Dynamic Expression of Lgr5, a Wnt Target Gene, in the Developing and Mature Mouse Cochlea
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The Wnt signaling pathway is a recurring theme in tissue development and homeostasis. Its specific roles during inner ear development are just emerging, but few studies have characterized Wnt target genes. Lgr5, a member of the G protein-coupled receptor family, is a Wnt target in the gastrointestinal and integumentary systems. Although its function is unknown, its deficiency leads to perinatal lethality due to gastrointestinal distension. In this study, we used a knock-in reporter mouse to examine the spatiotemporal expression of Lgr5 in the cochlear duct during embryonic and postnatal periods. In the embryonic day 15.5 (E15.5) cochlear duct, Lgr5-EGFP is expressed in the floor epithelium and overlapped with the prosensory markers Sox2, Jagged1, and p27(Kip1). Nascent hair cells and supporting cells in the apical turn of the E18.5 cochlear duct express Lgr5-EGFP, which becomes downregulated in hair cells and subsets of supporting cells in more mature stages. In situ hybridization experiments validated the reporter expression, which gradually decreases until the second postnatal week. Only the third row of Deiters’ cells expresses Lgr5-EGFP in the mature organ of Corti. Normal cochlear development was observed in Lgr5EGFP/EGFP and Lgr5EGFP/+ mice, which exhibited normal auditory thresholds. The expression pattern of Lgr5 contrasts with another Wnt target gene, Axin2, a feedback inhibitor of the Wnt pathway. Robust Axin2 expression was found in cells surrounding the embryonic cochlear duct and becomes restricted to tympanic border cells below the basilar membrane in the postnatal cochlea. Both Lgr5 and Axin2 act as Wnt targets in the cochlea because purified Wnt3a promoted and Wnt antagonist suppressed their expression. Their differential expression among cell populations highlights the dynamic but complex distribution of Wnt-activated cells in and around the embryonic and postnatal cochlea.
Keywordshair cells supporting cells Axin2 ß-catenin prosensory Deiters’ cells
In the developing inner ear, strict control of cell proliferation and differentiation is essential. The cochlear duct develops as a ventral outpouching of the otocyst around embryonic day 11 (E11). Starting at E13, cells in a specific region in the floor of the cochlear duct exit the cell cycle and subsequently differentiate into cells comprising the organ of Corti (Ruben 1967). Chen and Segil (1999) described this area as the zone of non-proliferating (ZNP) cells, which begin to express the cyclin-dependent kinase inhibitor p27(Kip1). While cell lineage analyses have not been conducted, most cells in the postnatal organ of Corti are presumed to derive from these ZNP cells, which include the cochlear prosensory domains (Chen et al. 2002; Chen and Segil 1999). As the wave of terminal mitoses progresses from the apex toward the basal turn of the cochlea between E12.5 and E16 (Lee et al. 2006; Ruben 1967), cellular differentiation within the prosensory domain expands from the mid-basal region toward the apical and basal poles (Rubel 1978). Multiple genes are expressed within the cochlear prosensory domain, including Jagged1, Islet1, Lunatic fringe, Prox1, Sox2, and Fgf20 (Bermingham-McDonogh et al. 2006; Dabdoub et al. 2008; Hayashi et al. 2008; Kiernan et al. 2005; Morrison et al. 1999; Morsli et al. 1998; Radde-Gallwitz et al. 2004). Wnt/ß-catenin signaling is involved in the specification of otic cell identity, dorsal patterning of the otocyst, as well as eventual formation of the vestibular organs (Hollyday et al. 1995; Jasoni et al. 1999; Lillevali et al. 2006; Ohyama et al. 2006; Riccomagno et al. 2005). However, few studies have examined the role of this signaling pathway in the developing cochlea in the late embryonic age. Several Wnt proteins and Frizzled receptors are expressed in the developing cochlear duct and postnatal organ of Corti (Dabdoub and Kelley 2005; Daudet et al. 2002; Sienknecht and Fekete 2008, 2009). Because the large number of possible ligand–receptor combinations and redundancies makes deciphering the exact individual functions of Wnt proteins and Frizzled receptors difficult, many investigators have taken advantage of Wnt target genes to identify cells with active Wnt signaling (Barolo 2006; Logan and Nusse 2004). Our study utilizes transgenic reporters to investigate the spatiotemporal expression of Wnt target genes in the mammalian cochlear duct to identify regions and cell types that display active Wnt/ß-catenin signaling. Here, we report the differential expression patterns of two Wnt target genes, Lgr5 and Axin2, within the embryonic and postnatal cochlea.
Lgr5EGFP-Ires-CreERT2/+ (Lgr5EGFP/+) mice (Barker et al. 2007) in a C57BL/6J background were purchased from the Jackson Laboratory (stock no. 008875; Bar Harbor, ME). Homozygous Lgr5-deficient mice die perinatally (Morita et al. 2004). Knock-in of the gene encoding enhanced green fluorescent protein (EGFP) into the first exon of Lgr5 results in the expression of EGFP that faithfully represents Lgr5 expression in the heterozygotes with no reported phenotypes (Barker et al. 2007). Axin2LacZ/+ mice in a CD1 background (Jho et al. 2002; Lustig et al. 2002) were a generous gift from R. Nusse (Stanford, CA). Axin2LacZ/+ heterozygous animals display no gross cochlear malformation and no auditory brainstem response (ABR) threshold shifts in comparison to wild-type littermates at postnatal day 30 (P30, unpublished data). Axin2LacZ/+ mice also utilize a faithful knock-in reporter, the LacZ gene encoding the bacterial ß-galactosidase gene. Besides normal cochlear morphology and ABR thresholds, no phenotype has been reported in heterozygous Axin2LacZ/+ mice (Soshnikova et al. 2003). At least three animals were examined at each developmental time point. The Stanford University Institutional Animal Care and Use Committee approved all experimental procedures.
Genotyping and RT-PCR and qPCR
Transgenic mice were genotyped using genomic DNA which was isolated by adding 200 μl 50 mM NaOH to cut tail tips, incubated at 98°C for 1 h, followed by the addition of 20 μl of 1 M HCl. We used the following genotyping primers: Lgr5: wild-type forward, 5′-CTGCTCTCTGCTCCCAGTCT-3′; reverse, 5′-ATACCCCATCCCTTTTGAGC-3′; mutant reverse, 5′-GAACTTCAGGGTCAGCTTGC-3′. Axin2: wild-type forward, 5′-AAGCTGCGTCGGATACTTGAGA-3′; reverse, 5′-AGTCCATCTTCATTCCGCCTAGC-3′; mutant reverse, 5′-TGGTAATGCTGCAGTGGCTTG-3′.
For reverse transcriptase polymerase chain reaction (RT-PCR) and quantitative polymerase chain reaction (qPCR), total RNA isolation (from P3 and P15 wild-type cochleae with the stria vascularis and modiolus microdissected away, or Lgr5-EGFP-positive and -negative cells isolated from P3 Lgr5EGFP/+ cochleae via flow cytometry) was carried out using Qiagen RNeasy mini extraction kits (Qiagen, Valencia, CA), followed by cDNA synthesis using SuperScript III First-Strand Synthesis System kits (Invitrogen, Carlsbad, CA). qPCR reactions were performed with SYBR Green PCR Master Mix (Applied Biosystems, Carlsbad, CA) on a 7900HT Fast Real-Time PCR System (Applied Biosystems). Each PCR reaction was carried out in triplicate and the relative quantification of gene expression analyzed using the ΔΔC T method with ß-actin as the endogenous reference (Livak and Schmittgen 2001).
Primer pairs were designed using the online Primer3 software available at http://frodo.wi.mit.edu/primer3/. Lgr5: forward, 5′-TCTTCACCTCCTACCTGGACCT-3′; reverse, 5′-GGCGTAGTCTGCTATGTGGTGT-3′; Sox2: forward, 5′-ATGAACGGCTGGAGCAACGGCA-3′; reverse, 5′-TCACATGTGCGACAGGGGCAGT-3′; Axin2: forward, 5′-ATGTTAGAGAGTGAGCGGCAGA-3′; reverse, 5′-CTTCAGCATCCTCCTGTATGGA-3′; Brn3.1: forward, 5′-ACCCAAATTCTCCAGCCTACAC-3′; reverse, 5′-GGCGAGATGTGCTCAAGTAAGT-3′; p27(Kip1): forward, 5′-AAGCACTGCAGAGACATGGAAG-3′; reverse, 5′-GTAGAAGAATCGTCGGTTGCAG-3′; GAPDH: forward, 5′-AACGGGAAGCCC-ATCACCATCTT-3′; reverse, 5′-CAGCCTTGGCAGCACCAGTGG-3′; ß-actin: forward, 5′-ACGGCCAGGTCATCACTATTG-3′; reverse, 5′-AGGGGCCGGACTCATCGTA-3′.
Heads from E15.5–P3 and otic bullae from P6–30 mice were isolated and fixed with 4% paraformaldehyde (Electron Microscopy Services, Hatfield, PA) in phosphate-buffered saline (PBS, pH 7.4) at 4°C overnight. For P9 or older animals, decalcification was performed with 0.5 mM EDTA in PBS for 1–3 days at 4°C. Tissues were then cryoprotected by successively incubating them in 10%, 20%, and 30% sucrose in PBS and then embedded in OCT compound (Sakura Finetek, Torrance, CA). Serial frozen sections of 10- to 12-μm thickness were made with a Leica CM3050 cryostat (Leica, Bannockburn, IL).
Cochleae from Lgr5EGFP/+ mice were isolated under sterile conditions with the anlage of the stria vascularis, modiolus, and tectorial membrane removed with fine forceps. Whole-mount cochleae were then placed onto 10-mm coverslips (Fisher Scientific, Pittsburgh, PA) pre-coated with CellTaK (BD Biosciences, San Jose, CA). Whole organs were cultured in DMEM/F12 (Invitrogen) supplemented with N2 (Invitrogen), B27 (Invitrogen), epidermal growth factor (20 ng/ml), insulin-like growth factor 1 (50 ng/ml), basic fibroblast growth factor (10 ng/ml), heparan sulfate (50 ng/ml), and ampicillin (50 μg/ml; all from Sigma, St. Louis, MO) in four-well Petri dishes (Greiner Bio-one, Monroe, NC). Purified Wnt3a (200 ng/ml) and Fz8CRD (25 μg/ml, generously provided by R. Nusse) were synthesized and purified as previously described (DeAlmeida et al. 2007; Willert et al. 2003). Culture media were replenished every 1–2 days.
Cochleae from P3 Lgr5EGFP/+ mice were dissected and incubated in 0.125% trypsin (Invitrogen) in PBS for 8 min at 37°C. The reaction was terminated with the addition of soybean trypsin inhibitor (6.7 mg/ml; Worthington Biochem, Lakewood, NJ). Following trituration, cells were passed through a 40-μm filter to achieve a single cell suspension. Propidium iodide (1 μg/ml, Sigma) was used to label nonviable cells. We routinely achieved 93–95% cell viability. Cell sorting was performed on a BD Aria II FACS cytometer (BD Bioscience). Cochlear cells from wild-type mice were used as controls, and no GFP signals were detected.
Immunohistochemistry, image acquisition, and image analyses
Tissues were immersed in blocking solution consisting of 5% goat or donkey serum, 0.1% tritonX-100, 1% bovine serum albumin (BSA), and 0.02% sodium azide (NaN3) in PBS at pH 7.4 for 1 h at room temperature. Incubation with primary antibodies that were diluted in blocking solution was done overnight at 4°C in a humidified chamber. The following day, tissues were rinsed with PBS and then incubated with secondary antibodies diluted in 0.1% tritonX-100, 0.1% BSA, and 0.02% NaN3 solution in PBS for 1 h at room temperature. After washing with PBS, tissues were mounted in antifade Fluorescence Mounting Medium (DAKO, Carpinteria, CA) and coverslipped. The following antibodies were used: anti-myosin7A (1:1,000, Proteus Bioscience, Ramona, CA; Oesterle et al. 2008); anti-p27(Kip1) (1:1,000, Fisher Scientific; Chen and Segil 1999); anti-Jagged1 (1:800, Santa Cruz Biotechnology, Santa Cruz, CA; Morrison et al. 1999); anti-Prox1 (1:1,000, Millipore; Bermingham-McDonogh et al. 2006); and anti-Sox2 (1:400 Santa Cruz Biotechnology; Hume et al. 2007). Monoclonal antibody against Lgr5 (1:1,000, clone FM4056) was generously provided by K. Masuda (Kyowa Hakko Kirin Company, Tokyo, Japan; Sasaki et al. 2010). The specificity of these antibodies has been confirmed in the referenced studies as well as by their respective suppliers. The secondary antibodies were conjugated with FITC, TRITC, or Cy5 (1:200, Jackson ImmunoResearch, West Grove, PA). Images were acquired using epifluorescence or confocal microscopy (Axioplan 2, Zeiss, Germany) and analyzed with Photoshop CS4 (Adobe Systems, San Jose, CA). Three-dimensional reconstruction of Z-stack images was performed using Volocity software (v5.3.0; Improvision, Waltham, MA).
In situ hybridization
In situ hybridization was performed as previously described (Hartman et al. 2009; Hayashi et al. 2007). Briefly, a digoxigenin-labeled probe was transcribed from a linearized Axin2 clone (bp 1-2397, a generous gift of F. Costantini at Columbia University, NY) and a full-length Lgr5 clone (clone ID 100062127) obtained from Open Biosystems (Huntsville, AL). Heads of embryos were collected from timed pregnant wild-type Swiss Webster mice. For P0 or older mice, brains were removed from half-heads. These tissues were fixed overnight at 4°C in modified Carnoy’s solution (60% ethanol, 11.1% formaldehyde, 10% glacial acetic acid), dehydrated through an ethanol series, prepared for paraffin embedding (2 × 15-min washes in xylene at RT and 3 × 30 min in paraffin at 65°C), and sectioned at 8 μm. Slides were incubated 2–4 h at 68°C, deparaffinized in xylene (2 × 10 min), rinsed in 100% ethanol, and air-dried at room temperature. Slides were then incubated in hybridization buffer for 1 h at 68°C before an overnight incubation at 68°C in hybridization buffer containing 1 μg/ml digoxigenin-labeled probe. After slides were washed at 68°C for 1 h in buffer containing 50% formamide, 0.5XSSC, and 1% SDS, they were washed for 1.5 h in buffer containing 50% formamide, 0.5XSSC, and 0.1% Tween. To remove formamide, the slides were washed twice in 0.5XSSC. Finally, slides were washed twice in PBS/0.1% Tween at room temperature. Hybridized probe was detected using anti-digoxigenin alkaline phosphatase-conjugated antibody (1:2,000, Roche Biochemical, Indianapolis, IN) and visualized with NBT/BCIP (Sigma). After in situ hybridization, sections were post-fixed for 30 min in 4% paraformaldehyde in PBS, rinsed, and mounted in Fluoromount G (Southern Biotech, Birmingham, AL).
Cochleae were isolated from Axin2LacZ/+ and wild-type mice. Following fixation in 4% paraformaldehyde in PBS for 1 h, cochleae were washed with 2 mM MgCl2 in PBS twice for 5 min each at room temperature. Next, cochleae were incubated in x-gal (1 mM) and x-gal mixer (1:40, v/v); x-gal mixer: 5 mM K3Fe(CN)6, 5 mM K4Fe(CN)3H2O, 0.01% sodium deoxycholate, 0.2% NP-40, 2 mM MgCl2 (in PBS, pH 7.4) for 1 h at 37°C. Tissues were rinsed with PBS prior to cryosectioning and immunostained as described above. Control experiments found no nonspecific staining with this procedure in wild-type cochlea.
Auditory brainstem responses
Mice were anesthesized using ketamine (100 mg/kg) and xylazine (10 mg/kg). ABR signals were measured with an Opti-Amp 8002 amplifier (Intelligent Hearing Systems (IHS), Miami, FL) from a needle electrode positioned at the ventral surface of the tympanic bulla referenced to an electrode placed at the vertex of the skull. A ground electrode was attached to the hind leg. The 8-, 16-, and 32-kHz tone bursts were created with stimulus intensity ranging from 10 to 80 dB SPL in 5-dB increments and delivered with high-frequency transducers (IHS). Measuring parameters were consistent with previously described procedures and results (Zheng et al. 1999).
Statistical analyses were conducted using Microsoft Excel (Microsoft, Redmond, WA) and Origin softwares (OriginLab, Northampton, MA). One-tailed, unpaired Student’s t test and one-way ANOVA were utilized, with p < 0.05 considered statistically significant.
Lgr5 expression in the embryonic cochlear duct
The Wnt target gene Lgr5 is upregulated in cells with active Wnt signaling in several organ systems, where Lgr5EGFP/+ transgenic mice have been utilized to report Lgr5 expression with EGFP (Barker et al. 2007; Jaks et al. 2008). In those systems, the reporter expression of Lgr5-EGFP has been validated to reflect Lgr5 expression and faithfully report active Wnt signaling. To characterize the expression pattern of active Wnt signaling in the developing cochlear duct, we analyzed the inner ears of the Lgr5EGFP/+ transgenic mice.
To further characterize the Lgr5-EGFP-expressing cells in the central portion of the cochlear duct floor, we examined other markers of the prosensory region. Jagged1, a ligand of the Notch signaling pathway, is localized to the prosensory region by E15 (Morrison et al. 1999). Its expression also overlapped with the medial band of Lgr5-EGFP-positive cells (E in Fig. 2). The cyclin-dependent kinase inhibitor p27(Kip1) is another marker of the prosensory domain. By E16, the expression of p27(Kip1) is restricted to the supporting cells of the sensory epithelium (Chen and Segil 1999). The central band of Lgr5-EGFP-positive cells partially overlapped with both the Jagged1- and p27(Kip1)-expressing domains (Fig. 2E–F). Nascent myosin7a-positive hair cells first appeared in the medial strip of Lgr5-EGFP-positive cells in E15.5 basal cochlear duct (Fig. 2G–G″).
Spatiotemporal expression of Lgr5 in the postnatal cochlea
Between P3 and P12, the level of Lgr5-EGFP expression gradually decreased in the inner pillar cells, inner phalangeal cells, and the lateral GER (Figs. 4E–I and 5B–D). During this early postnatal period, Lgr5-EGFP expression remained robust in the third row of Deiters’ cells. Overall, the mRNA levels of Lgr5 significantly decreased (74.3 ± 5.1%, p < 0.001) between P2 and P15 (Fig. 4M). In P12 or older cochleae, only the third row of Deiters’ cells expressed Lgr5-EGFP (Figs. 4I–L and 5D–E).
Lgr5 deficiency leads to perinatal lethality caused by gastric distension; however, its inner ear phenotype was unknown (Barker et al. 2007; Morita et al. 2004). We examined the cochleae of homozygous P1 Lgr5EGFP/EGFP mice and found normal organization of hair cells and supporting cells within the organ of Corti (Fig. 6D–E) and normal morphology of the lateral cochlear wall (data not shown), suggesting that Lgr5 is not essential for cochlear development. Because the homozygous mice have two copies of the reporter gene, cells that express low levels of Lgr5-EGFP are detectable in the homozygous genotype. Cell types expressing low levels of Lgr5-EGFP include the first two rows of Deiters’ cells, outer pillar cells, the lateral inner phalangeal cells, and all four rows of hair cells. These data indicate that Lgr5 expression is rapidly downregulated in specific supporting cells in the postnatal organ of Corti and becomes restricted to the third row of Deiters’ cells in the mature organ.
Wnt signaling regulates Lgr5 expression
Lgr5EGFP/+ phenotype and Lgr5-EGFP-positive cells
To further examine whether Lgr5-EGFP reporter activity reflects Lgr5 gene expression, we isolated the Lgr5-EGFP-positive cell population from P3 Lgr5EGFP/+ mice using flow cytometry (Fig. 8B). The EGFP-positive cell population constituted about 2.1% of the P3 cochlea. Analysis by quantitative PCR found that the EGFP-positive cells expressed significantly more copies of Lgr5 mRNA than Lgr5-EGFP-negative cells (73.6 ± 8.3-fold difference, p < 0.001; Fig. 8C). This analysis, along with the in situ hybridization (Fig. 4B) and immunochemistry data (Fig. 6B), supports the conclusion that Lgr5-EGFP reporter activity, like in other organs (Barker et al. 2007; Jaks et al. 2008), accurately reports Lgr5 expression in the cochlea. Because our data also suggest that Lgr5 expression is modulated by Wnt signals, we next compared its expression pattern to another Wnt target gene, Axin2.
Axin2 expression in the developing cochlear duct
The canonical Wnt pathway plays a critical role in organogenesis in several organ systems (Ikeya et al. 1997; Li et al. 2002; Majumdar et al. 2003; Yamaguchi et al. 1999). Studies examining the role of this signaling pathway during the development of the cochlear duct are just emerging. To augment our tools to identify Wnt-activated cells in the inner ear, we have hereby validated and characterized the expression of two Wnt target genes, Lgr5 and Axin2.
Previously, Wnt signaling was shown to mediate otic versus epidermal fate decisions during the formation of otic placode (Lillevali et al. 2006; Ohyama et al. 2006). In the developing chicken inner ear, Sienknecht and Fekete (2008, 2009) characterized dynamic expression patterns of multiple Wnt proteins, their respective family of Frizzled receptors, and inhibitory proteins in both prosensory and non-sensory cells between E4 and E15. Notably, several Wnt proteins are transiently expressed in the prosensory region. Forced activation of the Wnt pathway via the transfection of ß-catenin, the central mediator of active canonical Wnt signaling, or via the addition of exogenous Wnt3a to the developing cochlear duct prior to prosensory specification led to inner ear malformation and induced ectopic sensory patches (Stevens et al. 2003). These results point to a role of the Wnt pathway in prosensory domain formation and morphogenesis. Microarray analyses have demonstrated that several Wnt genes are expressed in the embryonic and neonatal mammalian cochlea (Chen and Corey 2002; Sajan et al. 2007). In the postnatal rat cochlea, Daudet et al. (2002) have identified transcripts of individual Wnt proteins and Frizzled receptors. Interpretation of the expression patterns of Wnt proteins and Frizzled receptors is difficult because of the myriad of possible combinations; therefore, many groups have used Wnt target genes to identify Wnt-activated cells (Barolo 2006).
Lgr5 is an orphan G protein-coupled receptor with a large leucine-rich domain, but lacks a clearly defined function (Hsu et al. 1998). Investigators have shown that Lgr5 expression is regulated by Wnt signaling and acts as a marker for endogenous stem cells in rapidly proliferating organs (Barker et al. 2007, 2010; Jaks et al. 2008; Ootani et al. 2009). Our data indicate that Lgr5 expression was dependent on endogenous Wnt proteins and promoted by exogenous Wnt3a, an established canonical Wnt protein (Willert et al. 2003). Based on these results, we conclude that Lgr5 is also a Wnt target in the inner ear. Other Wnt proteins can act as morphogens that control the planar cell polarity of hair cells during development via the non-canonical Wnt pathway (ß-catenin-independent; Dabdoub et al. 2003; Dabdoub and Kelley 2005; Qian et al. 2007); it is therefore possible for other Wnt proteins to regulate Lgr5 expression via this mechanism. Unlike the rapidly proliferating organs where Lgr5 has been previously characterized to mark somatic stem cells, the prosensory and sensory regions expressing Lgr5 are postmitotic (Chen and Segil 1999; Ruben 1967). Future cell tracing experiments will likely help elucidate the significance of Lgr5-positive cell lineage.
The observation that Lgr5 is dynamically expressed in the cochlear duct epithelium, including the prosensory region, raises the possibility that Wnt signaling may play a role in cell fate determination in the developing cochlear duct. Interestingly, Lgr5-EGFP expression rapidly disappears in hair cells and a subset of supporting cells. The notion that Wnt signaling plays a role in cell fate determination in the inner ear is supported by two previous studies: overexpression of ß-catenin, the central player of the canonical Wnt pathway, upregulates Atoh1 (Shi et al. 2010) and induces ectopic sensory patches (Stevens et al. 2003). While our data indicate that Lgr5 is not required for cochlear development, its expression likely reflects the pattern of Wnt-activated cells within the cochlear ductal epithelium.
The differential expression between Lgr5 and Axin2 at several developmental stages suggests that Wnt targets are cell type-dependent and indicates that other Wnt-activated cells exist outside the cochlear ductal epithelium. Such tissue-specific and development-specific behavior for Wnt target genes has also been reported in other organ systems (Barolo 2006; Barolo and Posakony 2002), and hence the rationale of examining multiple Wnt target genes using gain- and loss-of-function assays in the current study. Similar contextual expression patterns have been observed with the BATGAL reporter mouse, a TCF reporter (Barolo 2006; Maretto et al. 2003), and possibly explain the discrepancy between our data and those of Qian et al. (2007) who found no BATGAL expression in or surrounding the embryonic cochlear duct. TOPGAL is yet another transgenic TCF reporter mouse line that has been shown to label cells with active Wnt signaling in multiple tissue types (DasGupta and Fuchs 1999; Maretto et al. 2003). However, false-negative results have been reported with this mouse line since it occasionally failed to label tissues with known active Wnt signaling (Dessimoz et al. 2005; Geng et al. 2003; Riese et al. 1997). While we conclude that Lgr5-positive and Axin2-positiive cells represent Wnt-activated cells, the sum of both reporter activities still might not reveal the full degree of Wnt activity in the developing and maturing cochlea, which is a limitation of our study. While functional studies will be necessary to further delineate the role of Wnt signaling in the development and maturation of the cochlea, our study provides a framework for those studies as we show which cell types in the cochlear duct are actively receiving Wnt signals.
In conclusion, our study has identified a dynamic expression pattern for the Wnt target gene Lgr5 in both the embryonic and postnatal cochlear duct. Its expression pattern contrasted with that of another Wnt target gene, Axin2. While these two markers indicate the presence of active Wnt signaling in different cell types in the developing and mature inner ear, further analyses will be necessary to elucidate the functional role of Wnt signaling in the development and maintenance of the organ of Corti.
The authors thank Xin Hong Lim, Zahra Sayyid, and Catherine Ray for excellent technical assistance and Stefan Heller, Kazuo Oshima, Fuxin Shi, and Mirna Mustapha for advice and comments. This study was supported by NIDCD P30DC010363, a Stanford University Dean’s Fellowship (to R.C.), Stanford University Medical Scholars Program, Howard Hughes Medical Institute Training Fellowship (both to T.A.J.), NIDCD R01DC009991 (to O.B-McD), the American Otological Society, the Triological Society, a Percy Memorial Award, and NIDCD K08DC011043 (all to A.G.C.).
Conflict of Interest
The authors have no conflict of interest to declare.
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