1 Medical Genetics, Department of Medical Biochemistry, Institute of Anatomy and
Cell Biology, Göteborg University, Box 440, SE-405 30 Göteborg,
Sweden
2 MRC Institute of Hearing Research, University Park, Nottingham NG7 2RD,
UK
3 The Electron Microscopy Unit, Institute of Anatomy and Cell Biology,
Göteborg University, Box 440, SE-405 30 Göteborg, Sweden
4 Department of Molecular Biology, Institute of Anatomy and Cell Biology,
Göteborg University, Box 440, SE-405 30 Göteborg, Sweden
Author for correspondence (e-mail:
sven.enerback{at}medgen.gu.se)
Accepted 8 January 2003
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SUMMARY |
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Key words: Forkhead, Foxi1, Pendrin, Deafness, Inner ear, Mouse
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INTRODUCTION |
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Here we describe the Foxi1-/- (Fkh10) mutant in which the endolymphatic compartment is severely dilated. We show that these mice are deaf and lack an endocochlear potential, indicating a primary defect in fluid homeostasis in the inner ear. This is associated with an expansion of the endolymph compartment followed by rupture of the endolymphatic epithelium. Based on morphological, physiological and genetic analysis we propose a molecular mechanism by which the endolymphatic inner ear fluid is allowed to accumulate to abnormal levels.
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MATERIALS AND METHODS |
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Histology and hybridization
The morning that vaginal plugs were detected was designated embryonic day
0.5 (E0.5). Tails from embryos were collected for genotyping using PCR as
described above. Embryos were fixed overnight in PBS with 4% paraformaldehyde
at 4°C. For histological examination, embryos were dehydrated and embedded
in paraffin wax and 6 µm sections were cut. Tissue sections were stained
with Haematoxylin and Eosin. In situ hybridization on whole-mount mouse
embryos (Rosen and Beddington,
1993) and in situ hybridization of cryosections
(Bostrom et al., 1996
) were
performed with digoxigenin-labeled antisense cRNA probes. No signals were
observed when control sense probes were used. We used plasmids to generate
probes for: Pds (Everett et al.,
1999
), Pax2 (Torres
et al., 1996
), ErbB3
(Britsch et al., 1998
),
Eya1 (Xu et al.,
1999
), Hmx3 (Wang et
al., 1998
), Jag1
(Morrison et al., 1999
),
Otx1 (Acampora et al.,
1996
) and Coch
(Robertson et al., 1997
). For
histology and in situ hybridization experiments inner ears from at least seven
mice of each genotype (Foxi1+/+,
Foxi1+/- and Foxi1-/-) of CD-1
background and three of each genotype of B16 background were analyzed.
Immunohistochemistry
For BrdU labeling, pregnant female mice were injected intraperitoneally
with BrdU (1 ml of 10 mM BrdU per 100 g body weight; Roche). Injected mice
were sacrificed 2 hours later and processed for immunohistochemistry,
according to protocols supplied by the manufacture. Kits (Roche) were used to
identify apoptotic cells by the TUNEL assay according to the manufacturer's
instructions. For immunohistochemistry, BrdU labeling experiments and TUNEL
assay inner ears from at least four mice of each genotype
(Foxi1+/+, Foxi1+/- and
Foxi1-/-) of CD-1 background were analyzed.
Paint-fill
Paint-filling of inner ears (E11.5: Foxi1+/+;
n=5, Foxi1+/-; n=3,
Foxi1-/-; n=3, E12.5:
Foxi1+/+; n=3, Foxi1+/-;
n=8, Foxi1-/-; n=3, E16.5:
Foxi1+/+; n=6, Foxi1+/-;
n=11, Foxi1-/-; n=5) was performed in a
similar way to that described for chicken and mouse embryos
(Martin and Swanson, 1993).
Briefly, mice (E11.5-E16.5) were decapitated and fixed in Bodian's fixative
(75% ethanol, 5% formalin, 5% glacial acetic acid) overnight and dehydrated
twice in each of the following solutions: 75%, 95%, 100% ethanol (minimum 2
hours for each wash). Heads were bisected and cleared overnight in methyl
salicylate. The endolymphatic compartment of the inner ear was injected via
either the common crus and/or cochlea using a pulled glass capillary pipette
(20-40 µm diameter) filled with 1% gloss paint in methyl salicylate. The
ears were then dissected free of the skull and photographed in methyl
salicylate. Temporal bone biopsies were cleared in methyl salicylate for
analysis of otoconial crystals.
Three-dimensional reconstruction
Three-dimensional reconstructions from inner ears were made using
approximately 300 serial sections (6 µm) from an entire inner ear. The
inner lumen of the ear was traced and sensory areas, identified by the typical
pseudostratified appearance of the epithelium, were outlined in different
colors. Areas of interest from each section were traced, aligned and
reconstructed into three-dimensional images essentially as has been described
earlier (Wu and Oh, 1996). For
data processing we used the computer software Spyglass Slicer/T3D (Research
System,
http://www.researchsystems.com/noesys).
Four skulls of each genotype (Foxi1+/+,
Foxi1+/- and Foxi1-/-) of CD-1
background were sectioned and all four in each group had identical phenotypes.
Two of each genotype were used for three-dimensional reconstruction.
Skeleton staining
The embryos were fixed in 95% ethanol and stained with Alizarin Red to
reveal bone and Alcian Blue to reveal cartilage
(McLeod, 1980). They were
subsequently cleared by trypsin digestion and KOH treatment, and stored in
glycerol.
Scanning electron microscopy
Heads of E14.5 embryos were fixed immediately after decapitation in a
mixture of 2% paraformaldehyde, 2.5% glutaraldehyde, 0.1% sodium azide in 0.05
M sodium cacodylate buffer, pH 7.2. The heads were embedded in 7% agar and
serial sections were obtained with an oscillating tissue slicer (Leica VT
1000S; section thickness set at 200 µm, horizontal orientation). The slices
were examined in a tissue preparation microscope to identify levels with
opened endolymphatic structures. Selected sections were further treated with a
repeated sequence of 1% osmium tetroxide and a saturated thiocarbohydrazide
solution (OTOTO method), followed by dehydration in ethanol and infiltration
in hexamethyldisilazane (HMDS). The specimens were dried by evaporation of the
HMDS in a fume hood and were flat mounted on aluminum stubs. They were
examined in a Zeiss 982 Gemini field emission scanning electron microscope
without prior thin film metal coating.
Electrophysiology
Mice (n=5 controls (2 Foxi1+/+, 3
Foxi1+/-); 5 Foxi1-/-, aged 29-30
days; and n=6 Foxi1+/+; 6
Foxi1+/-; 5 Foxi1-/- aged 67-68 days)
were anaesthetized with urethane, the middle ear was opened, and a recording
electrode was placed on the round window of the cochlea. A closed sound system
in the external ear canal was used to deliver calibrated tonebursts (15
mseconds duration, 1 mseconds rise/fall time, 100 mseconds interstimulus
interval, average 200 repetitions). Thresholds for detection of a cochlear
nerve compound action potential (CAP) response were obtained using 2-3 dB
steps. The endocochlear potential (EP) was measured in the same animals as
well as in additional ones (Foxi1+/+, n=12;
Foxi1+/-, n=15; Foxi1-/-,
n=14) using a micropipette electrode inserted into the basal turn
scala media through the lateral cochlear wall
(Steel and Smith, 1992).
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RESULTS |
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Morphological analysis of inner ear development from E11.5 to
E13.5
From earlier experiments, it was known that otic vesicle formation at E9.5
is unaffected in Foxi1 null mutants
(Hulander et al., 1998). To
investigate the morphology in wild type and Foxi1-deficient mice
during later stages of inner ear development, we compared the histology using
sections from E11.5 (not shown), E12.5 (not shown) and E13.5
(Fig. 1). At E11.5 formation of
the endolymphatic appendage, the cochlear anlagen and the vertical plate of
the vestibular system (future anterior and posterior semicircular ducts) is
seemingly unaffected in mice that lack Foxi1 (not shown). From
E12.5-13.5, we find a normal cochlea as well as normal semicircular ducts in
both wild-type and Foxi1-/- mice (not shown). At E11.5,
the combined facial and vestibulo-cochlear ganglion (cranial nerve ganglion
VII and VIII) is present and at E12.5 to E13.5 the vestibulo-cochlear ganglion
(VIII) has formed in both wild type and null mutants. To assess the general
morphology of the membranous labyrinth we used a paint-fill method in which a
thin needle was inserted into the most cranial part of the common crus, formed
by the anterior and posterior semicircular ducts
(Martin and Swanson, 1993
).
Subsequently white gloss paint was injected to visualize the endolymphatic
compartment. The future endolymphatic and semicircular ducts as well as
saccule and cochlea showed no signs of abnormality at E11.5 and E12.5
(Fig. 2). As illustrated (Figs
1,
2) no apparent difference in
morphogenesis can be seen at early stages of inner ear development.
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DISCUSSION |
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In mice that lack Foxi1 expression, an expansion of the
endolymphatic chamber becomes evident at E16.5 with the endolymphatic duct/sac
as the most expanded structure (Figs
4,5,6).
The absence of Pds gene expression in the epithelium of this
structure (Fig. 12B)
establishes Foxi1 as an upstream regulator of Pds gene
expression at this location. There are two possible reasons for the lack of
Pds expression in the endolymphatic duct/sac: Foxi1 may in a
direct way regulate Pds expression, or without Foxi1, cells
that normally express Pds may be missing. Pds gene
expression in the cochlea and vestibulum is unaffected by the lack of
Foxi1, suggesting that expression at these locations is regulated by
other factors. Both Coch and Jag1 expression are also
dependent on Foxi1 in the endolymphatic duct/sac while expression at
other sites in the developing inner ear is not
(Fig. 12D-H). In the inner ear
of wild-type mice, Pds gene expression is first visualized in the
prospective endolymphatic duct/sac epithelium at approximately E13. Two days
later at E15 Pds gene expression is also found in the non-sensory
regions adjacent to the maculae of the saccule and utricle as well as in the
cells beneath the spiral prominence on the lateral wall of the external sulcus
in the cochlea (Everett et al.,
1999). It is interesting to note that the expansion of the
endolymphatic compartment, in Foxi1-/- embryos, starts
between E13.5 and E16.5 (Figs
4,
5,
6) the same time span
during which Pds gene expression first becomes detectable in
wild-type mice (Everett et al.,
1999
). Taken together these findings support the idea that the
inner ear phenotype in Foxi1-/- mice is, at least to some
extent, due to lack of pendrin expression in the endolymphatic duct/sac
epithelium. This hypothesis is further supported by the fact that patients
with Pendred syndrome display enlargement of the endolymphatic duct and sac as
a constant feature (Phelps et al.,
1998
). As a consequence of this, it seems that Pds gene
expression at inner ear locations other than the endolymphatic duct/sac
epithelium is not sufficient to substitute for the lack of Pds
expression in the endolymphatic duct/sac epithelium and hinder the abnormal
expansion of the endolymphatic compartment. Alternatively, the expression at
these locations, initiated at E15, is not capable of reversing the presumably
established state of dysfunction in the endolymphatic duct/sac epithelium,
involving not only Pds gene expression but also Coch and
Jag1. The exact order of events underlying the phenotype described
here is not resolved. It is however possible that Notch-mediated patterning of
the endolymphatic epithelium, supported by early (E9.5) Foxi1
expression, is disturbed because of a lack of Jag1 and that this
leads to lack of a specialized cell type that expresses pendrin and serves as
a major site for endolymph resorption
(Fig. 13). Alternatively, it
is also conceivable that lack of pendrin expression leads to a state of
epithelial dysfunction not compatible with Jag1 and Coch
expression, at a later stage of inner ear development. Such dysfunction could
not be too pronounced since expression of both E-cadherin and ZO-1, markers of
biologically active epithelium, are unaffected by the absence of
Foxi1 expression (not shown). Nevertheless a gene regulatory
hierarchy has been established in which Foxi1 is upstream of Pds,
Coch and Jag1. Further support for our suggestion that abnormal
Pds expression may underlie the pathology we observe in
Foxi1 mutants comes from the Pds mutant mouse, which shows a
similar early expansion of the endolymphatic compartments including
endolymphatic duct and sac and resultant malformation
(Everett et al., 2001
).
Only minor differences in TUNEL (Fig.
9) and BrdU (not shown) labeling can be demonstrated. The
abnormalities revealed by bone and cartilage staining
(Fig. 10) are interesting
since epithelial-mesenchymal interactions are necessary for condensation of
the periotic-mesenchyme during formation of the otic capsule
(Frenz and Van De Water, 1991;
Van de Water and Ruben, 1971
).
Results from bone and cartilage staining demonstrate an altered gross
morphology of the otic capsule in Foxi1 null embryos at E18.5. This
may be due to increased endolymphatic pressure, leading to expansion of the
endolymphatic compartment, resulting from defective pendrinmediated chloride
ion resorption. It should be noted that a defect in epithelial-mesenchymal
interaction could not be ruled out as the primary defect of the abnormal otic
capsule. However, it seems unlikely since Foxi1 expression at E16.5
is restricted to the endolymphatic duct/sac epithelium, which is located
outside the otic capsule. One consequence of altered function of the
endolymphatic duct/sac epithelium is most likely a change in ionic composition
of the endolymph as demonstrated by lack of proper crystallization of the
otoconia (Fig. 8). The complete
hearing loss together with an endocochlear potential (EP) of approximately 0
mV in Foxi1 null mutants (Fig.
11A,B) suggest a severe `ion channelopathy' possibly in
combination with a rupture of the endolymphatic epithelium allowing peri- and
endolymph, to mix. However, we cannot decisively determine which of these two
factors is the major cause of deafness. We would like to speculate that the
findings of (i) a progressive thinning of the endolymphatic epithelium (Figs
4,
7), (ii) lack of a
perilymphatic compartment in the cochlea
(Fig. 7D), (iii) expansion of
endolymphatic compartments in the vestibulum
(Fig. 7C) and (iv) an EP of 0
mV (Fig. 11B) all are
compatible with a more or less total rupture of the endolymphatic epithelium.
Nevertheless, it is most likely that the initial event is an expansion of the
endolymphatic compartment due to a defective ionic composition, possibly
including an increased osmolarity of the endolymph, which in turn severely
affects the ability of stria vascularis to maintain a proper EP. A rupture of
the endolymphatic epithelium at P12, as suggested by the results in
Fig. 7, would further hamper
this function.
The molecular mechanisms governing transformation of the near spherical
otocyst into a highly complex sensory organ the inner ear
remain to a large extent obscure. One obvious task during this process is to
guide morphogenetic events that produce the intricate three-dimensional
structure of the mature inner ear. In many instances during embryogenesis,
formation of complex structures is achieved through preprogrammed alterations
in rate of cell proliferation and/or apoptosis, e.g. limb formation. However,
a recent study addressing this in the developing inner ear of chicken failed
to demonstrate a clear increase in cell proliferation during outgrowth of
endolymphatic duct and canal plates (Lang
et al., 2000). Instead differences in the number of cells in
various regions of the otic vesicle was noted, suggesting that cells were
redistributed. For instance, the dorsal aspect of the otic vesicle (giving
rise to the endolymphatic duct and sac) was found to undergo thinning of the
epithelial surface with only a slightly increased cell proliferation
(Lang et al., 2000
). It is
intriguing that several aspects of the inner ear phenotype seen in
Foxi1-/- mice bear resemblance to that seen in
Hoxa1-/- and Fgf3-/- animals
(Chisaka et al., 1992
;
Lufkin et al., 1991
;
Mansour et al., 1993
). During
early embryonic development the endolymphatic duct/sac fails to form in
Hoxa1-/- and Fgf3-/- mice and the
inner ear develops into large irregular cavities or has a distended and
swollen appearance, respectively. This finding underscores the importance of
an intact endolymphatic duct/sac, for normal inner ear development. Although
there are many differences between Foxi1, Hoxa1 and Fgf3
mutants, they might all reflect the consequences of inappropriate endolymph
turnover resulting from a lack of (Hoxa1-/-,
Fgf3-/-) or inadequate function
(Foxi1-/-) of the endolymphatic duct/sac epithelium. The
phenotypic differences could be explained by the temporal appearance of these
defects, early at the otic vesicle stage in Hoxa1 null mutants and
late in the case of Foxi1-/- mice. These models also
emphasize the need for tightly regulated endolymph secretion/resorption during
normal inner ear development. It is possible that normal inner ear development
is dependent upon `pressure driven' events. This idea is supported by the
finding that the initial events in outgrowth of the endolymphatic duct are not
correlated with increased cell proliferation but rather thinning of the
epithelial surface (Lang et al.,
2000
).
The endolymphatic duct and sac are crucial for maintaining proper osmotic
pressure and ionic composition of the endolymph. These structures can function
as an expansion chamber and expand beyond the inner ear compartment into the
interdural space. In this way variations in the production of endolymph can be
tolerated without any significant increase of inner ear pressure
(Barbara et al., 1988a;
Everett et al., 1999
). This is
emphasized by the fact that one experimental approach to induce inner ear
hydrops is by obliteration of the endolymphatic duct/sac
(Kimura and Schuknecht, 1965
).
The endolymphatic duct/sac epithelium is also important for absorption of
endolymph (Everett et al.,
1999
) several ion transporters are expressed here such as
the chloride transporter pendrin (Everett
et al., 1999
). The relevance of this for the pathogenesis of human
inner ear diseases is exemplified by conditions such as
Ménière's disease and Pendred syndrome which are both, to some
extent, associated with inner ear hydrops (i.e. accumulation of endolymph in
the inner ear resulting in hampered inner ear function) and an expanded
endolymphatic duct/sac (Everett et al.,
1999
; Phelps et al.,
1998
; Schuknecht and Gulya,
1983
). We would also like to point out that mutations in the
COCH gene have been linked to Ménière's disease
(Fransen et al., 1999
). The
finding of a specific cell type, with projecting microvilli, in the
endolymphatic duct/sac epithelium that most likely expresses Foxi1, Pds,
Coch and Jag1 raises the intriguing possibility that these FORE
(forkhead related) cells
(Fig. 13) are important for
maintaining proper volume and/or composition of the endolymph. The FORE cell,
here genetically defined, has many similarities with a morphologically
identified cell type called `mitochondria-rich' cell.
(Qvortrup and Bretlau, 2002
).
We would like to speculate that the FORE cell could prove to play a role in
diseases characterized by altered endolymph turnover/composition, e.g. Pendred
syndrome and Ménière's disease.
FOXI1 (also known as FKHL10), the human homologue of
Foxi1, has been mapped to the chromosomal localization of 5q32-34
(Larsson et al., 1995). To
this date no human disease has been linked to mutations in this gene. As much
as 10% of all hereditary deafness could be due to Pendred syndrome
(Fraser, 1965
) and as many as
23% of the patients with a clinical diagnosis of Pendred syndrome lack
mutations in the PDS gene
(Reardon et al., 2000
). This
implies that mutations in FOXI1 could prove to be a cause of human
deafness.
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ACKNOWLEDGMENTS |
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Footnotes |
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