doi: 10.1242/10.1242/dev.00376
Lack of pendrin expression leads to deafness and expansion of the endolymphatic compartment in inner ears of Foxi1 null mutant mice
Malin Hulander1,
Amy E. Kiernan2,*,
Sandra Rodrigo Blomqvist1,*,
Peter Carlsson4,
Emma-Johanna Samuelsson3,
Bengt R. Johansson3,
Karen P. Steel2 and
Sven Enerbäck1,
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
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Fig. 1. Inner ear histology of Foxi1+/+ and
Foxi1-/- mice at E13.5 (A-D). Lateral (L) and anterior (A)
orientations as marked in A. At E13.5 (A,B), normal vestibulocochlear ganglion
(VIII), lateral (LSD) and posterior (PSD) semicircular ducts are formed in
both Foxi1+/+ and Foxi1-/- animals.
There is also a developing cochlea of normal appearance (C,D). Scale bar: 100
µm.
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Fig. 2. Paint-filled inner ears of E11.5 and E12.5 embryos. No major differences
can be demonstrated when the three (Foxi1+/+,
Foxi1+/- and Foxi1-/-) genotypes are
compared. ssc (superior semicircular canal), psc (posterior semicircular
canal), cd (cochlear duct) ed (endolympahtic duct) and sac (saccule) are
indicated. Scale bar: 250 µm.
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Fig. 3. Whole-mount in situ hybridization of E11.5 wild-type embryo. Foxi1
has an otic vesicle-specific expression pattern (left panel, red arrowhead).
At higher magnification (right panel), the contours of the future
endolymphatic duct can be seen (arrowheads). Scale bars: 100 µm.
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Fig. 4. Histology of inner ears from Foxi1+/+ and
Foxi1-/- embryos at E16.5. (A,B) The lateral (LSD) and
posterior (PSD) semicircular ducts as well as the endolymphatic duct (ED) are
expanded in the mutant ear. (C,D) In the cochlea of
Foxi1-/- ear the basal turn (BT) is larger and the apical
turn (AT) has formed an apical cyst (AC). Scale bar: 200 µm for A,B; 100
µm for C,D.
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Fig. 5. Paint-filled inner ears from E16.5 mice. Superior (ssc), lateral (lsc),
posterior (psc) semicircular canals, endolymphatic duct (ed), utricle (ut),
saccule (sac) and cochlear duct (cd) are all clearly expanded in
Foxi1-/- embryos (C,D) whereas
Foxi1+/+ (A) and Foxi1+/- (B) inner
ears appear normal. Scale bar: 1 mm.
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Fig. 6. Three dimensional reconstruction of E16.5 inner ears using 6 µm tissue
sections. Lateral and medial views are shown. am, ampulla; other abbreviations
as in Figs
1,2,3,4.
Sensory patches are marked in the cochlea (dark blue), saccule and utricle
(light blue), and in the ampullae (purple). In spite of the enormous expansion
in the Foxi1-/- inner ear, the sensory patches can be
identified at approximately the correct locations. In the tissue sections used
in this experiment the sensory areas appear normal in all three genotypes (not
shown). Scale bar: 100 µm.
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Fig. 7. Histology of inner ears of P12 Foxi1+/+ and
Foxi1-/- mice. Nasal (N) and caudal (C) orientations as
marked in A. In Foxi1-/- mice the entire inner ear is
enlarged with large irregular cavities in place of the delicate inner ear
structures of the wild-type ear. S, saccule; U, utricle; ME, middle ear;
asterisks mark the basal turn of the cochlea in A. (C) In the mutant the
endolymphatic sac (ES) seems to compress the cerebellum and the posterior part
of the ES (arrowheads) is expanded considerably in the posterior/dorsal
direction (double headed arrow) compared with the wild type (A). The temporal
bone (TB, arrowheads) is also clearly reduced. (B,D) The normal cochlea with
its modiolus (Mo, in B) is absent in the mutant (D) and in its place is an
enlarged cavity with an apical cyst (AC). Scale bars: in A, 100 µm for A,C;
in B, 100 µm for B,D.
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Fig. 8. Morphology of the otoconia, at E16.5, in Foxi1+/- (A)
and Foxi1-/- (B) inner ears. The white utricular (uo) and
saccular (so) otoconia are not formed properly (arrows) in
Foxi1-/- mice (B). This finding suggests a defect in
proper crystallization of calcium carbonate crystals normally found in the
otoconia. In this mouse strain (CD-1), the perimeter of the oval window (ow)
has a white color. Scale bar: 250 µm.
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Fig. 9. Cryosections of the major inner ear structures of E16.5
Foxi1+/+ (A,C) and Foxi1-/- (B,D) mice
were labeled using the TUNEL protocol (C,D). (A,B) The ampulla (AM), utricle
(UT), endolymphatic duct (ED) and common crus (CC) are enlarged in the mutant.
Only one minor difference was detected in the TUNEL staining: a small
mesenchymal cell population adjacent to the expanded ED (white arrowheads in
D) showed increased labeling. (The same cells are indicated with red
arrowheads in B.) (E) The number of TUNEL-positive cells were counted in
anatomically corresponding sites in 5 Foxi1+/+ and 5
Foxi1-/- inner ears (represented by the the boxed area in
C and D). Numbers expressed as means ±s.d. Scale bar: 100 µm.
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Fig. 10. Heads stained for bone (dark purple) and cartilage (blue) at E18.5.
Superior semicircular canal (SSC), tympanic ring (TR) and the basioccipital
bone (BO) are marked. In Foxi1+/+ and
Foxi1+/- an arrowhead indicates the ossification centers
of the middle ear bones. Normal morphology is seen in
Foxi1+/+ and Foxi1+/- mice;
Foxi1-/- animals display a clearly aberrant integration of
the otic capsule in the temporal bone as evident by excessive ectopic
cartilage condensation/bone formation in both the cochlea (arrowheads) and the
vestibulum (arrows). Scale bar: 200 µm.
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Fig. 11. (A) A compound action potential response, measured from the round window of
the cochlea. Foxi1+/+ and Foxi1+/-
littermates showed thresholds (±s.e.m.) that were very similar to each
other, but relatively high compared with other mouse strains especially at
high frequencies, suggesting that the genetic background of these mice (mostly
CD-1) was associated with poor hearing. Foxi1-/- mice
showed no cochlear responses of any sort, up to maximum sound output used;
indicated by the black triangles at the top of the graph (n=7,
Foxi1+/+; n=9, Foxi1+/-;
n=10, Foxi1-/-). (B) Mean endocochlear potentials
(EP; ±s.e.m.) from wild type, heterozygote and null mutant littermates
aged 29 to 129 days. There was no difference in the EPs in wild types and
heterozygotes, but homozygous mutants showed potentials close to zero
(n=14, Foxi1+/+; n=18,
Foxi1+/-; n=19,
Foxi1-/-).
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Fig. 12. In situ hybridization using probes for Foxi1, Pds, Coch and
Jagged1 on inner ear sections from E16.5. (A) Foxi1 and
Pds have similar expression patterns in wild-type endolymphatic
duct/sac epithelium. At this developmental stage Foxi1 expression is
restricted to the endolymphatic duct/sac epithelium. (B) On
Foxi1-/- inner ear sections (B) Pds expression is
not detectable in the endolymphatic epithelium (right arrowhead) while it is
expressed in the utricle (left arrowhead). Higher magnifications (on the
right) of both Foxi1+/+ and Foxi1-/-
epithelium also demonstrate a lack of Pds expression in
Foxi1-/- epithelium. (C) Pds is expressed
(arrowheads) in the cochlea (top row) and in the epithelium adjacent to
sensory areas of the utricle (bottom row) in both Foxi1+/+
and Foxi1-/- mice. (D) Coch expression is missing
in the endolymphatic duct/sac epithelium in Foxi1-/-
animals (arrowheads) whereas the expression is unaffected in the common crus
(CC). A typical punctate pattern is seen in wild-type endolymphatic duct/sac
epithelium. (E) In spite of altered morphology, Coch is expressed in
other inner ear structures such as cochlea (CO), saccule/utricle (SU) and
ampulla (AM) in subepithelial cell layers in the Foxi1-/-
inner ear. (F) The expression in the cochlea also seems to be similar in
Foxi1+/+ and Foxi1-/- mice. (G) In
Foxi1-/- inner ears Jagged1 expression is missing
in the endolymphatic sac/duct epithelium (ED, arrowheads) but the expression
is not affected in the utricle (U). In wild-type mice, Jagged1 has a
punctate expression pattern in the endolymphatic duct/sac epithelium. (H)
Jagged1 expression can be detected in the cochlea of both
Foxi1+/+ and Foxi1-/- mice
(arrowheads). Scale bars: 100 µm.
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Fig. 13. Scanning electron micrographs of endolymphatic duct/sac epithelium (E14.5)
from Foxi1+/+ and Foxi1-/- mice.
Arrows (in wild type) indicate two cells of a type we call forkhead related
(FORE) cells, that are missing in Foxi1-/- epithelium.
Scale bar: 10 µm.
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© The Company of Biologists Ltd 2003
