DEVELOPMENT AND DISEASE RESEARCH ARTICLE
The sensory epithelium of the mammalian cochlea, the organ of
Corti (OC), comprises at least six distinct cell types arranged into
precise rows that extend along the entire length of the cochlear
spiral. The OC contains four rows of hair cells (Fig. 1A): three
rows of outer hair cells (OHCs) supported by underlying Deiter’s
cells (DCs) and flanked on the lateral edge by a several rows of
Hensen’s cells (HeCs), and one row of inner hair cells (IHCs) with
underlying phallangeal cells. Separating the two types of hair cells
are parallel rows of non-sensory pillar cells (PCs) (Fig. 1B). When
mature, PCs form the boundaries of a triangular fluid-filled space
referred to as the tunnel of Corti (Fig. 1C) (Raphael and
Altschuler, 2003). The tunnel of Corti and the PCs that form this
structure are unique to the mammalian auditory system and are
found in no other vertebrate class. Defects in PC development
result in significant hearing impairment (Colvin et al., 1996).
Despite their crucial role in cochlear function, the factors that
regulate PC formation are poorly understood. Previous work has
demonstrated that ongoing activation of one of the fibroblast growth
factor receptors, Fgfr3, is required for PC development (Colvin et
al., 1996; Mueller et al., 2002). Ectopic activation of Fgfr3 in vitro
by treatment with Fgf2 induces an overproduction of PCs,
suggesting that the relative level of ligand available for Fgfr3
activation plays a key role in regulating PC number and position
within the OC (Mueller et al., 2002). Fgfr3 is one of four related
receptors that bind to members of the fibroblast growth factor family.
All Fgf receptors are transmembrane proteins that contain a tyrosine
kinase (TK) domain in their intracellular region. Fgfr activation is
mediated through binding of one of at least 23 known Fgfs and a
sulfated glycosaminoglycan such as heparin sulfate. Binding of Fgf
ligand and heparin leads to receptor dimerization, cross-activation
of the TK domains and downstream signaling through the MAP
kinase signaling pathway (Mohammadi et al., 2005).
Within the developing cochlea, Fgfr3 is initially expressed at
~E16 in a broad pool of progenitor cells located directly adjacent to
developing IHCs (Mueller et al., 2002; Peters et al., 1993), the first
cells to differentiate within the epithelium (Sobin and Anniko,
1984). Based on the spatiotemporal pattern of expression, it seems
likely that Fgfr3 is expressed in progenitors that will ultimately
develop as PCs and OHCs, as well as HeCs and DCs. As
development proceeds, Fgfr3 is downregulated in progenitors that
develop as OHCs, HeCs and DCs, but is maintained in PCs (Mueller
et al., 2002; Pirvola et al., 1995). RNA expression analysis using
quantitative PCR has suggested that the Fgfr3c splice variant is the
predominant isoform expressed in the cochlea (Pickles, 2001). In
addition, ligand-binding assays indicate that the ‘c’ isoform of Fgfr3
binds to the Fgf8b isoform with high affinity (MacArthur et al.,
1995; Olsen et al., 2006; Ornitz et al., 1996). The Fgf8b ligand has
been shown to have important regulatory roles during pattern
formation, differentiation and cell growth throughout the developing
embryo and nervous system (Olsen et al., 2006). Quantitative RT-
PCR analysis has indicated that it is expressed in the embryonic
cochlear sensory epithelium (Pickles, 2001). Here, we demonstrate
that Fgf8is expressed in a pattern that is consistent with an inductive
role in PC development and that changes in the levels of Fgf8, or in
Fgfr3 activation, lead to corresponding changes in the number and
differentiation of PCs.
Fgf8 induces pillar cell fate and regulates cellular patterning
in the mammalian cochlea
Bonnie E. Jacques1,2, Mireille E. Montcouquiol1,*, Erynn M. Layman1, Mark Lewandoski3
and Matthew W. Kelley1,†
The mammalian auditory sensory epithelium (the organ of Corti) contains a number of unique cell types that are arranged in
ordered rows. Two of these cell types, inner and outer pillar cells (PCs), are arranged in adjacent rows that form a boundary
between a single row of inner hair cells and three rows of outer hair cells (OHCs). PCs are required for auditory function, as mice
lacking PCs owing to a mutation in Fgfr3 are deaf. Here, using in vitro and in vivo techniques, we demonstrate that an Fgf8 signal
arising from the inner hair cells is the key component in an inductive pathway that regulates the number, position and rate of
development of PCs. Deletion of Fgf8 or inhibition of binding between Fgf8 and Fgfr3 leads to defects in PC development, whereas
overexpression of Fgf8 or exogenous Fgfr3 activation induces ectopic PC formation and inhibits OHC development. These results
suggest that Fgf8-Fgfr3 interactions regulate cellular patterning within the organ of Corti through the induction of one cell fate
(PC) and simultaneous inhibition of an alternate fate (OHC) in separate progenitor cells. Some of the effects of both inhibition and
overactivation of the Fgf8-Fgfr3 signaling pathway are reversible, suggesting that PC differentiation is dependent upon constant
activation of Fgfr3 by Fgf8. These results suggest that PCs might exist in a transient state of differentiation that makes them
potential targets for regenerative therapies.
KEY WORDS: Organ of Corti, Hair cell, Fgfr3, Mouse
Development 134, 3021-3029 (2007) doi:10.1242/dev.02874
1Section on Developmental Neuroscience, Porter Neuroscience Research Center, 35
Convent Dr, Room 2A-100, National Institute on Deafness and Other
Communication Disorders, National Institutes of Health, Bethesda, MD 20892, USA.
2University of Maryland College Park, Department of Biology, College Park, MD,
USA. 3Genetics of Vertebrate Development Section, Cancer and Developmental
Biology Laboratory, National Cancer Institute, Frederick, MD, USA.
*Present address: Equipe AVENIR 2 ‘Neurosciences Dévelopementales’ IFR8, INSERM,
Institut F. Magendie des Neurosciences, 33077 Bordeaux Cedex, France
†Author for correspondence (e-mail: email@example.com)
Accepted 29 May 2007
MATERIALS AND METHODS
In situ hybridization
In situ hybridization (ISH) was performed as described previously (Wu and
Oh, 1996) for Fgf8 and Fgfr3 on 12 ?m frozen sections or whole organs
from cochleae isolated at E15, E16 and P0. A probe specific to exons 2 and
3 of Fgf8(the region excised by Cre in the Fgf8?2,3n/flox;Foxg1cre/+mutants)
was also used on E16-18 cochleae from Fgf8?2,3n/flox;Foxg1cre/+mutantsand
their wild-type littermates to demonstrate excision of the targeted region.
Generation of Fgf8? ?2,3n/flox; Foxg1cre/+mutants and analysis of
pillar cell defects
Animals with a targeted deletion of Fgf8 in the forebrain, retina and inner
ear were generated by crossing Fgf8flox/floxfemales with Fgf8?2,3n/+;
Foxg1cre/+males. Mice carrying these alleles have been described previously
(Meyers et al., 1998; Hebert and McConnell, 2000). Mutant progeny of the
genotype Fgf8?2,3n/flox;Foxg1cre/+were visually identified based on obvious
defects in the development of the forebrain (Storm et al., 2003). Siblings
were of the genotypes Fgf8+/flox; Foxg1cre/+, Fgf8+/flox; Foxg1+/+or
Fgf8?2,3n/flox; Foxg1+/+and served as normal littermate controls. Cochleae
were dissected from mutants and littermate controls at E15.5, E16 and E19,
and fixed in either 4% paraformaldehyde (PFA) or 3% glutaraldehyde/2%
PFA overnight. Following fixation, the cochleae were dissected and the OC
were exposed and labeled with cell type-specific antibodies: anti-myosin VI
(Proteus Biosciences) 1:1000; anti-p75ntr(Chemicon) 1:1000; anti-?-actin
(Sigma) 1:200. Secondary antibodies were conjugated to one of the
following: Alexa 350, Alexa 488, Alexa 546 or Alexa 633 (Molecular
Probes). In addition, filamentous actin was labeled using phalloidin at 1:200
conjugated to either Alexa 488 or Alexa 633 (Molecular Probes). Specimens
were then flat-mounted and the total length of the cochlear duct was
measured. The cochlea was then divided into four equal sections, each
representing a quarter of the total length of the cochlear duct, and the
distances between the inner hair cells and first row of outer hair cells (ITO
distances) were determined in each region (n=5 animals; greater than 50
cells counted per region). All animal care and procedures were approved by
the Animal Care and Use Committee at NIH and complied with the NIH
guidelines for the care and use of animals.
Measurement of ITO distance
The inner-to-outer (ITO) distance is defined as the distance between the
lateral edge of the IHC and the medial edge of the first row OHC. This is the
distance encompassed by the inner pillar head. Digital images of the OC
were captured for each sample using a Zeiss 510 LSM confocal laser-
scanning microscope. Measurements of ITO distances were taken at three
specific points along the length of the cochlear duct of each sample, roughly
at 25%, 50% and 75% of the distance from the most basal region and moving
towards the apex. A minimum of 15 ITO measurements were made at each
of the three locations. Cell counts were also taken of each cell type in the
Temporal bones from control and Fgf8?2,3n/+; Foxg1cre/+littermates were
fixed in 3% glutaraldehyde/2% paraformaldehyde, tissue was dehydrated in
ethanol and then embedded in Immunobed (Polysciences). Cochleae were
oriented to generate mid-modiolar sections, cut at 5 ?m and stained with
Explant cultures of embryonic cochleae were established as described
previously (Montcouquiol and Kelley, 2003) and maintained for 6 DIV.
E13.5 explants were incubated for 24 hours before exposure to growth
factors or antibodies that were diluted in culture medium to the stated final
concentrations along with 0.1% DMSO and 1 ?g/?l heparin. Anti-Fgf8b,
75-150 ?g/ml; anti-Fgf5, 75-150 ?g/ml; Fgf17, 300 ng/ml (all from R&D
systems). Antibodies and proteins were used at 100 times the ND50and
ED50, to ensure penetration through the reticular lamina, a strong ionic
barrier that exists at the lumenal surface of the OC.
Full-length cDNA for murine Fgf8b was kindly provided by Elizabeth
Grove, University of Chicago (Fukuchi-Shimogori and Grove, 2001). Fgf8b
was excised from its original vector using BamHI and then directly ligated
into the pAM/CAG-IRES_EGFP vector at the BamHI site. Orientation was
determined by sequencing. Empty pAM/CAG-IRES_EGFP vector and
pAM/CAG-IRES_EGFP containing full-length Fgf8b in the reverse
orientation were used as controls. Electroporation of cochlear explants was
carried out as previously described (Jones et al., 2006); n>30 for each vector
Images of electroporated explants were obtained using a Zeiss LSM510
confocal microscope. All samples were obtained during the same session
using the same laser power and detection settings. To quantify the effects of
overexpression of Fgf8, a rectangle (225 ?m?110 ?m) was oriented such
that the short dimension of the rectangle was parallel with the line of PCs in
the region being measured. The rectangle was positioned so that its strial
edge aligned with third row OHCs. Thus, the rectangle included a 110 ?m
stretch of the OC with the adjacent region of the greater epithelial ridge
containing transfected cells. Control and experimental regions were obtained
and then thresholded for both green and red pixels. The total number of
pixels of each color was then determined as a percentage of the total of
number of pixels within the entire rectangle.
Expression of Fgf8 and Fgfr3 in the organ of Corti
PCs develop on the medial edge of an Fgfr3-expression domain
located directly adjacent to the IHCs. Given the crucial role of Fgfr3
in PC formation (Mueller et al., 2002; Pirvola et al., 1995), it seemed
likely that hair cells located adjacent to developing PCs might act as
a source of ligand for Fgfr3. Furthermore, Fgf8, a high affinity
ligand for Fgfr3, has been reported to be expressed by the IHCs of
the adult cochlea (Pirvola et al., 2002; Shim et al., 2005). To
Development 134 (16)
Fig. 1. Anatomy of the mouse organ of Corti. (A) Diagram of the
lumenal surface of the organ of Corti (OC) at P0. A row of alternating
inner hair cells (light green) and phalangeal cells (gray) are bordered by
one row of inner PCs (red) and one row of outer pillar cells (PCs, pink).
Outer PCs project between first-row outer hair cells (OHCs, olive green).
Second and third row OHCs are separated by Deiter’s cells (DC, blue).
Hensen’s cells (HeC, gray) form the lateral edge of the OC. (B,C) Cross-
sections of the OC at (B) P0 and (C) adult.
Montcouquiol, M. and Kelley, M. W. (2003). Planar and vertical signals control
cellular differentiation and patterning in the mammalian cochlea. J. Neurosci.
Mueller, K. L., Jacques, B. E. and Kelley, M. W. (2002). Fibroblast growth factor
signaling regulates pillar cell development in the organ of corti. J. Neurosci. 22,
Olsen, S. K., Li, J. Y. H., Bromleigh, C., Eliseenkova, A. V., Ibrahimi, O. A.,
Lao, Z., Zhang, F., Linhardt, R. J., Joyner, A. L. and Mohammadi, M. (2006).
Structural basis by which alternative splicing modulates the organizer activity of
FGF8 in the brain. Genes Dev. 20, 185-198.
Ornitz, D. M., Xu, J., Colvin, J. S., McEwen, D. G., MacArthur, C. A., Coulier,
F., Gao, G. and Goldfarb, M. (1996). Receptor specificity of the fibroblast
growth factor family. J. Biol. Chem. 271, 15292-15297.
Pauley, S., Wright, T. J., Pirvola, U., Ornitz, D., Beisel, K. and Fritzsch, B.
(2003). Expression and function of FGF10 in mammalian inner ear development.
Dev. Dyn. 227, 203-215.
Peters, K., Ornitz, D., Werner, S. and Williams, L. (1993). Unique expression
pattern of the FGF receptor 3 gene during mouse organogenesis. Dev. Biol. 155,
Pickles, J. O. (2001). The expression of fibroblast growth factors and their
receptors in the embryonic and neonatal mouse inner ear. Hear. Res. 155, 54-
Pirvola, U., Cao, Y., Oellig, C., Suoqiang, Z., Pettersson, R. F. and Ylikoski, J.
(1995). The site of action of neuronal acidic fibroblast growth factor is the organ
of Corti of the rat cochlea. Proc. Natl. Acad. Sci. USA 92, 9269-9273.
Pirvola, U., Ylikoski, J., Trokovic, R., Hebert, J. M., McConnell, S. K. and
Partanen, J. (2002). FGFR1 is required for the development of the auditory
sensory epithelium. Neuron 35, 671-680.
Puligilla, C., Feng, F., Ishikawa, K., Bertuzzi, S., Dabdoub, A., Griffith, A. J.,
Fritzsch, B. and Kelley, M. W. (2007). Disruption of fibroblast growth factor
receptor 3 signaling results in defects in cellular differentiation, neuronal
patterning, and hearing impairment. Dev. Dyn. 236, 1905-1917.
Raphael, Y. and Altschuler, R. A. (2003). Structure and innervation of the
cochlea. Brain Res. Bull. 60, 397-422.
Shim, K., Minowada, G., Coling, D. E. and Martin, G. R. (2005). Sprouty2, a
mouse deafness gene, regulates cell fate decisions in the auditory sensory
epithelium by antagonizing FGF signaling. Dev. Cell 8, 553-564.
Sobin, A. and Anniko, M. (1984). Early development of cochlear hair cell
stereociliary surface morphology. Arch. Otorhinolaryngol. 241, 55-64.
Storm, E. E., Rubenstein, J. L. and Martin, G. R. (2003). Dosage of Fgf8
determines whether cell survival is positively or negatively regulated in the
developing forebrain. Proc. Natl. Acad. Sci. USA 100, 1757-1762.
White, P. M., Doetzlhofer, A., Lee, Y. S., Groves, A. K. and Segil, N. (2006).
Mammalian cochlear supporting cells can divide and trans-differentiate into hair
cells. Nature 441, 984-987.
Wu, D. K. and Oh, S.-H. (1996). Sensory organ generation in the chick inner ear.
J. Neurosci. 16, 6454-6462.
Zheng, J. L. and Gao, W. Q. (2000). Overexpression of Math1 induces robust
production of extra hair cells in postnatal rat inner ears. Nat. Neurosci. 3, 580-
Fgf8 induces pillar cell fate in the cochlea