efficiency within the region of IPCs showed efficient recombination ranging from 75% to 82% in both models (Fig 2A and 2B).We further validated β-Catenin deletion using cochlear immunostaining against β-Catenin 48 ).On the other hand, β-Catenin staining was present in the transcription deletion model (β-Catenin fl/dm ; Lgr5 CreERT2/+ ; Rosa tdTomato/+ ), as β-Catenin cell adhesion function is expected to be preserved in this model.Analysis of β-Catenin deficient cochlea showed loss of p75 staining from many recombined (tdTomato+) cells within the region of IPCs in both conditional deletion models when compared to controls (Fig 2A and 2B).In complete deletion cochleae, the average percentages of recombined IPCs that lost the p75 staining were significantly higher (39.7% (base), 50.6% (middle), and 31.2%(apex)) than those in controls (2.6% (base), 2.7% (middle), and 1.6 (apex), Fig 2C) (n = 3, mean, Student's t-test p<0.05).Similarly, in transcription deletion cochleae, the average percentages of recombined IPCs that lost the p75 staining were significantly higher (36.7% (base), 36.3% (middle), and 29.3% (apex)) compared to controls (2.3% (base), 0% (middle), and 1.6% (apex), Fig 2D) (n = 3, mean, Student's t-test p<0.05).Of note, we observed that some recombined cells in IPC region maintained p75 staining, which is likely because not all recombined cells have deleted β-Catenin.This is supported by the fact that some recombined cells maintained β-Catenin expression (S2A and S2B Fig) .Nevertheless, these results affirm that β-Catenin transcriptional activity is required for establishing IPC fate.Next, to assess IPC to OPC conversion, we analyzed the percentage of recombined IPCs that lost p75 and gained CD44 expression.Because CD44 expression has not yet matured in the apical turn of E18.5 cochlea, we analyzed the basal and middle turns only.In complete deletion cochleae, 23.6% (basal turn) and 32.3% (middle turn) of recombined IPCs lost p75 and gained CD44 staining, whereas none was observed in controls.Similarly, transcription deletion cochleae, some recombined IPCs lost p75 and gained CD44 staining (24.7% (basal turn) and 15.6% (middle turn)) whereas none was detected in controls (n = 3/group, mean, Student's t-test p<0.05) (Fig 2E and 2F).These results affirm that β-Catenin transcriptional activity is required for establishing IPC fate.Furthermore, these results indicate that a subset of β-Catenin deficient cells that failed to differentiate to IPCs had likely acquired an OPC fate.

Single cell RNA sequencing of β-Catenin-deficient supporting cells shows loss of IPC identity
To understand the molecular identity of the β-Catenin-deficient supporting cells within the IPC region, we utilized single cell RNA (scRNA) sequencing of cochlear duct cells from dm-cKO (β-Catenin dm/fl ; Fgf20 Cre/+ ) P0 pups and littermate controls (β-Catenin fl/+ ; Fgf20 Cre/+ ).We pooled both cochleae of one control pup at P0 and analyzed 2,830 cells with 58,718 mean reads per cell, and a sequence saturation of 73.5%.Around 87% of the reads were mapped to the mouse genome and a total of 18,757 genes were detected.We analyzed control sample data using unsupervised graph-based clustering in Loupe Browser v.6 (10X genomics) to cluster similar transcriptome signatures from control cochlear cells.We were able to identify unique transcriptomic signatures belonging to specific cell populations based on previously published markers.Hair cells markers such as Barhl1, Pou4f3, Pvalb were enriched in hair cell clusters [43], Lfng, Fgfr3 and Prox1 [20] were enriched in supporting cells (S3A-S3C Fig) .For the top 20 enriched genes per cluster, see Table 1.For the full list of differentially expressed genes, please refer to S1 Table .Using single cell sequencing data from the control sample, we further selected the Prox1 + supporting cell population and re-clustered this population to identify specific subclusters within the supporting cells at P0. Unsupervised graph-based clustering identified 3 different clusters within supporting cells at P0 (Fig 4A, top panel).IPCs formed a separate cluster as indicated by p75 expression, while the other 2 clusters were partially connected.To identify the identity of the other 2 clusters, we examined CD44 (OPC marker) and Lgr5 (marker of 3 rd row of Deiters' cells and IPCs) expression (Fig 4A and 4B).Data showed that the 3 rd row of Deiters' cells (DC3) clustered separately from the 1 st and 2 nd rows of Deiters' cells (DC1/2).OPC cells (CD44+ cells) were located within the DC1/2 cluster, indicating that OPCs' molecular signature is similar to the DC1/2 signature at P0 (Fig 4A).We analyzed differentially expressed genes (DEGs) among the 3 clusters (IPCs, DC3 & DC1/2) and identified enriched genes within each cluster (see Table 2 for the top 20 genes per cluster and S2 Table for the full list) which constitute the molecular signature of each cluster at P0.
We next analyzed P0 dm-cKO cochlear cells using the same methods mentioned above.We pooled both cochleae from one pup and were able to analyze a total of 2,991 cells, with mean reads of 50 .Forty-four percent of downregulated genes in dm-cKO belong to the IPC signature, including Igfbpl1, p75, Npy and Cryab, while 44% of upregulated genes in dm-cKO belong to the DC3 signature, including Srp9, Frmd4a, Hnrnpu and Mgst3.These results suggest a loss of the IPC signature upon deletion of β-Catenin transcriptional activity.For the top DEGs, please refer to Table 3.We next analyzed enriched gene ontologies within upregulated and downregulated gene sets in dm-cKO vs control Prox1+ supporting cells using the Database of Annotation, Visualization, and Integrated Discovery (DAVID) [44,45].Within the downregulated genes, we found genes associated with canonical Wnt signaling (fzd4 and klf4), MAPK signaling pathway genes (p75, Nr4a1, and Map2k1), and few transcription factors (Hes6, Nr4a1, Fosb, and Klf4).Enriched gene ontologies within upregulated genes included multiple genes involved in cell differentiation (Enah, Itga8, Hnrnpu, Lrrc8a, Edf1 and Prrc2b) (S4A and S4B Fig) .To validate the loss of IPC signature in dm-cKO supporting cells, we immunostained cochlear samples from both models against a recently identified IPC marker Npy [43], and found it to be significantly reduced in all turns of dm-cKO cochlea (Fig 5C).Through fluorescence in situ hybridization, we validated that another IPC maker (Lgr6) was downregulated in dm-cKO supporting cells (Fig 5D).Lastly, we found ectopic expression of the OPC marker Tuba1b in the IPC region in dm-cKO cochleae (Fig 5E ), providing additional evidence for the IPC-to-OPC identify shift.

β-Catenin cell autonomous role in establishing IPC identity
To investigate if β-Catenin functions in a cell-autonomous or non-cell-autonomous manner to establish IPC identity, we utilized an Npy Cre mouse line to delete β-Catenin from IPCs, as Npy expression is restricted to IPCs and is expressed as early as E16.5 [43].Upon crossing the Npy Cre line with a Rosa tdTomato line, tdTomato expression (as a marker of Cre recombination) was restricted to IPCs and glial cells in the cochlea at P0 (S5A Fig) .To confirm IPC-specific deletion of β-Catenin, we generated an IPC-specific β-Catenin conditional knockout along with the tdTomato reporter gene (β-Catenin fl/fl ; Rosa tdTomato/+ ; Npy Cre/+ ).Analysis of P1 cochlea from this conditional knockout showed reduced levels of β-Catenin in the majority of IPCs (S5B Fig) .To test the effects of IPC-specific β-Catenin deletion, we generated and    Npy Cre/+ ) did not show statistically significant changes, a trend of reduction in p75 expression and occasional ectopic CD44+ cells were observed (Fig 6D -6F).Overall, our results strongly suggest a cell-autonomous role of β-Catenin in establishing IPC identity.

Discussion
Supporting cells serve multiple crucial roles in the mammalian cochlea, contributing to the structural integrity of organ of Corti during sound stimulation and maintaining the optimal micro-environment required for normal hair cell function [3,46,47].Pillar cells are supporting cells required to form the tunnel of Corti, which is unique to the mammalian cochlea and is required for normal hearing [16,22,48].FGF8-FGFR3 signaling pathway has been implicated for pillar cell development [22][23][24][25][26][27], but the mechanism of IPC versus OPC fate determination is still unknown.This study shows multiple lines of evidence that β-Catenin transcriptional activity is required for IPC identity.
Using two independent β-Catenin deletion models (complete deletion and transcriptional deletion) along with multiple Cre drivers, we show that lack of β-Catenin causes cells within the IPC region to lose IPC signature genes (such as p75, Npy, and Lgr6) and gain OPC genes (such as CD44 and Tuba1b) ( Fig 7) indicating that the β-Catenin transcriptional role is involved in establishing IPC versus OPC identity.It is not clear why cells in the IPC region lose p75 expression while gaining expression of CD44.We postulate that the OPC fate might be the default fate for pillar cells, and only when β-Catenin transcriptional activity is turned on, the IPC fate is established.The phenotype of ectopic OPCs was more evident in the complete deletion model compared to the transcriptional deletion model (Fig 1I and 1J), suggesting that cell adhesion may also play a role in pillar cell development.We have observed an increase in the density of Prox1+ supporting cells in these models that is not accompanied by change in hair cell density, indicating that changes in cochlear duct length is not contributing to the phenotype.Immunostaining for the proliferation marker Ki67 did not detect a difference in mutant cochleae, thereby ruling out a change in proliferation of supporting cell progenitors.We postulate that there might be a fate change from lesser epithelial ridge (LER) cells to supporting cells following β-Catenin deletion, which warrant further examination in future experiments.Lineage tracing of β-Catenin-deficient cells showed a loss of p75 and gain of CD44 expression from a subset of recombined cells within the IPC region in both complete and transcriptional deletion models.Not all recombined cells showed this phenotype, suggesting that β-Catenin activity may not be the sole determinant of IPC fate.Nevertheless, our findings confirm the requirement of β-Catenin transcriptional role for IPC identity.
Using a constitutively active β-Catenin model, we tested whether β-Catenin is sufficient for inducing IPC fate.Data show no ectopic IPCs outside their respective cochlear domain, but that some IPCs proliferated to create clusters of IPCs (Fig 3).This data shows that IPCs are capable of β-Catenin-induced proliferation during development, yet β-Catenin is not sufficient to induce IPC fate in supporting cells.Our data aligns with recently published work showing that β-Catenin stabilization increases proliferation in cochlear epithelial cells [49].
In addition to using p75 and CD44 as markers for IPCs and OPCs, respectively [50,51], we analyzed the molecular signature of pillar cells in β-Catenin transcriptional deletion model to confirm the loss of IPC identity and to identify targets downstream β-Catenin during supporting cell development.Single cell transcriptomic analysis at P0 showed that supporting cells lacking the β-Catenin transcriptional function failed to establish the unique IPC molecular signature as identified in the controls.Although we utilized one biological sample per condition for the scRNA sequencing experiment, we have validated a subset of DEGs using immunostaining and in situ hybridization to support IPC identity loss.Analyzing supporting cells from the β-Catenin transcriptional deletion model, we observed a significant loss of IPC specific genes and gain of other supporting cells-specific genes including Deiters' and/or OPC marker genes.Downregulated genes in β-Catenin deficient supporting cells did not show canonical Wnt target genes such as Axin2, Lgr5 or Sp5, which may be due to the late time point of the scRNA sequencing sample (P0) relative to the time of IPC specification and differentiation (around E15) [23][24][25].A valid follow up experiment is to investigate the expression of Wnt target genes within the supporting cells from β-Catenin deletion models at earlier time points (E15.5-16.5).Nevertheless, P0 single cell transcriptome analysis shows the ultimate impact of β-Catenin deletion on pillar cell fates.
Finally, we limited β-Catenin deletion to IPCs to determine its autonomous role in IPC fate establishment.IPC-specific conditional β-Catenin deletion resulted in a similar phenotype of IPC to OPC marker conversion within IPCs.Notably, this phenotype was more evident in the apex than in the middle and basal turns.We suggest that pillar cell specification follow a basal to apical gradient similar to hair cell differentiation.Basal cells may have already established IPC identity at the time of Cre-mediated β-Catenin deletion.This may explain why we found fewer p75 to CD44 conversions in the basal and middle turns compared to the apex.Nevertheless, the occurrence of converted cells and CD44/p75 double positive cells in all turns in the cochlea suggests a cell autonomous role of β-Catenin in IPC identity establishment.
While this work has shown β-Catenin transcriptional activity requirement for IPC fate, there are still multiple questions that need to be addressed.First, which Wnt(s) is required for IPC fate?It has been shown that Wnt5a and Wnt7a are expressed in cochlear duct at E14.5 [52,53] and play a role in regulating planar cell polarity.However, it is not clear if these ligands also contribute to canonical Wnt signaling activity in supporting cells.Deleting Wnt ligands during pillar cell specification could help answer this question.Second, how is Wnt receptor expression spatially and temporally regulated to ensure restricted Wnt signaling activity to IPCs?A few Wnt receptors, such as Fzd1 and Fzd2, are expressed within the supporting cells during development (reviewed in [54]) and may contribute to IPC fate establishment.Third, does canonical Wnt/β-Catenin signaling interact with FGF8/FGFR3 signaling to establish pillar cell fate?All these questions are unanswered and further research is needed to address them.
One of the most significant applications of Wnt/β-Catenin signaling role in IPC fate is for hair cell regeneration.Activation of Wnt signaling within IPC was sufficient to drive proliferation, as shown in this study.Since proliferation is a prerequisite for regeneration after damage in avian cochlea [55], activating proliferation in established IPCs can be the source of regenerating cells that can be further programmed to hair cells through gene manipulation [56][57][58].It is still unclear what is the molecular signature of proliferating IPCs upon Wnt activation, and whether these cells carry the programming potential to become hair cells.

Conclusion
This work provides a thorough comparison of supporting cell phenotypes in β-Catenin complete deletion models versus β-Catenin transcriptional deletion models (Fig 7).We show using multiple lines of evidence that β-Catenin transcriptional activity during supporting cell development is required to establish IPC identity.Postnatal roles of β-Catenin in IPC maturation and maintenance remain to be further tested with similar experiments involving cochlea from later postnatal stages.

Ethics statement
This study was carried out in accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health.All protocols were approved by the Animal Care and Use Committees at Stanford University, and University of Nebraska Medical Center (IACUC numbers 18606 and 16-005-02-EP, respectively).

Proliferation assay
Pregnant females were injected with Ethynyl deoxyuridine (EdU) at 400μg/g body weight two hours ahead of collecting the embryos.Embryos were collected and prepared for cryosectioning.Staining for incorporated EdU along was performed according to Click-iT EdU Cell Proliferation Kit (Invitrogen C10340) manufacturer protocol.Stained sections were then imaged on a Zeiss LSM 700 confocal microscope.

Single cell RNA sequencing
A P0 pup was euthanized, and cochleae were dissected in sterile cold HBSS, then placed in Dispase (1U/mL) in DMEM/F-12 (Stem cell Technologies) for 15 min at 37˚C.Cochlear ducts from both sides were dissected and pooled into a single tube, incubated in 0.25% trypsin-EDTA for 15 min at 37˚C with gentle trituration every 5 min, followed by trypsin inactivation by adding an equal volume of DMEM/F-12.Dissociated cells were then passed through a 30μm strainer, pelleted at 300g and then resuspended in 100μl of cold PBS supplemented with 1% fetal bovine serum (FBS, Gibco: 26140079).Single cells were captured and lysed, and mRNAs were reverse transcribed into cDNAs using a 10X Genomics Chromium Controller at University of Nebraska Medical Center Sequencing core.cDNA libraries were prepared using Chromium Single Cell 3 0 Reagents according to the manufacturer's instructions.Libraries were sequenced on an Illumina NextSeq.Sequences were aligned to the Ensembl mouse MM10 assembly using Cell Ranger 2.1.1 analysis software (10× Genomics).Processing of the Cell Ranger output data was done with Loupe Browser v.6 (10X Genomics).Gene expression-based clustering information, including t-SNE projections and differential gene expression were done utilizing Loupe Browser v.6.The raw data from the scRNA sequencing is deposited in gEAR database and can be accessed through the following link: https://umgear.org/index.html?multigene_plots=0&layout_id=d4505add&gene_symbol_exact_match=1&gene_symbol=sox2

RNA fluorescence in situ hybridization (FISH)
Cryosections (10μm) were used for RNA FISH analysis following the manufacturer's protocol (Molecular instruments, HCR RNA-FISH protocol for fresh frozen [61].

Quantification and statistics
Data analysis was done using Image J software Version 1.53n [62].To measure HC and SC density, at least 400μm regions of the base, middle, and apex of whole mount immunostained cochleae were counted and normalized to 100μm.Two independent investigators performed manual counts blindly after separation of individual channels, selection of images/regions without artifacts, conversion to 8-bit images, manually setting of the threshold to reduce background, selection of the region length/area to measure, then manually counting the positive cells using ImageJ cell counter tool.Cells were called positive for a specific staining based on signal to noise ratio.Cell counts were averaged across manual counts and presented on graphs.For each experiment, the numbers of samples (n) are indicated.The P value for difference between samples was calculated using multiple unpaired two-tailed Student's t-test, and P < 0.05 was considered as significant.False Discovery Rate (FDR) was used to adjust for multiple comparisons using two-stage step-up.For ScRNA sequencing data analysis, Cell Ranger 2.1.1 analysis software (10X Genomics) was used.

5 .
Constitutively active β-Catenin cochlea exhibited multiple foci of p75+ cell clusters within the IPC region, yet no ectopic IPC formation was found outside the IPC region (Fig 3A).Some cells around the p75+ foci showed CD44 expression disruption (Fig 3A).Quantification of p75+ cell density showed a significant and modest increase in the basal and middle turn of constitutively active β-Catenin mutants versus controls (27.6 cells versus 20.1 /100 μm and 26.9 cells versus 20.9 cells/100 μm, respectively) (Fig 3B).The density Rosa tdTomato/+ cochlea compared to control showing similar results as (A).(C-D) Quantification of the percentage of recombined IPCs that are p75-in each turn from both models.(E-F) Quantification of the percentage of recombined IPCs that are p75-and CD44+ in each turn from both models.Scale bar = 50μm.n = 3, mean±SE, Student's t-Test pvalue indicated.https://doi.org/10.1371/journal.pgen.1010925.g002PLOS GENETICS of CD44+ cells decreased in the basal turn of the constitutively active β-Catenin mutant cochlea (11.1 cells/100 μm versus 14 cells/100 μm in control) (n = 3, mean, Student's t-test p <0.05) (Fig 3C)

Fig 4 .
Fig 4. β-Catenin transcriptional activity within supporting cells is required for inner pillar cell gene expression signature.(A) t-SNE plots representing graph-based clustering of scRNA sequencing data from Prox1+ supporting cell population at P0 in control (β-Catenin fl/+ ; Fgf20 Cre/+ : top panel), dm-cKO (β-Catenin dm/fl ; Fgf20 Cre/+ ; middle panel) and aggregate of both samples (bottom panel) show 3 different cell clusters in control, identified based on known markers of each population (IPCs in blue, 3 rd row of Deiters' cells in black, and OPCs together with 1 st and 2 nd rows of Deiters' cells in red).dm-cKO plots (middle panel) shows only 2 cell clusters (color-coded) with decreased p75 expression and increased CD44 expression when compared to control.Upon aggregating both samples (lower panel), control IPCs make a tight cluster expressing p75 (black oval), while cells from dm-cKO are in a transitional zone relative to the rest of Prox1+ cells (red oval) (Loupe Browser v6.0, 10x Genomics).(B) Schematic diagram of organ of Corti cells depicting the 3 different clusters of supporting cells found in control (C) Quantification of the percentage of IPCs (Prox1+p75+), OPCs (Prox1+CD44+), and Deiters' cells (Prox1+, p75-, and CD44-) relative to total Prox1+ cells in control versus dm-cKO cochlea at P0 showing a decrease in IPCs and gain of OPCs in dm-cKO.(D) Quantification of the percentage of Lgr5+ cells (marker of 3 rd row of Deiters' cells and IPCs) relative to total Prox1+ cells in control versus dm-cKO cochlea at P0. https://doi.org/10.1371/journal.pgen.1010925.g004

Fig 5 .
Fig 5. β-Catenin transcriptional activity regulates gene expression within supporting cells to establish inner pillar cell gene expression signature.(A) Heat map of differentially expressed genes within Prox1+ cells at P0 in control (β-Catenin fl/+ ; Fgf20 Cre/+ ) versus dm-cKO (β-Catenin dm/fl ; Fgf20 Cre/+ ) showing top up-and downregulated genes (Loupe Browser v6.0, 10x Genomics).(B) Pie charts of the percentage of differentially expressed genes that are crossreferenced to the molecular signatures of the 3 identified clusters of supporting cells at P0: IPCs, 3 rd row of Deiters' cells, and OPCs along with 1 st and 2 nd rows of Deiters' cells.The biggest percentage of downregulated genes belongs to the IPC gene signature.(C) Validation of IPC identity loss in dm-cKO using whole mount immunostaining of E18.5 cochlear epithelium (basal turn) showing Npy expression (IPC marker) in red along with Sox2 (supporting cells marker) in green.Cochleae from dm-cKO mice shows lack of Npy staining when compared to control.(D) Fluorescence in-situ hybridization for IPC marker Lgr6 mRNA along with immunostaining for CD44 at P0 showing loss of Lgr6 expression in dm-cKO (yellow arrowhead) (F) Fluorescence in-situ hybridization for potential OPC marker (Tuba1b) along with immunostaining for Myo7a (HC marker) in P0 showing gain of Tuba1 expression in IPC region in dm-cKO.Scale bar = 100μm.https://doi.org/10.1371/journal.pgen.1010925.g005

Fig 7 .
Fig 7. A schematic diagram for β-Catenin transcriptional versus cell adhesion function in supporting cell development.Intact β-Catenin cell adhesion and transcriptional functionalities during supporting cell development are required for normal cell arrangement and establishment of IPC identity (top panel).Lack of both β-Catenin functionalities causes disruption in supporting cell arrangement, disruption of medial-lateral compartment as well as loss of IPC identity (middle panel).Lack of β-Catenin transcriptional function only, while cell adhesion function is intact, causes loss of IPC identity, with subsets of IPCs either expressing OPC markers, expressing both IPC and OPC markers, or neither, indicating lost IPC identity.https://doi.org/10.1371/journal.pgen.1010925.g007