Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2011 Mar 1.
Published in final edited form as: Dev Dyn. 2010 Mar;239(3):1019–1026. doi: 10.1002/dvdy.22236

Expression patterns of FGF receptors in the developing mammalian cochlea

Toshinori Hayashi 1, Catherine A Ray 1, Christa Younkins 1, Olivia Bermingham-McDonogh 1,*
PMCID: PMC2933402  NIHMSID: NIHMS225977  PMID: 20131355

Abstract

Many studies have shown the importance of the fibroblast growth factor (FGF) family of factors in the development of the mammalian cochlea. There are four fibroblast growth factor receptors (FGFR1-4) and all four are expressed in the cochlea during development. While there are examples in the literature of expression patterns of some of the receptors at specific stages of cochlear development there has been no systematic study. We have assembled a full analysis of the patterns of receptor expression during cochlear development for all four Fgfrs using in situ hybridization. We have analyzed the expression patterns from E13.5 through post-natal ages. We find that Fgfr1, 2 and 3 are expressed in the epithelium of the cochlear duct and Fgfr4 is limited in its expression to the mesenchyme surrounding the duct. We compare the receptor expression pattern to markers of the sensory domain (p27kip1) and the early hair cells (math1).

Keywords: hair cell, support cell, pillar cell, inner ear, sensory epithelium, the organ of Corti

Introduction

The mammalian cochlea contains the primary sensory cells for the auditory system. The mature organ of Corti contains a single row of inner hair cells and three rows of outer hair cells, as well as various types of supporting cells. A great deal has been learned about the normal development of this structure and in the past ten years the molecular mechanisms that regulate its development have begun to be elucidated. The cochlea, in the mouse, forms initially from an out-pocketing of the ventro-medial otocyst at embryonic day (E) 11–12 (Morsli et al., 1998). The precursors of the hair cells and the associated support cells exit the cell cycle between E12–E16, in a apical to basal gradient, and most are generated by E14 (Ruben, 1967). After the cells in the sensory region have completed their last mitotic division, they begin their differentiation as hair cells and support cells in the mid-basal region; the hair cells and support cells continue to differentiate in a temporal gradient that extends both basally and apically, such that the mature complement of hair cells and support cells is achieved in the mouse by E17 to E18 (Ruben, 1967; Sher, 1971; Lim and Anniko, 1985).

Many studies have shown the importance of fibroblast growth factors (FGFs) and their receptors at various stages of cochlear development. 23 different Fgfs and 4 FGF receptors (Fgfr1-4) have been identified in the mouse and human (Ford-Perriss et al., 2001; Itoh, 2001; Ornitz and Itoh, 2001; Itoh and Ornitz, 2004; Zhang et al., 2006; Itoh and Ornitz, 2008). In mice, FGF3, FGF10 and FGF8 have been implicated in the early inductive events of the otic vesicle (Mansour et al., 1993; Alvarez et al., 2003) (Wright and Mansour, 2003; Ladher et al., 2005; Martin and Groves, 2006; Ohyama et al., 2007). Mice deficient in both Fgf3 and Fgf10 fail to form otic vesicles. Similar defects in the early stages of otocyst development are present in mice with targeted deletion of a specific isoform of Fgfr2 (FGFR2 IIIB) (Pirvola et al., 2000) and it has been proposed that FGF10 and FGF3 act as the ligands for FGFR2 in otic placode formation and patterning (Pauley et al., 2003; Wright et al., 2003). In the sensory specification phase of the organ of Corti, FGF20 and FGFR1 play a key role (Pirvola et al., 2002; Hayashi et al., 2008). Tissue specific deletion of Fgfr1 results in severe defects in the development of both hair cells and support cells, and those sensory cells that develop are found in small clusters (Pirvola et al., 2002). Inhibition of FGF20 at this stage causes a reduction in the number of the hair cells and support cells similar to that in the Fgfr1 deletion (Hayashi et al., 2008). At a later stage of embryonic and neonatal development of the organ of Corti, FGF8, acting through FGFR3, is required for pillar cell differentiation (Colvin et al., 1996; Hayashi et al., 2007; Jacques et al., 2007; Puligilla et al., 2007).

Many lines of evidence have shown the importance of FGF signaling in cochlear development. Although, Wright et al. analyzed expression of FGF receptors at early stages of otic vesicle development (Wright et al., 2003), there is no report that systematically examines the detailed expression patterns of the 4 receptors throughout the later stages of cochlear development. Therefore, we undertook a comprehensive analysis using in situ hybridization of Fgfr1, Fgfr2, Fgfr3 and Fgfr4 at embryonic, neonatal and adult stages of cochlear development. These expression patterns confirm and extend previous reports (Peters et al., 1993; Pirvola et al., 1995; Colvin et al., 1996; Pirvola et al., 2000; Pickles, 2001; Pirvola et al., 2002; Hayashi et al., 2007; Puligilla et al., 2007; Hayashi et al., 2008) and highlight the many critical roles of these receptors in cochlear development.

Results and Discussion

At the earliest stage of cochlear development we analyzed, E13.5, the cochlear duct has not fully extended and so the typical three turns are not yet present. This stage of cochlear development is characterized by the gradual cessation of proliferation in the prosensory region and the specification of hair and support cell progenitors in the presumptive organ of Corti. The cochlear duct expresses both Fgfr1 and Fgfr2 at this stage (Figure 1A, B), while Fgfr3 and Fgfr4 are not expressed in the epithelium (Figure 1C, D). All probes used in this study will detect both splices forms of the receptors. The expression of Fgfr1 appears diffuse through the epithelium of the cochlear duct and in the adjacent mesenchyme. Despite the diffuse pattern of staining, this expression is specific as evidenced by the highly specific patterns of expression obtained elsewhere in the embryo (see Supplemental Figure 1A). The expression of Fgfr2 is confined to the non-sensory regions of the cochlear duct (Figure 1B) and is also expressed in the developing otic capsule. The expression of the receptors is similar at E14.5, though now the three turns of the cochlea are apparent (Figure 1E, F). In addition, at this age, Fgfr1 expression is highest in the ventral aspect of the duct; this is particularly apparent in the apical part of the cochlear duct (Figure 1E, 3). Fgfr2 expression at E14.5 resembles that at E13.5, and Fgfr3 and Fgfr4 are not expressed in the epithelial cells of the duct (Figure 1C, D, G and H). We confirmed that the all probes showed the specific patterns using E13.5 head and E14.5 eye sections (Supplemental figure1). We use “ventral” to refer to the part of the ductal epithelium that contains the prosensory domain and “lateral” and “medial” to refer to the position relative to the modiolus of the cochlea, which is the central axis of the cochlea.

Figure 1. Expression patterns of Fgf receptors in the E13.5, E14.5 and E15.5 cochlea.

Figure 1

Open in a new tab

A–D show micrographs of adjacent sections of the middle region of the E13.5 cochlear duct showing in situ localization of Fgfr 1-4, respectively. E–H show adjacent sections of the E14.5 cochlea. Levels (half-turns) of the cochlear duct are numbered from the base (turn 1) to the apex (turn 3). I–L show adjacent sections of the E15.5 cochlea with the same numbering as at E14.5. Scale bars= 100μm.

Figure 3. Comparison of Fgfr1 and 3 with Sox2 at E15.5.

Figure 3

Open in a new tab

A–C show expression of Sox2, Fgfr1 and Fgfr3 respectively. A’–C’ are higher magnification images of the basal turn shown boxed in A–C. The dark staining areas marked with asterisks are nonspecific background.

The expression of Fgfr1 in the presumptive sensory domain is consistent with previous data demonstrating a critical role for this receptor in prosensory specification. Pirvola’s group demonstrated that mice with a conditional deletion in Fgfr1 in developing cochlea have severe defects in the development of the hair and support cells (Pirvola et al., 2002). We have previously reported similar defects when FGF signaling is inhibited with SU5402, a pan-FGFR inhibitor. Moreover, inhibition of FGF20, a ligand specifically expressed in the prosensory domain at this stage, has similar effects as genetic or small molecule inhibition of FGF signaling (Hayashi et al., 2008). Since none of the other FGF receptors is expressed in the presumptive sensory domain at this stage, it is likely that FGF20 acts through FGFR1 in the process of prosensory specification.

During the next stage of cochlear development, there is a transition from the sensory specification phase to a maturation phase. The hair cells and support cells begin their differentiation in the mid-basal turn at E15.0 and the differentiation proceeds towards the apex and also towards the extreme base. By E15.5, the pattern of expression of the receptors begins to change. Fgfr1 is still more highly expressed in the ventral aspect of the duct in the more apical turns (Figure 1I), but towards the base of the cochlea, the expression becomes concentrated into a cluster of cells at the lateral aspect of the prosensory domain in the region of the developing outer hair cells (Figure 1I, Figure 2A’). Fgfr2 expression is similar to the pattern at E14.5 (Figure 1J). Fgfr4 expression also begins at E15.5; however, this receptor is not expressed in the epithelium, but rather in the mesenchymal cells adjacent to and surrounding the duct (Figure 1L, arrowheads). Figure 2 shows the relationship between the Fgfr1 and Fgfr2 expression and the sensory epithelial domain (labeled with p27kip1) and the first developing inner hair cells (labeled with Math1-GFP). The Math1-GFP hair cells are immediately adjacent to the medial aspect of the more highly expressing Fgfr1+ cells (Figure 2A and A’), and co-extensive with the p27kip1+ domain at this age (Figure 2B and B’). By contrast, Fgfr2 is expressed in cells just lateral to the sensory epithelial domain (Figure 2C and C’). Comparison of the expression patterns for Fgfr1 and Fgfr3 with Sox2 shows that Fgfr3 is expressed at the lateral edge of Sox2 expression (Figure 3A, A’ and C, C’) whereas Fgfr3 overlaps more with Fgfr1 expression (Figure 3B, C and B’, C’).

Figure 2. Temporal and spatial relationship between Fgfr1 and Fgfr2 expression and the sensory epithelium.

Figure 2

Open in a new tab

Shown are micrographs of adjacent sections of the E15.0 cochlea from a Math1-GFP transgenic mouse taken at low magnification (A, B, C) and high magnification of the 2nd turn (A’B’C’). The cochlear turns are numbered from the base (1) to apex (4). Following in situ hybridization for Fgfr1 or Fgfr2, the sections were processed for immunofluorescence with antibodies against p27kip1 (red) to identify the sensory domain and GFP (green) to label the developing hair cells. The arrow points to the GFP+ cells in A’, B’ and C’, while the p27kip1 domain is shown as a black bar. Scale bars= 100μm

Also at E15.5, Fgfr3 begins to be expressed in the developing sensory region, specifically in the basal two turns of the cochlea (Figure 1K, arrows). The expression of Fgfr1 and Fgfr3 overlaps at this stage. This can be best observed by examination of the expression of these receptors in relation to the developing inner hair cells, labeled with Math1 (Figure 4). The panels in Figure 4A, A’ show expression of Fgfr1, Figure 4B, B’, the expression of Math1 on adjacent sections, and Figure 4C, C’ the expression of Fgfr3, also on adjacent sections. The in situ expression patterns were pseudo-colored and overlapping images were generated and are shown in Figure 4D, E, and F. From these images, the overlap between the highest region of expression of Fgfr1, and that of Fgfr3 can be seen, and it appears that the Fgfr3 expressing cells are a nested subset of the high Fgfr1 expressing cluster. The position of these cells immediately adjacent to the first Math1 expressing cells (inner hair cells) suggests that the presumptive pillar cells, outer hair cells and Deiters’ cells express the two receptors at this stage of development.

Figure 4. Temporal and spatial relationship between Fgfr1and Fgfr3 expression and the developing hair cells.

Figure 4

Open in a new tab

Shown are micrographs of adjacent sections of E15.0 cochlea after in situ for Fgfr1 (A, A’), Math1 (B, B’) or Fgfr3 (C, C’). Panels D–F are pseudo-colored combinations to show the relationship among the expression patterns. The expression domain of Fgfr3 appears to be nested within the expression domain of Fgfr1 (F), while Math1 expression in the developing inner hair cell medial to both Fgfr1 (D) and Fgfr3 (E) suggests that both these receptors are expressed in the developing outer hair cells and supporting cells. In panels A, B, and C, the cochlear turns are numbered from the base (1) to apex (4). The mid- basal turns (turn 2) are shown in panels A’, B’ and C’. Scale bars= 100μm

As early as E15, there exists the possibility that Fgfr3 may be able to compensate for loss of Fgfr1, since there is substantial overlap in their expression. As noted above, loss of function of Fgfr1 leads to defects in both hair cell and support cell development, and therefore Fgfr3 cannot replace the early functions of Fgfr1. In fact, rather than a decline in the number of hair ells and support cells, targeted deletion of Fgfr3 leads to mice with an over-production of hair cells and support cells in the apical two thirds of the cochlea (Hayashi et al., 2007; Puligilla et al., 2007), suggesting that FGFR3 may actually interact to reduce signaling through FGFR1. One way this could occur is if activation of FGFR3 led to increased Sprouty2 expression, which could then reduce the effectiveness of signaling through both the FGFR1 and the FGFR3; however, we found no changes in the expression of Sprouty2 in our analysis of Fgfr3 mutants (Hayashi et al., 2007). An alternative possibility is that FGFR1 and FGFR3 compete for the FGF ligands. While our expression data show that these two receptors are expressed in the same cells at this stage of development, and the genetic evidence indicates the presence of an interaction, the nature of that interaction remains elusive.

At E16.5, Fgfr1 expression continues to be highest in the cells in the lateral part of the prosensory domain, the region that will give rise to outer hair cells, pillar cells and Deiters’ cells. However, this receptor is still expressed in most of the rest of the ductal epithelium as well (Figure 5A and A’). Fgfr2, by contrast, is expressed in a very similar region of the epithelium as that at earlier embryonic stages, lateral to the prosensory region (Figure 5B and B’). By E16.5, Fgfr3 expression is no longer confined to the base (Figure 5C and C’), but is now extended through most of the cochlea (though still absent from the most apical turn). At each point along the base-to-apex axis, Fgfr3 is expressed in a subset of cells in the sensory domain, the differentiating outer hair cells, pillar cells and Deiters’ cells. At E18.5, most hair cells and support cells have differentiated (and can be labeled with antibodies to Prox1 and Myo6+, respectively), except at the apical tip. Figure 5E–H show the expression of the receptors at this stage, in relation to the Myo6 labeled hair cells (Figure 5E’–H’). By E18.5, Fgfr1 is expressed only weakly in the sensory region, although expression is maintained and even up-regulated in other regions of the epithelium, most notably the greater epithelial ridge (GER) and Hensen’s cells (Figure 5A, A’, E). The expression of Fgfr3 is still robust in the sensory epithelium (Figure 4C, C’, G), and is now more clearly confined to the pillar cells and Deiters’ cells. Fgfr2 shows the same expression pattern as at earlier stages (Figure 5B, B’, F), and Fgfr4 is not expressed in the epithelium, but is maintained in the surrounding mesenchyme (Figure 5D, D’, H).

Figure 5. Expression patterns of Fgf receptors in the E16.5 and E18.5 cochlea.

Figure 5

Open in a new tab

A–D show sister sections of the E16.5 cochlea at low magnification; the levels (half-turns) of cochlear duct are numbered from the base (1) to apex (4). The basal turns (turn 1) are shown in panels A’, B’, C’ and D’. E–H show the basal turns (turn 1) from sections of the E18.5 cochlea. The inner hair cells (arrows in E’–H’) and the outer hair cells (arrowheads in E’–H’) were labeled with anti-Myo6 antibody after in situ hybridization. Scale bars= 100μm

After birth, the cells of the organ of Corti continue to mature. The tunnel of the organ of Corti flanked by the pillar cells, opens first in the base at approximately postnatal day 7 (P7) and the animals begin to hear after P10. At P0 and P3 (Figures 6 and 7, respectively) the expression of Fgfr1 is now nearly absent in the sensory domain. However, the signal is up-regulated in the GER (presumptive inner sulcus and border cells) and at the lateral edge of the sensory epithelium in Hensen’s cells and possibly Claudius’ cells (Figures 6 and 7, A and A’). The expression of Fgfr2 is similar to that at earlier stages, occupying a domain lateral to the developing sensory epithelium with high levels of expression in the developing Claudius’ cells, outer sulcus and spiral prominence (Figures 6 and 7, B and B’). This receptor appears to be expressed at lower levels in the developing stria vascularis and Reissner’s membrane (Figures 6 and 7, B and B’). At both P0 and P3, Fgfr3 shows robust expression in the pillar cells and Deiters’ cells, though at P3 the expression appears to be stronger in the pillar cells than the Deiters’ cells (Figures 6 and 7, C, C’). After P7, obtaining reliable signal from in situ hybridization is difficult due to the calcification of the cochlear capsule. We could only obtain consistent results with Fgfr3 (see Supplemental Figure 2). We found Fgfr3 to be robustly expressed in the pillar cells and Deiters’ cells into adulthood. The role of Fgfr3 in the differentiation of the pillar cells is now well established. FGF8 expressed by the inner hair cells activates FGFR3 in the adjacent pillar cells and this is necessary and sufficient for their differentiation. Over-activation of this signaling, either by ectopic expression of Fgf8 or addition of FGF17 to explant cultures of cochlea (Jacques et al., 2007), or by targeted deletion of the inhibitor of FGF signaling Sprouty2, causes an increase in the number of pillar cells (Shim et al., 2005). In addition mutations in Fgfr3 which cause over-activation also result in increased numbers of pillar cells (Mansour et al., 2009). Our results are consistent with this interpretation, since at the stages of pillar cell differentiation, Fgfr3 is the only FGF receptor expressed in these cells.

Figure 6. Expression patterns of Fgf receptors in the P0 cochlea.

Figure 6

Open in a new tab

Panels A–D show sister sections of the P0 cochlea. The levels (half-turns) of cochlear duct are numbered from the base (1) to apex (4). The basal turns (turn 1) are shown at higher magnification in panels A’, B’, C’ and D’. Scale bars= 100μm

Figure 7. Expression patterns of Fgf receptors in the P3 cochlea.

Figure 7

Open in a new tab

Panels A–D show sister sections of the P3 cochlea. The levels (half-turns) of the cochlear duct are numbered from the base (1) to apex (5). The basal turns (turn 1) are shown in panels A’, B’, C’ and D’. The inner hair cells (arrows in A’–D’) and the outer hair cells (arrowheads in A’–D’) were labeled with anti- Myo6 antibody after in situ hybridization (A”–D”). Scale bars= 100μm

One of the more striking findings of our analysis is the robust and consistent expression of Fgfr2 in the presumptive stria vascularis and spiral prominence from E13.5 to P3. A similar conclusion was reached by Pickles using microdisection and RT-PCR in newborn mice (Pickles, 2001), and Pirvola’s group showed a similar pattern at E16 (Pirvola et al., 2000). Although a role for Fgfr2 in early cochlear development and otic induction has been well established, it is not known whether it has a more specific function in the development of the stria or spiral prominence. Conditional deletions of Fgfr2 in the myelinating glial cells of the spiral ganglia leads to progressive hearing loss (Wang et al., 2009). FGF16 is expressed very specifically in the area of the developing spiral prominence from E14.5 (Hatch et al., 2009) so this may be an important ligand for FGFR2. Conditional deletions of this receptor specifically in the non-sensory regions of the cochlear duct may shed light on potential additional roles for this receptor in inner ear development. In the region of the developing spiral prominence the Fgfr2 receptor could be deleted using the Fgf16-Cre developed in the Mansour lab (Hatch et al., 2009).

A summary of the expression of the FGF receptors during key stages of cochlear development is shown in Figure 8. It is clear that the two important receptors as far as sensory cell development are Fgfr1 and Fgfr3. Based on its expression pattern it is likely that Fgfr2 plays a role in the development of outer sulcus, spiral prominence and stria vascularis. Expression of Fgfr4 was confined to the mesenchyme at all stages of development and the morphological development of the cochlea is normal in Fgfr4−/− animals (Bermingham-McDonogh, unpublished data). Our results, together with those of a number of investigators, have identified several key roles for FGF signaling during cochlear development. The results of this study further consolidate our understanding of these signaling molecules and suggest that there are additional, undiscovered roles for FGFs in auditory development.

Figure 8. Schematic of Fgfr expression through development.

Figure 8

Open in a new tab

The expression of the four receptors is color-coded and shown at four developmental stages. Fgfr1 expression is depicted in blue with the darker blue representing more robust expression. Fgfr2 is represented in orange with the dots of color depicting less expression. Fgfr3 is shown in purple and Fgfr4 is depicted with grey lines, this expression is throughout the mesenchyme immediately surrounding the duct. The black dotted line denotes the developing sensory region within the epithelium.

Experimental Procedures

Mice

All mice were housed by the Department of Comparative Medicine; all procedures were carried out in accordance with the guidelines of the animal care and use committee at the University of Washington. Timed pregnant and non pregnant female mice (Swiss-Webster) were purchased from Harlan (Indianapolis, IN). To obtain Math1-GFP expressing embryos, male mice carrying the Math1-GFP transgene were mated with Swiss-Webster females. Pregnant mothers were euthanized and we used the Theiler staging system (Theiler, 1989) to accurately stage the embryos at the time of harvest (http://genex.hgu.mrc.ac.uk/Atlas/intro.html). For the postnatal animals, P0 is defined as the day of birth.

In situ hybridization

Whole heads of embryos were fixed in a modified Carnoy’s solution for 6 hours at room temperature. The samples were washed and dehydrated in 100% ethanol overnight at 4°C, and then were embedded in paraffin and 6 μm sections were collected. At least 3 animals were examined at each time point. The hybridization was carried out according to Hayashi et. al. (Hayashi et al., 2007) and Digoxigenin (DIG)-labeled probes were prepared according to the manufacturer’s manual for DIG-11-UTP (Roche, Indianapolis, IN). The in situ product was visualized using anti-DIG alkaline phosphatase conjugated secondary antibody (Roche) and NBT (nitroblue tetrazolium)/BCIP (5-bromo-4-chloro-3-indolyl phosphate). After color development, the slides were fixed with 4% PFA for 1 hour. cDNA coding for mouse Fgfr1 and Fgfr2 were gift from Dr. P. Soriano (Mt. Sinai school of medicine of New York university, NY). NT 1-2526 of Fgfr1 clone (Gene Bank ID; NM_010206) was sub-cloned into pCRII vector (Invitrogen). cDNA of Fgfr2 (NM_010207, nts 891–2634) was sub-cloned into pCRII. Fgfr3 clone was purchased from OpenBiosystems Inc. Huntsville, AL ( clone ID5708838), probe was prepared using Xho1 restriction endonuclease which generates a probe of 2206 NTs. The probes used for Fgfr1-3 will not distinguish between the two splice variants. cDNA for mouse Fgfr4 was gifted from Dr. A. McMahon (Harvard University, Boston, MA), and sub-cloned into pBlueScriptII (NM_010207, nts 891–2634). Math1 clone was purchased from OpenBiosystems Inc (2,144 bp, clone ID 6530849). cDNA encoding Sox2 (530 bp) was a gift from Dr. Kondoh (Osaka University, Japan).

Immunofluorescence

Paraffin embedded and sectioned tissue was used after de-waxing or after in situ hybridization. Tissue sections were incubated with 10% fetal bovine serum and 1% nonfat dry milk in PBS/0.1% Triton X-100 (PBST) for 30 minutes. Sections were incubated overnight at 4°C with the following primary antibodies and dilutions: rabbit anti-Myo6,1:1000 (Proteus Biosciences, Ramona, CA); mouse anti-p27kip1, 1:100 (BD Transduction Laboratories, San Diego, CA); or chicken anti-GFP, 1:200 (Abcam, Cambridge, MA). The sections were rinsed with PBST (PBS with 0.1%Triton X100), incubated for 4 hours with a fluorescent-conjugated secondary antibody, rinsed with PBST, and mounted in Fluoromount G (Southern Biotechnology, Birmingham, AL).

Supplementary Material

Supp Fig s1. Supplementary Figure 1. Fgfr expression in mouse embryos.

To verify the specificity of the probes used for in situ hybridization, we tested them on sections of developing brain. Fgfr1, Fgfr2, and Fgfr3 are all highly expressed in the lateral ventricle, as reported in previous publications. Fgfr4 is not highly expressed in the brain, but is highly expressed in the extra-ocular muscles. Co; Cochlea, LV; lateral ventricle; L; lens; R; Retina; OM; ocular muscle. Scale bars=100μm

Supp Fig s2. Supplemental figure 2: Fgfr3 is expressed in the P21 cochlea.

A and B show sections of the mid-apical tunes of P21 cochlea. In panel B, the organ of Corti was photographed at the high-magnification. The asterisks in panel B indicate the nuclei of the Deiters’ cells. TM; tectorial membrane. OHC; outer hair cells. PCs; pillar cells. DCs; Deiters’ cells. Scale bar in A = 100 μm Scale bar in B = 50μm

Acknowledgments

We thank Dr. Jane Johnson for the Math1-GFP mouse strain. We also thank Drs. P. Soriano (Mt. Sinai school of medicine of New York University, NY) and A. McMahon (Harvard University, Boston, MA) and Dr. H. Kondoh (Osaka University, Japan) for providing us with plasmids. We thank Drs. Thomas Reh, Joe Brzezinski and Anna del la Torre for comments on this manuscript and to members of the Reh lab for helpful discussions. This work was supported by National Institutes of Health Grants DC005953, P30DC00466, and the Hearing Regeneration Initiative.

References

  1. Alvarez Y, Alonso MT, Vendrell V, Zelarayan LC, Chamero P, Theil T, Bosl MR, Kato S, Maconochie M, Riethmacher D, Schimmang T. Requirements for FGF3 and FGF10 during inner ear formation. Development. 2003;130:6329–6338. doi: 10.1242/dev.00881. [DOI] [PubMed] [Google Scholar]
  2. Colvin JS, Bohne BA, Harding GW, McEwen DG, Ornitz DM. Skeletal overgrowth and deafness in mice lacking fibroblast growth factor receptor 3. Nat Genet. 1996;12:390–397. doi: 10.1038/ng0496-390. [DOI] [PubMed] [Google Scholar]
  3. Ford-Perriss M, Abud H, Murphy M. Fibroblast growth factors in the developing central nervous system. Clin Exp Pharmacol Physiol. 2001;28:493–503. doi: 10.1046/j.1440-1681.2001.03477.x. [DOI] [PubMed] [Google Scholar]
  4. Hatch EP, Urness LD, Mansour SL. Fgf16(IRESCre) mice: a tool to inactivate genes expressed in inner ear cristae and spiral prominence epithelium. Dev Dyn. 2009;238:358–366. doi: 10.1002/dvdy.21681. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Hayashi T, Cunningham D, Bermingham-McDonogh O. Loss of Fgfr3 leads to excess hair cell development in the mouse organ of Corti. Dev Dyn. 2007;236:525–533. doi: 10.1002/dvdy.21026. [DOI] [PubMed] [Google Scholar]
  6. Hayashi T, Ray CA, Bermingham-McDonogh O. Fgf20 is required for sensory epithelial specification in the developing cochlea. J Neurosci. 2008;28:5991–5999. doi: 10.1523/JNEUROSCI.1690-08.2008. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Itoh N. FGFs as multifunctional signaling molecules: diversity of structure and function. Seikagaku. 2001;73:525–535. [PubMed] [Google Scholar]
  8. Itoh N, Ornitz DM. Evolution of the Fgf and Fgfr gene families. Trends Genet. 2004;20:563–569. doi: 10.1016/j.tig.2004.08.007. [DOI] [PubMed] [Google Scholar]
  9. Itoh N, Ornitz DM. Functional evolutionary history of the mouse Fgf gene family. Dev Dyn. 2008;237:18–27. doi: 10.1002/dvdy.21388. [DOI] [PubMed] [Google Scholar]
  10. Jacques BE, Montcouquiol ME, Layman EM, Lewandoski M, Kelley MW. Fgf8 induces pillar cell fate and regulates cellular patterning in the mammalian cochlea. Development. 2007;134:3021–3029. doi: 10.1242/dev.02874. [DOI] [PubMed] [Google Scholar]
  11. Ladher RK, Wright TJ, Moon AM, Mansour SL, Schoenwolf GC. FGF8 initiates inner ear induction in chick and mouse. Genes Dev. 2005;19:603–613. doi: 10.1101/gad.1273605. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Lim DJ, Anniko M. Developmental morphology of the mouse inner ear. A scanning electron microscopic observation. Acta Otolaryngol Suppl. 1985;422:1–69. [PubMed] [Google Scholar]
  13. Mansour SL, Goddard JM, Capecchi MR. Mice homozygous for a targeted disruption of the proto-oncogene int-2 have developmental defects in the tail and inner ear. Development. 1993;117:13–28. doi: 10.1242/dev.117.1.13. [DOI] [PubMed] [Google Scholar]
  14. Mansour SL, Twigg SR, Freeland RM, Wall SA, Li C, Wilkie AO. Hearing loss in a mouse model of Muenke syndrome. Hum Mol Genet. 2009;18:43–50. doi: 10.1093/hmg/ddn311. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Martin K, Groves AK. Competence of cranial ectoderm to respond to Fgf signaling suggests a two-step model of otic placode induction. Development. 2006;133:877–887. doi: 10.1242/dev.02267. [DOI] [PubMed] [Google Scholar]
  16. Morsli H, Choo D, Ryan A, Johnson R, Wu DK. Development of the mouse inner ear and origin of its sensory organs. J Neurosci. 1998;18:3327–3335. doi: 10.1523/JNEUROSCI.18-09-03327.1998. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Ohyama T, Groves AK, Martin K. The first steps towards hearing: mechanisms of otic placode induction. Int J Dev Biol. 2007;51:463–472. doi: 10.1387/ijdb.072320to. [DOI] [PubMed] [Google Scholar]
  18. Ornitz DM, Itoh N. Fibroblast growth factors. Genome Biol. 2001:2. doi: 10.1186/gb-2001-2-3-reviews3005. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Pauley S, Wright TJ, Pirvola U, Ornitz D, Beisel K, Fritzsch B. Expression and function of FGF10 in mammalian inner ear development. Dev Dyn. 2003;227:203–215. doi: 10.1002/dvdy.10297. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Peters K, Ornitz D, Werner S, Williams L. Unique expression pattern of the FGF receptor 3 gene during mouse organogenesis. Developmental Biology. 1993;155:423–430. doi: 10.1006/dbio.1993.1040. [DOI] [PubMed] [Google Scholar]
  21. Pickles JO. The expression of fibroblast growth factors and their receptors in the embryonic and neonatal mouse inner ear. Hear Res. 2001;155:54–62. doi: 10.1016/s0378-5955(01)00247-7. [DOI] [PubMed] [Google Scholar]
  22. Pirvola U, Cao Y, Oellig C, Suoqiang Z, Pettersson RF, Ylikoski J. The site of action of neuronal acidic fibroblast growth factor is the organ of Corti of the rat cochlea. Proc Natl Acad Sci U S A. 1995;92:9269–9273. doi: 10.1073/pnas.92.20.9269. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Pirvola U, Spencer-Dene B, Xing-Qun L, Kettunen P, Thesleff I, Fritzsch B, Dickson C, Ylikoski J. FGF/FGFR-2(IIIb) signaling is essential for inner ear morphogenesis. J Neurosci. 2000;20:6125–6134. doi: 10.1523/JNEUROSCI.20-16-06125.2000. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Pirvola U, Ylikoski J, Trokovic R, Hebert J, McConnell S, Partanen J. FGFR1 Is Required for the Development of the Auditory Sensory Epithelium. Neuron. 2002;35:671. doi: 10.1016/s0896-6273(02)00824-3. [DOI] [PubMed] [Google Scholar]
  25. Puligilla C, Feng F, Ishikawa K, Bertuzzi S, Dabdoub A, Griffith AJ, Fritzsch B, Kelley MW. Disruption of fibroblast growth factor receptor 3 signaling results in defects in cellular differentiation, neuronal patterning, and hearing impairment. Dev Dyn. 2007;236:1905–1917. doi: 10.1002/dvdy.21192. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Ruben RJ. Development of the inner ear of the mouse: a radioautographic study of terminal mitoses. Acta Otolaryngol Suppl. 1967;220:1–44. [PubMed] [Google Scholar]
  27. Sher AE. The embryonic and postnatal development of the inner ear of the mouse. Acta Otolaryngol Suppl. 1971;285:1–77. [PubMed] [Google Scholar]
  28. Shim K, Minowada G, Coling DE, Martin GR. Sprouty2, a mouse deafness gene, regulates cell fate decisions in the auditory sensory epithelium by antagonizing FGF signaling. Dev Cell. 2005;8:553–564. doi: 10.1016/j.devcel.2005.02.009. [DOI] [PubMed] [Google Scholar]
  29. Theiler K. The House Mouse: Atlas of Mouse Development. New York: Springer-Verlag; 1989. [Google Scholar]
  30. Wang SJ, Furusho M, D’Sa C, Kuwada S, Conti L, Morest DK, Bansal R. Inactivation of fibroblast growth factor receptor signaling in myelinating glial cells results in significant loss of adult spiral ganglion neurons accompanied by age-related hearing impairment. J Neurosci Res. 2009 doi: 10.1002/jnr.22164. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Wright TJ, Hatch EP, Karabagli H, Karabagli P, Schoenwolf GC, Mansour SL. Expression of mouse fibroblast growth factor and fibroblast growth factor receptor genes during early inner ear development. Dev Dyn. 2003;228:267–272. doi: 10.1002/dvdy.10362. [DOI] [PubMed] [Google Scholar]
  32. Wright TJ, Mansour SL. Fgf3 and Fgf10 are required for mouse otic placode induction. Development. 2003;130:3379–3390. doi: 10.1242/dev.00555. [DOI] [PubMed] [Google Scholar]
  33. Zhang X, Ibrahimi OA, Olsen SK, Umemori H, Mohammadi M, Ornitz DM. Receptor specificity of the fibroblast growth factor family. The complete mammalian FGF family. J Biol Chem. 2006;281:15694–15700. doi: 10.1074/jbc.M601252200. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supp Fig s1. Supplementary Figure 1. Fgfr expression in mouse embryos.

To verify the specificity of the probes used for in situ hybridization, we tested them on sections of developing brain. Fgfr1, Fgfr2, and Fgfr3 are all highly expressed in the lateral ventricle, as reported in previous publications. Fgfr4 is not highly expressed in the brain, but is highly expressed in the extra-ocular muscles. Co; Cochlea, LV; lateral ventricle; L; lens; R; Retina; OM; ocular muscle. Scale bars=100μm

NIHMS225977-supplement-Supp_Fig_s1.tif (28.2MB, tif)
Supp Fig s2. Supplemental figure 2: Fgfr3 is expressed in the P21 cochlea.

A and B show sections of the mid-apical tunes of P21 cochlea. In panel B, the organ of Corti was photographed at the high-magnification. The asterisks in panel B indicate the nuclei of the Deiters’ cells. TM; tectorial membrane. OHC; outer hair cells. PCs; pillar cells. DCs; Deiters’ cells. Scale bar in A = 100 μm Scale bar in B = 50μm

NIHMS225977-supplement-Supp_Fig_s2.tif (2.7MB, tif)

RESOURCES