We have previously shown that treatment with Fgf2, a strong ligand for all four Fgf-receptors, delays cell structural development and decreases stiffness at the OHC and PC surfaces in vitro.11 To explore which Fgf receptor(s) could mediate the organization of the cytoskeleton and control the maturation of cell mechanical properties, we analyzed Fgfr3-deficient mice kindly provided by Dr. David Ornitz (Washington University in St. Louis, Missouri, USA). All genotypes were confirmed with PCR, which confirmed that the genetic deletion leads to a loss of both ligand binding and transmembrane domains of Fgfr3.14 Mice were bred on a C57 black 6 background to generate Fgfr3−/− animals, which are viable but have a 60 dB hearing loss compared with wild-type littermates.14 To determine whether defects in OHC function might underlie the hearing loss in these mutants, we first assessed the electromotility of OHCs, a phenomenon crucial for cochlear amplification.15 We measured OHC voltage-dependent (nonlinear) capacitance, which results from translocation of the electrical charge across the plasma membrane during conformational changes of OHC membrane motors and is considered to be a “signature” of electromotility.16 Organ of Corti explants were dissected at postnatal day 4 (P4) and cultured for 4 to 5 days in vitro before measuring outer hair cell capacitance with whole-cell patch-clamp recording techniques as described previously.17 There was no statistically significant difference in nonlinear capacitance in OHCs of Fgfr3−/− and Fgfr3+/− mice (Fig. 1A). To compare between different cells, the capacitance was normalized to the cell surface area. No significant differences were observed in the maximal charge translocated across the plasma membrane, average linear (voltage-independent) capacitance, and the potential at the peak of nonlinear capacitance (Fig. 1B). We also noticed apparently normal mechano-electrical transduction in Fgfr3−/− OHCs (data not shown). Therefore, we conclude that Fgfr3 deficiency does not affect the normal development of the sensory and motor functions of OHCs.
Figure 1. Outer hair cell electromotility is normal in Fgfr3 knockout mice. (A) Voltage-dependent (nonlinear) component of OHC membrane capacitance in two representative cells approximately at the middle of the cochlea from control heterozygous (left) and Fgfr3 deficient (right) mice. Data were fit to the derivative of the Boltzmann function (solid line), where . C0 is the voltage-independent (linear) capacitance, Qmax is the electrical charge transferred across the plasma membrane upon transition of the cell from fully extended to fully contracted state, Vp is the potential at the peak of Cm(V), z is the effective valence of a charge moving from the inner to the outer aspect of the plasma membrane, k is Boltzmann’s constant, T is absolute temperature (293 K), and e is the electron charge. (B) Boltzmann fit parameters (mean ± s.e.m.) in Fgfr3−/− (5 cells from 3 animals) and Fgfr3+/− (4 cells from 4 animals) OHCs. The errors of pClamp capacitance measurement algorithm were corrected offline as previously described17 to account for a non-ideal ratio of the access resistance to the membrane resistance. To compare between OHCs, the voltage-dependent component of capacitance was divided by the surface area of the plasma membrane with the formula ; where Xm(V) is the specific nonlinear voltage-dependent capacitance of the plasma membrane in µF/cm2 and Xlb = 1 µF/cm2 is the specific capacitance of a lipid bilayer.
Despite normal OHC electromotility, Fgfr3−/−mice have no detectable distortion product otoacoustic emissions,7 which indicates that the loss of cochlear amplification is due to other factors. For example, defects to surrounding supporting cell structure may disrupt mechanical properties of the cochlear partition.18 Therefore, we assessed how the loss of Fgfr3 impacts pillar cell structure. Cochleae from P3 mutants and heterozygous littermates were prepared for electron microscopy as previously described.11 Consistent with previous findings,2,11 three types of supporting cells, the inner phalangeal cell, inner pillar cell, and outer pillar cell, were clearly present at the lumenal surface of the sensory epithelium between IHCs and the first row OHCs of Fgfr3+/− mice (Fig. 2A). Micrographs of Fgfr3 deficient mice showed a decrease in the supporting cell surface area between IHCs and OHCs relative to heterozygous littermates at P3 (Fig. 2A). In apical regions of Fgfr3 mutants, PCs protruding to the lumenal surface of the sensory epithelium were also smaller and lacked the characteristic bundles of microtubules (Fig. 2B) previously described in normally developing PCs.11 To quantify this difference in tissue architecture, we calculated the average inner hair cell-to-outer hair cell (ITO) distance as previously described19 for 2 basal and 2 apical samples from 2 animals for each condition at 1 µm, 3 µm, and 5 µm distances from the lumenal surface of the pillar cell. Overall, a 50% decrease in the average ITO distance was observed at all three distances from the lumenal surface of the sensory epithelium (Fig. 2C). This difference could not be statistically tested with our small sample size. Therefore we also examined the lumenal surface of this epithelium by labeling P3 Fgfr3-mutant and heterozygous littermate supporting cells as previously described11 with an antibody against calcium binding protein S100-A1 and phalloidin, which labels actin filaments. In these whole-mount preparations, we also observed a decreased distance between IHCs and the first row of OHCs in Fgfr3-deficient mice (Fig. 2D). These data indicate that while the same numbers of cells are present between the IHC and first row of OHCs in mutant and heterozygous cochleae, fewer cells protrude to the lumenal surface between the IHC and first row of OHCs in Fgfr3-deficient mice.
Figure 2. Fgfr3 knockout mice have underdeveloped pillar cells and decreased cell surface stiffness. (A) Representative electron micrographs of P3 cross-sections at the base of the cochlea show three supporting cells between IHCs and OHCs in Fgfr3+/− (top) and Fgfr3−/− (bottom). (B) Transmission electron micrographs of P3 cross-sections in the base and apex of the cochlea show microtubules (asterisks) in supporting cells between IHCs and OHCs lacking in Fgfr3−/− relative to Fgfr3+/− mice. (C) Average ITO distance (mean ± s.e.m.) measured from 4 micrographs each from 2 samples in mutant (hatched bars) compared with heterozygous (solid bars) mice. (D) Representative confocal z-projections of P3 basal cochleae labeled with an antibody against calcium binding protein S100-A1 expressed in supporting cells (red), and Phalloidin which stains filamentous actin (green), show the decreased distance between IHCs and OHCs in Fgfr3−/− relative to Fgfr3+/− mice. Double arrow lines represent 5 μm distance across the lumenal surface of the epithelium. (E) Average Young's modulus (mean ± s.e.m.) is significantly decreased when measured at the surface of both OHCs and PCs in Fgfr3−/− relative to Fgfr3+/− mice (*p-value < 0.05; student’s t-test). Note that explants from 10–12 mice were examined for each condition. Scale bar, 10 μm, applies to (A). Scale bar, 2 μm, applies to (B). Scale bar, 10 μm, applies to (D). IHC, inner hair cell; OHC, outer hair cell; IPC, inner pillar cell; OPC, outer pillar cell; IPh, inner phalangeal cell, KO, Kölliker’s organ; DC, Deiters’ cell;
It is known that Fgf-signaling has direct implications on development of the actin cytoskeleton,20 and that decreased Fgf-signaling can lead to patterning defects during cochlear development.7,9,13,14 To examine the effects of Fgfr3-deficiency on cell surface mechanical properties, we measured the stiffness at different points across the surface of the sensory epithelium. Average Young’s modulus for regions corresponding to OHCs and PCs was calculated as previously described11 and compared between Fgfr3 knockout mice and heterozygous littermates. Results indicate that at P3, OHC Young’s modulus (mean ± s.e.m) was decreased from 6.45 ± 0.95kPa to 2.59 ± 0.61kPa in Fgfr3−/− mice and PC Young's modulus was decreased from 3.95 ± 1.06kPa to 1.68 ± 0.38kPa in Fgfr3−/− mice relative to heterozygous littermates (Fig. 2E). Together, these results suggest that Fgfr3 does regulate microtubule formation in PCs, and that microtubules may contribute a significant part of the developing Young’s modulus at the cell surface.
Since Fgfr3−/− mice have fewer microtubules in PCs, we set out to determine whether acute modulation of Fgf signaling would also influence microtubule formation in wild type mice. Therefore, cochlear explant cultures were established at E16, P0, and P3, and maintained 18 h in vitro with one of the following: Fgf2 and heparin sulfate to bind and activate Fgf-receptors, SU5402 to inhibit Fgf-receptor tyrosine kinase activity,21 or vehicle control as previously described.11 To confirm the effectiveness of these treatments, an antibody against p75 neurotrophin receptor (p75ntr) was used to assess differentiation of PCs, which showed increased p75ntr expression and supporting cell de-differentiation induced by Fgf2 treatment, and decreased p75ntr expression under SU5402 treated conditions as previously described.11,19 At postnatal day 0 (P0), β-Tubulin I&II localized to the lateral side of the OHC below the lumenal surface (Fig. 3A), and is consistent with previous expression profiling of β-Tubulin isotypes during development in vivo.22,23 By comparison with control cultures, Fgf2- and SU5402-treated cultures showed decreased β-Tubulin I&II fluorescence (Fig. 3A). To quantify this difference, OHCs were counted as positive or negative for β-Tubulin I&II fluorescence (Fig. 3A), as untreated OHCs contained β-Tubulin I&II at all time-points measured. Then the percent of positively labeled OHCs per culture (mean ± s.e.m.) was calculated and compared between control and treated conditions. Cultures treated with Fgf2 had a significantly lower percentage of β-Tubulin I&II labeled cells at E16 and P3 when compared with control conditions, while SU5402-treated cultures showed a significant decrease in β-Tubulin I&II only at P3 (Fig. 3B). These data suggest that disruptions to Fgf-signaling lead to decreased levels of β-Tubulin in apical regions, which, over time, may contribute to the observed patterning disruption and auditory deficits in Fgfr3-deficient cochleae.
Figure 3. Both stimulation and antagonism of Fgf-signaling pathway disrupts microtubule formation in the developing cochlea. Cochlear explant cultures were treated with either SU5402 or Fgf2 and heparin sulfate for two days in vitro. (A) Confocal Z-projections of whole-mount preparations in apical regions of the cochlea labeled with anti- β-Tubulin I&II antibody (red) and direct-conjugated 488 Phalloidin (green) at postnatal day 0 (P0) show that β-Tubulin I&II localizes to OHCs (numbered 1–9). However cultures treated with FGF2 or SU5402 show fewer β-Tubulin I&II positive OHCs (numbered 1–6). β-Tubulin I&II immunolabeled kinocilia are observed in all culture conditions (arrows). Scale Bar 20 µm. (B) Quantification of 100 OHCs repeated in apical regions of 5 samples for each condition shows that β -Tubulin I&II is significantly decreased in Fgf2-treated cultures (black bar) at embryonic day 16 (E16) and postnatal day 3 (P3), and in SU5402-treated cultures (gray bar) at P3 relative to controls (white bar) (data shown as mean ± s.e.m., *p-value < 0.05; **p-value < 0.01; student’s t-test). (C) Clustergram displaying normalized fold increase/decrease (red/green) of 84 cytoskeletal regulators. Eighteen clusters of 2 or more genes were determined using hierarchical clustering in RT2 ProfilerTM PCR array data analysis software (Qiagen). (D) Fold change gene expression of cytoskeletal regulators in Fgf2- and SU5402-treated conditions normalized to fold-change in control cultures. Significant differences are observed in fold change expression of Cytoplasmic linker-associated protein 2 (Clasp2) in Fgf2 treated cultures normalized to control conditions, while cytoplasmic tyrosine kinase adaptor molecule Crk, cytoplasmic Fragile X Mental Retardation-1 interacting protein 1 (Cyfip1), Microtubule-associated protein End-Binding family member 2 (Mapre2), and Mark2, an enzyme in the ELKL motif kinase family of small serine/threonine protein kinases are significantly differentially expressed in SU5402-treated cultures when normalized to control cochleae.
To identify potential downstream transcriptional targets of Fgf-dependent microtubule development in OHCs and PCs, cochleae were cultured and treated as previously described11 for 2 days in vitro with one of the following: Fgf2, SU5402, or vehicle control. Six cochleae per condition were pooled for RNA extraction and cDNA synthesis using an RT2 First Strand Kit (Qiagen). A gene expression panel of 84 cytoskeletal regulators was screened and repeated in triplicate for each condition using a commercially available PCR array (PAMM-088ZA, Qiagen). To express similarity among relative gene expression levels, a hierarchical clustering method was used (RT2 ProfilerTM Software, Qiagen). This method applied the correlation coefficient as a similarity metric, as well as the absolute expression level and an average linkage method, to calculate the average distances between all pairs of genes. Based on this method, 18 clusters of genes were linked for these tissue samples (Fig. 3C), and one cluster of genes in particular (Aurkc, Msn, Cdk5, and Fscn2), which is involved in actin regulation and cell-cycle progression,24,25 showed increased expression when treated with SU5402, and decreased expression when treated with Fgf2, relative to control conditions (Fig. 3C). Since no change in proliferation was observed in Fgfr3-deficient conditions7,19 we examined the fold expression change normalized to a house-keeping gene panel of β2 microglobulin, glyceraldehyde 3-phosphate dehydrogenase, glucuronidase-β, and heat-shock protein-90, for each gene, and found significant differences between control and treated conditions in microtubule regulators (Fig. 3D). Cytoplasmic linker associated protein 2 (Clasp2) showed a significant 3-fold increase in expression in Fgf2 relative to control conditions (Fig. 3D). The protein product of this gene has been shown to stabilize a subset of microtubules26 indirectly through binding with CLIPs and End-Binding (EB) proteins.27,28 In SU5402-treated cochleae, statistically significant increases in expression of Crk, Cyfip1, Mapre2, and Mark2 were observed relative to control cultures (Fig. 3D, p-value < 0.05). Cytoplasmic tyrosine kinase adaptor molecule, Crk, had a relative 5-fold higher expression in SU5402-treated cultures. Crk is a member of an adaptor protein family that binds to several tyrosine-phosphorylated proteins,29,30 including Epidermal Growth Factor (EGF) receptor, upon stimulation with EGF.31 These expression data suggest that a loss in Fgf-receptor tyrosine kinase activity may have a different regulatory cascade from stimulation of Fgf-receptors that can lead to upregulation of genes for adaptor molecules involved in receptor tyrosine kinase activities, as has been seen for mitogen-inducible genes.20 Microtubule-associated protein RP/EB family member 2, Mapre2, had 3-fold higher gene expression in SU5402-treated relative to control conditions. Mapre2 encodes the protein EB-2, which binds the plus-end of growing microtubules.32 Enzyme in the ELKL Motif Kinase (EMK) family member Microtubule affinity regulating kinase 2, Mark2, had 4-fold higher gene expression in SU5402-treated relative to control conditions; it is a serine/threonine protein kinase involved in the control of cell polarity and is known to interact with doublecortin33 and KIF13B.34 The one exception to the characterization of cytoskeletal regulators involved in microtubule formation was Cyfip1, encoding a clathrin heavy chain binding protein that showed a 4-fold increase in gene expression and has been shown to interact with the product of the Fragile X Mental Retardation (FMR) gene.35 FMR protein was first found to be responsible for Fragile X Syndrome, and it has been suggested that the phenotype observed in the central nervous system36 is due to a disruption in protein synthesis, and, more recently, indirectly associated with remodeling of the actin cytoskeleton.37 In summary, both stimulation and antagonism of the Fgf-signaling pathway alters the gene expression profile of different candidate microtubule regulators in the developing cochlea in vitro.
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