The role of Six1 in mammalian auditory system development
Weiming Zheng,
Li Huang,
Zhu-Bo Wei,
Derek Silvius,
Bihui Tang and
Pin-Xian Xu*
McLaughlin Research Institute for Biomedical Sciences, 1520 23rd Street
South, Great Falls, MT 59405, USA
*
Author for correspondence (e-mail:
pxu{at}po.mri.montana.edu)
Accepted 23 May 2003
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SUMMARY
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The homeobox Six genes, homologues to Drosophila sine oculis
(so) gene, are expressed in multiple organs during mammalian
development. However, their roles during auditory system development have not
been studied. We report that Six1 is required for mouse auditory
system development. During inner ear development, Six1 expression was
first detected in the ventral region of the otic pit and later is restricted
to the middle and ventral otic vesicle within which, respectively, the
vestibular and auditory epithelia form. By contrast, Six1 expression
is excluded from the dorsal otic vesicle within which the semicircular canals
form. Six1 is also expressed in the vestibuloacoustic ganglion. At
E15.5, Six1 is expressed in all sensory epithelia of the inner ear.
Using recently generated Six1 mutant mice, we found that all
Six1+/- mice showed some degree of hearing loss because of
a failure of sound transmission in the middle ear. By contrast,
Six1-/- mice displayed malformations of the auditory
system involving the outer, middle and inner ears. The inner ear development
in Six1-/- embryos arrested at the otic vesicle stage and
all components of the inner ear failed to form due to increased cell death and
reduced cell proliferation in the otic epithelium. Because we previously
reported that Six1 expression in the otic vesicle is Eya1
dependent, we first clarified that Eya1 expression was unaffected in
Six1-/- otic vesicle, further demonstrating that the
Drosophila Eya-Six regulatory cassette is evolutionarily conserved
during mammalian inner ear development. We also analyzed several other otic
markers and found that the expression of Pax2 and Pax8 was
unaffected in Six1-/- otic vesicle. By contrast,
Six1 is required for the activation of Fgf3 expression and
the maintenance of Fgf10 and Bmp4 expression in the otic
vesicle. Furthermore, loss of Six1 function alters the expression
pattern of Nkx5.1 and Gata3, indicating that Six1
is required for regional specification of the otic vesicle. Finally, our data
suggest that the interaction between Eya1 and Six1 is
crucial for the morphogenesis of the cochlea and the posterior ampulla during
inner ear development. These analyses establish a role for Six1 in
early growth and patterning of the otic vesicle.
Key words: Six1, Auditory system, Inner ear, Regional specification, Mouse, Eya1, Pax2, Fgf3, Fgf10, Bmp4, Nkx5.1, Gata3
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INTRODUCTION
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The mammalian auditory system includes three distinct parts: the outer, the
middle and the inner ears. Despite the complexity and multiple functions, the
ear forms one anatomical unit that serves both hearing and equilibrium. The
earliest morphological evidence for inner ear development is the otic placode,
a thickened area of surface ectoderm on each side of the hindbrain. The otic
placode invaginates to form the otic cup and vesicle, which subsequently
undergoes proliferative growth and eventually differentiates into different
regions of the inner ear. In the mouse, the basic architecture of the inner
ear is fully established by E14.5 (Morsli
et al., 1998
).
A large number of otic genes, including transcription factors, secreted
factors, receptors, cell adhesion proteins and others have been described;
however, functional importance in early morphogenetic processes has only been
demonstrated for some genes (Fekete and Wu,
2002
). The homeobox-containing genes such as the NK-related
homeobox gene Nkx5.1 and the paired-box gene Pax2 are
expressed in complementary patterns in the otic vesicle, with Nkx5.1
dorsolaterally and Pax2 ventromedially
(Herbrand et al., 1998
).
Mutation in the Nkx5.1 gene results in agenesis of the semicircular
canals and circling behavior (Hadrys et
al., 1998
), while mutation in the Pax2 gene leads to
agenesis of the cochlea (Torres et al.,
1996
). The GATA family zinc-finger gene Gata3 shows
reciprocal relationships with Pax2 in the regional patterning of the
early otocyst and cellular patterning within the sensory epithelia and ears of
Gata3-null mouse mutants remain cystic, with a single extension of
the endolymphatic duct (Karis et al.,
2001
; Lawoko-Kerali et al.,
2002
). The eyes absent gene Eya1, which encodes a
transcription coactivator, is also expressed early in the otic epithelium and
the inner ear development in Eya1 knockout mice arrests at the otic
vesicle stage (Xu et al.,
1999a
). This is the first described mouse mutant lacking all
sensory areas of the inner ear. Secreted factors like the Bmp-family of
Tgfß-like polypeptides, Fgfs and receptor molecules like the Fgfr2 IIIb
and Fgfr1 are also expressed in the otic epithelium and serve as signaling
molecules in early otic development (Chang
et al., 1999
; Ohuchi et al.,
2000
; Pirvola et al., 2000; Noramly and Grainger, 2002;
Pirvola et al., 2002
).
Nonetheless, it is largely unknown how these genes function and respond to the
inductive signals from neighboring tissues in the morphogenetic processes of
inner ear development.
The murine homeobox Six gene family has been identified on the basis of
sequence homology with the Drosophila sine oculis (so) gene.
At present, six members (Six1-Six6) of the Six gene family have been
isolated and they are suggested to interact with Pax and Eya genes based on
their wide co-expression in many tissues during mammalian organogenesis and
development (Oliver et al.,
1995a
; Oliver et al.,
1995b
; Kawakami et al.,
1996
; Chen et al.,
1997
; Pignoni et al.,
1997
; Xu et al.,
1997a
; Xu et al.,
1997b
). However, their functional roles during mammalian inner ear
development have not been studied. In this study, we analyzed the expression
of Six1 during inner ear development and its role in mouse auditory
system development. In the developing inner ear, Six1 is expressed in
all sensory epithelia. Inactivation of the Six1 gene led to
malformation of the auditory system involving the outer, middle and inner
ears. The inner ear development in Six1-/- embryos
arrested at the otic vesicle stage and all components of the inner ear failed
to form because of increased cell death and reduced cell proliferation in the
otic epithelium. Molecularly, Six1 is not required for the expression
of Eya1, Pax2 and Pax8 in the otic epithelium. By contrast,
Six1 is required for the normal expression of Fgf3, Fgf10, Bmp4,
Gata3 and Nkx5.1 in the otic vesicle, indicating that
Six1 is required for the regional specification of the otic vesicle.
Finally, we provide evidence for a genetic interaction between Eya1
and Six1 during inner ear development. These analyses indicate that
similar to Eya1, Six1 is not required for the initiation of otic
placode morphogenesis to form otic vesicle, but is required for the normal
growth and regional specification of the otic vesicle.
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MATERIALS AND METHODS
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Animals and genotyping
Eya1/Six1 double heterozygous mice were generated by crossing mice
carrying mutant alleles of Eya1 and Six1
(Six1lacZ) and genotyping of mice and embryos was
performed as previously described (Torres
et al., 1995
; Xu et al.,
1999a
; Xu et al.,
2002
; Laclef et al.,
2003
).
ABR testing and ear morphologic analyses
We used a computer-assisted evoked potential system to obtain ABR
thresholds for tone pips at 5, 8, 11, 16, 22, 32 and 45 kHz (tone pip duration
5 mseconds); repetition rate 30/secons and averaged responses to 512 pips of
alternating polarity.
Adult ears were sectioned after paraffin wax embedding (8 µm) for
morphological analysis as described (Xu et
al., 1999a
). We examined 10 heterozygotes in both 129/Sv and
C57BL/6J backgrounds and compared them with sections from five 129/Sv and
three C57BL/6J wild-type mice.
The latex paintfilling of the ears at E16.5 and 17.5 was performed as
described (Morsli et al.,
1998
). The paintfilled inner ears were dissected out and
photographed.
Phenotype analyses and in situ hybridization
Embryos for histology and in situ hybridization were dissected out in PBS
and fixed with 4% paraformaldehyde (PFA) at 4°C overnight. Embryonic
membranes were saved in DNA isolation buffer for genotyping. Histology was
performed as described (Xu et al.,
1999a
). To visualize Six1lacZ expression,
mutant embryos were stained with X-gal and sectioned as described
(Xu et al., 2002
). To reveal
the middle ear ossicles, we performed skeletal staining of cartilage and bone
as described (Peters et al.,
1998
).
For in situ hybridization, we used four wild-type or mutant embryos at each
stage for each probe as described (Xu et
al., 1997a
).
TUNEL assay and BrdU labeling
TUNEL assay was performed as described
(Xu et al., 1999a
). To label
the proliferating cells, timed pregnant mice at E8.5 and 9.5 were injected
i.p. twice at 2-hour intervals with 5-bromodeoxyuridine (BrdU, Sigma) and
embryos were collected as described (Xu et
al., 1999b
). Paraffin wax embedded sections of 6 µm were
prepared and denatured with 4N HCl for 1 hour at 37°C. Mouse anti-BrdU
monoclonal antibody and goat anti-mouse IgG coupled with HRP or Cy3 were used
for detection. The number of proliferating cells was counted in serial
sections from each otic placode or vesicle, and at least five embryos (10
ears) of each genotype were counted.
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RESULTS
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Six1 expression in the developing inner ear
As the inserted lacZ transgene displayed an expression pattern
identical to the Six1 mRNA distribution obtained by in situ
hybridization, we analyzed the expression of Six1 gene during inner
ear development in Six1lacZ heterozygotes by staining for
ß-galactosidase activity (Fig.
1). During inner ear development, Six1 expression was
first detected in the ventral region of the otic pit at around E8.75
(Fig. 1A). Its expression
domain expands as otic development proceeds and by E9.5, Six1
expression became restricted to the middle and ventral otic vesicle within
which the vestibular and auditory epithelia form respectively
(Fig. 1B,C). By contrast,
Six1 expression is excluded from the dorsal region within which the
semicircular canals form (arrows, Fig.
1A-C). Six1 is also expressed in the vestibuloacoustic
ganglion (gVIII, Fig. 1D).
During subsequent stages of inner ear development, Six1 is expressed
in all sensory epithelia. In the vestibule, Six1 is expressed in the
developing cristae, saccule and utricle
(Fig. 1D-F). In the utricle and
saccule, Six1 is expressed throughout the neuroepithelium at E12.5
(Fig. 1D and data not shown)
and by E15.5 its expression appears to be restricted to the hair cells in the
middle of the epithelium (Fig.
1F). In the cochlea, Six1 is expressed throughout the
future greater and lesser epithelial ridge (GER and LER) of the cochlear duct
at E12.5 (Fig. 1G); however,
its expression became weaker in a region within which the organ of Corti
begins to differentiate at this stage (bracket,
Fig. 1G). In mice, the
progenitors of hair and supporting cells in the primordial organ of Corti
become postmitotic between E12.5 and E14.5
(Ruben, 1967
). At E14.5-E15.5
when the cochlear duct has made 1.5 turns, hair and supporting cell
differentiation initiates in the mid-basal region of the cochlea and hair cell
differentiation proceeds until the entire length of the sensory epithelium is
patterned into one inner row and three outer rows of hair cells at E17.5-E18.5
(Sher, 1971
;
Lim and Anniko, 1985
;
Chen et al., 2002
). In the apex
of E15.5 cochlea, Six1 expression was weakly detected in the
supporting cells and started to appear in the inner hair cell (arrowhead,
Fig. 1H). By contrast, strong
Six1 expression was observed in the GER and LER flanking the
developing organ of Corti. In the basal cochlea where the development of the
organ of Corti is more advanced than in the apex, Six1 is expressed
in the outer and inner hair cells (arrows and arrowhead,
Fig. 1I), but was undetectable
in the supporting cells beneath the hair cells. Strong Six1
expression was maintained in some cells in the GER. In addition, Six1
is expressed in the region from which the stria normally develops. Therefore
in the developing organ of Corti, Six1 is initially expressed in the
progenitors of hair and supporting cells but its expression disappears when
the progenitor cells exit cell cycle and later on it is expressed in the
terminally differentiated hair cells. Taken together, our data show that
Six1 is predominantly expressed in all sensory regions of the inner
ear, suggesting a role for Six1 in the morphogenesis of sensory
organs during mammalian inner ear development.

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Fig. 1. Six1 expression during inner ear development. E8.75 to 15.5
Six1lacZ heterozygous embryos or inner ears were stained
with X-gal for Six1lacZ and sectioned through the inner
ear region. (A) A transverse section showing Six1 expression in the
otic pit (op) at E8.75. (B) A transverse section showing Six1 expression in
the otic cup (oc) at E9.0. (C) A transverse section showing Six1
expression in the middle and ventral otic vesicle (ov) at E9.5. Note
Six1 is excluded from the dorsal otic pit and vesicle (arrows). (D) A
transverse section at E12.5 showing Six1 expression in the saccular
region and the vestibuloacoustic ganglion (gVIII). For A-D, dorsal is upwards.
(E) A section showing Six1 expression in the primordia of lateral
(la) and anterior (aa) Crista ampullaris. Arrow indicates the origin of the
anterior ampulla on the other side. (F) A section showing Six1
expression in the hair cells (hc) of the utricle (u). (G) In the cochlea,
Six1 is expressed throughout the future greater and lesser epithelial
ridge (GER and LER). Note its expression level is reduced in an area that will
become the organ of Corti (bracket) at E12.5. (H) In E15.5 cochlea,
Six1 is expressed weakly in the supporting cells and first appeared
in the inner hair cell (arrowhead) in the apex. Strong Six1
expression was also observed in the cells flanking the developing organ of
Corti in the GER and LER. (I) In the base of E15.5 cochlea, Six1 is
expressed in the outer (arrow) and inner (arrowhead) hair cells. It is also
expressed in some cells in the GER. In addition, it is expressed in the
thinner part of the cochlea duct that will probably differentiate into the
stria vascularis (sv). (J) A latex paintfilled E15.5 cochlea showing the
apical and basal regions. Scale bars: 50 µm.
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Six1+/- mice show a conductive hearing loss
Most of the 129 Six1 heterozygous mice had certain degree of
hearing loss, as determined by auditory-evoked brainstem response (ABR)
threshold measurements (n=26; Fig.
2A). Of the 13 Six1+/- mice analyzed, eight
had hearing loss in both ears, two had hearing loss in one ear and three
showed mild hearing loss in both ears. Similar observation was obtained in
C57BL/6J background.

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Fig. 2. ABR threshold measurements and pathological structures in
Six1+/- adult mouse ears. (A) Average
threshold±s.e.m. for wild-type and Six1+/- ears in
backgrounds 129/Sv and C57BL/6J (129/Sv, n=6 wild type, 13
Six1+/-; C57BL/6J, n=6 wild type, 10
Six1+/-). Averages used 90 dB SPL for thresholds beyond
the upper limit of the sound system. The hearing loss was variable among mice
and ears. Of the 13 Six1+/- mice tested, eight mice had
severe hearing loss in both ears (threshold shifted by 50 dB between 15 and 32
kHz), two mice had mild hearing loss in both ears (threshold shifted by 20
dB), whereas three mice had normal hearing in the right ears and >70 dB
loss in the left ears. Broken lines, C57BL/6J strain; unbroken lines, 129/Sv
strain. (B-G) Histological analysis in wild type and
Six1+/- ears. (B) Wild-type ear showing the stapes seated
in the oval window (not visible because apposed by the stapes footplate, sf),
and one arch of the stapes (s). The stapedial artery (a) is normally
positioned. Part of the long process of the malleus (lp) is also present in
this section. nVII, the VIIth cranial nerve. (C,D) Six1+/-
ears showing that the footplates of the stapes are seated in the oval window.
However, the VIIth nerve passed abnormally close to the oval window and the
stapes or filled up the middle ear space near the oval window (arrows). Part
of the stapes arches and the long process of the malleus are present in these
sections. (E) Wild-type superior region of the middle ear space showing the
junction of the malleus (ma) and incus (in). tm, tympanic membrane. (F,G)
Six1+/- superior region of the middle ears showing the
junction of the malleus and incus. The middle ear space was small and
partially filled with loose connective tissue (arrow). The tympanic membrane
was also abnormal (arrowheads). ABR threshold from these ears demonstrated a
severe hearing loss. Scale bars: 100 µm.
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To determine whether the hearing loss is conductive, sensorineural or both,
we sectioned the Six1+/- ears associated with hearing
loss. All Six1+/- ears with hearing losses revealed middle
ear abnormalities: typically, a failure of the ossicles to complete a sound
transmission path from the tympanum to the oval window. Although the stapes
attached to oval window, the VIIth cranial nerve passed abnormally between the
oval window and stapedial artery or over the surface of the cochlea under the
stapedial artery (Fig. 2C,D and
data not shown). The middle ears also showed morphologically abnormal
ossicles, including stapes with a small lumen and the middle ear space was
filled up with loose connective tissues (arrow,
Fig. 2F and data not shown).
The latter could be due to secondary inflammation. The middle ear space
appeared to be small (Fig.
2C,D,F,G) and the tympanic membrane was also abnormal (arrowheads,
Fig. 2F,G). In four cases from
3 animals, there were no stapedial arteries (data not shown). In the inner
ear, the cochlear spiral of all heterozygotes was well formed, although four
out of 22 Six1+/- ears (three out of 11 embryos) showed
slightly shortened cochlea by latex paintfilling at E16.5 (see
Fig. 8 and
Table 1). Nonetheless, our
analyses clearly show that there is a failure of sound transmission in the
middle ear.

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Fig. 8. Enhancement of inner ear defects in Eya1/Six1 double mutants.
(A-D) Medial (A-C) and lateral (D) views of paintfilled inner ears of
wild-type (A), Six1+/- (B), and
Six1+/-/Eya1+/- compound heterozygous
(C,D) embryos at E16.5. (A) All structures of the inner ear reached their
mature shape at E16.5. The cochlea completed 1.75 turns by this stage (inset).
aa, anterior ampulla; asc, anterior semicircular canal; cc, common crus; co,
cochlea; csd, cochleosaccular duct; ed, endolymphatic duct; es, endolymphatic
sac; la, lateral ampulla; lsc, lateral semicircular canal; pa, posterior
ampulla; psc, posterior semicircular canal; s, saccule; u, utricle; usd,
utriculosaccular duct. (B) A Six1+/- inner ear showing a
malformed saccule (asterisk), absence of the endolymphatic sac and a truncated
endolymphatic duct (arrowhead). The cochlea only completed 1.5 turns (arrow
and inset). (C,D) Inner ears from
Six1+/-/Eya1+/- double heterozygotes
showing severe cochlear defects (arrows), absence of posterior ampulla
(asterisk) and truncated posterior semicircular canal. The cochlea only
completed less than one turn and their distal tips were enlarged and
mal-shaped (insets). (E-H) Frontal histological sections at comparable levels
of X-gal-stained E9.5 embryos of Eya1/Six1 double heterozygous (E)
and double homozygous embryos (F-H). Eya1/Six1 double heterozygote
shows restricted Six1lacZ expression in the otic vesicle
(ov). In the double homozygotes, the otic vesicles appeared to be formed in
the correct position but severely hypoplastic. Because
Six1lacZ is Eya1 dependent, it is not expressed
in Eya1-/-/Six1-/- otic vesicle
(arrows) but is ectopically turned on in rhombomeres 2, 4 and 6 (r4 and r6).
Scale bars: 100 µm.
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Six1 is required for normal growth of the otic vesicle
Auditory system abnormalities in Six1 homozygotes involve the
outer, middle and inner ears (Fig.
3). The outer ear revealed malformed auricles, preauricular pits
and malformed eardrums (Fig.
3A,B and data not shown). In the middle ear, the incus was present
but malformed or fused with the malleus (arrowheads,
Fig. 3D). The short process of
the malleus was typically absent (arrow,
Fig. 3D), as was the stapes,
while the tympanic cavity is present in Six1 homozygotes (data not
shown). In the inner ear, although the otic vesicle forms, it appears to be
smaller and abnormal at E10.5 (arrow, Fig.
3F). By E12.5, no inner ear structure or sometimes only severely
malformed vestibule-like structure was observed in Six1-/-
embryos (arrow, Fig. 3H). The
malformed vestibule-like structure observed in some
Six1-/- embryos at E12.5 also failed to develop further
(data not shown). In addition, the vestibuloacoustic (gVIII) and petrosal
(gIX) ganglia were absent (asterisk and arrowhead,
Fig. 3H). Thus, Six1
plays a direct role in the normal development of the mammalian auditory
system.

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Fig. 3. Auditory system development in Six1 homozygotes. (A,B) All
Six1 homozygotes die at birth and exhibit severe auditory system
defects involving the outer (arrow), middle and inner ears, as well as other
defects. (C,D) Microdissected middle ear ossicles from E18.5 wild-type and
Six1-/- embryos. In the mutant, the incus (in) is
malformed and fused with the malleus (ma) (arrowheads) and the stapes (st) is
absent. The short process (sp) of the malleus is often missing (arrow) and the
long process (lp) is also shortened. (E,F) Transverse sections of E10.5
Six1 heterozygous and homozygous embryos stained with Hematoxylin and
Eosin showing the developing otic vesicle (ov) and the vestibuloacoustic
ganglion (gVIII). In Six1-/- embryos, although the otic
vesicle formed, it appeared much smaller and abnormal (arrow) and the gVIII is
absent (arrowhead). (G,H) Transverse sections of E12.5 wild-type and
Six1 mutant embryos stained with X-gal for
Six1lacZ and counterstained with diluted Hematoxylin
showing the developing inner ear, Six1lacZ expression in
the utricle and saccule region, semicircular canals, cranial ganglia gIX,
gVIII and gV in the heterozygotes. However, in Six1-/-
embryos, only malformed semicircular canal-like structure was observed
(arrow). Other inner ear structures are not formed and gIX (arrowhead) and
gVIII (asterisk) are absent in the homozygotes. (I,J) TUNEL analysis of
transverse sections through the ear region of Six1+/- and
Six1-/- embryos at E9.5. Numerous apoptotic cells are only
detected in the lateral wall of Six1-/- otic vesicle
(arrow). Scale bars: 100 µm.
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The failure of inner ear development in Six1-/- embryos
was associated with an increased cell death as detected by TUNEL analysis.
Although apparent morphological difference of the otic vesicle was not
observed between Six1-/- and wild-type embryos at E9.5
(data not shown), numerous apoptotic cells in the lateral wall of
Six1-/- otic vesicle were detected (arrow,
Fig. 3J). At E10.5, apoptotic
cells were also increased in the medial region of Six1-/-
otic vesicle (data not shown). Thus, Six1 is required for otic
epithelial cell survival.
We next tested whether Six1-/- otic epithelial cells
proliferate appropriately by assaying BrdU incorporation in the mutant otic
placode and vesicle at E8.5 and 9.5, before apparent morphological alteration
was seen in Six1-/- embryos. Four hours after BrdU
injection, BrdU-labeled cells were seen throughout the otic placode in
wild-type embryos (Fig. 4A).
However, in Six1-/- embryos, the number of BrdU-labeled
cells was reduced in the otic placode (arrowhead,
Fig. 4B). By E9.5,
BrdU-positive cells were largely reduced in the dorsal half of
Six1-/- otic vesicle (above arrowheads,
Fig. 4D). Using an image
analysis system, we next counted the number of BrdU-positive cells from 10
wild-type and 10 Six1-/- ears at each stage on serial
sections to determine the labeling index
(Fig. 4E). At E8.5, the number
of BrdU-positive cells in Six1-/- otic placode was 80% of
wild-type embryos (Fig. 4E). By
E9.5, the number of BrdU-positive cells in Six1-/- otic
vesicle was reduced to 50% of that in wild-type embryos
(Fig. 4E). As the epithelial
cells in the lateral wall of Six1-/- otic vesicle undergo
abnormal apoptosis from E9.5 (Fig.
3J), to further clarify whether the reduction of cell
proliferation in E9.5 Six1-/- otic vesicle is due to
abnormal cell death, we determined the labeling index from the lateral and
medial half of the otic vesicle, respectively. In the lateral half, the number
of BrdU-labeled cells in Six1-/- otic vesicles was 60% of
that in wild-type embryos (E9.5L, Fig.
4E). Similarly, in the medial half of the otic vesicle, although
no abnormal apoptosis was observed in Six1-/- embryos at
E9.5, the number of BrdU-labeled cells was reduced to 40% of that seen in
wild-type embryos (E9.5M, Fig.
4E). Thus, Six1 is required for normal growth of the otic
vesicle by regulating cell proliferation during early otic development.

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Fig. 4. Six1 controls proliferation of otic epithelial cells during early
inner ear development. (A-D) Transverse sections of otic regions from E8.5
(A,B) and E9.5 (C,D) wild-type and Six1-/- embryos showing
BrdU-labeled cells (orange). BrdU-positive cells were slightly reduced in
Six1-/- otic placode (OP) at E8.5 (B) and by E9.5,
BrdU-positive cells were largely reduced in the dorsal region of
Six1-/- otic vesicle (OV, above arrowheads in D). (E) The
labeling index was determined by counting the number of BrdU-positive cells
from each otic placode or vesicle. Ten ears for each genotype were counted and
the numbers were averaged. At E8.5, the number of BrdU-labeled cells in
Six1-/- otic placode was 80% of wild-type embryos. By
E9.5, the number of BrdU-labeled cells in Six1-/- otic
vesicle was reduced to 50% of that in wild-type embryos. In
Six1-/- embryos, the number of BrdU-positive cells in the
lateral otic vesicle was reduced to 60% (E9.5L), while in the medial half the
number was reduced to 40% of that in wild-type embryos (E9.5M). Scale bars: 50
µm.
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Eya1, Pax2 and Pax8 expression does not
require Six1 function during early otic development
To determine the molecular defects in early otic development of
Six1-/- animals, we first examined whether the expression
of the Eya and Pax gene families depends upon Six1. Studies in
Drosophila indicate that eya is epistatic to so and
both genes reside within the same genetic and molecular pathway downstream of
the Pax6 gene ey (Halder
et al., 1998
). As we previously found that Six1 mRNA
expression was undetectable in Eya1-/- embryos
(Xu et al., 1999a
), we first
analyzed the Six1lacZ expression by X-gal staining to
further confirm this observation. Six1lacZ expression was
also undetectable in Eya1-/- otic epithelium, further
demonstrating that Six1 expression in the otic epithelium is
Eya1 dependent (Fig.
5A,B). We next analyzed the expression of Eya1 in
Six1-/- embryos to further clarify their regulatory
relationship during early otic development. Eya1 is normally
co-expressed with Six1 in the otic epithelium and its expression was
unaffected in Six1-/- otic vesicles at E9.5 and E10.5
(Fig. 5C,D and data not shown).
This further confirms that Eya1 functions upstream of Six1
and that the Eya-Six regulatory pathway elucidated in Drosophila eye
imaginal disc is evolutionarily conserved in early mammalian otic development.
Because we previously found that the expression of both Pax2 and
Pax8 was unaffected in Eya1-/- otic epithelium
(Xu et al., 1999a
), we next
examined whether the Pax gene expression in the otic epithelium is also
Six1 independent. In Six1-/- embryos,
Pax2 and Pax8 are expressed in the otic placode and vesicle
at normal levels at E8.5-E10.5 (Fig.
5E-H and data not shown). This indicates that similar to Eya1,
Six1 is also not required for the expression of both Pax2 and
Pax8 in the otic epithelium.

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Fig. 5. Eya1, Pax2 and Pax8 expression in otic epithelium is
Six1-independent. (A,B) Six1lacZ is normally
expressed in otic vesicle (ov) and its expression was undetectable in
Eya1-/- embryos. (C-H) Eya1 (C,D), Pax2
(E,F) and Pax8 (G,H) expression levels in otic vesicle were
unaffected in Six1-/- embryos. Scale bars: 100 µm. nt,
neural tube.
|
|
Six1 is required for the activation of Fgf3
expression and the maintenance of Fgf10 and Bmp4 expression
in the otic vesicle
We next examined the expression of several other well-characterized
molecular markers in the otic epithelium at E8.5-10.5. Fgf3, a member
of the Fgf superfamily of secreted signals, begins to be expressed in
the ventrolateral wall of the otic vesicle at E9.5 and in the delaminating
neuroblasts and gVIII (Fig. 6A
and data not shown). Inactivation of Fgf3 results in inner ear
defects and its expression in the otic vesicle is Eya1 dependent
(Mansour et al., 1993
;
Xu et al., 1999a
). Similarly,
Fgf3 expression was undetectable in Six1-/- otic
vesicle from E9.5 (Fig. 6B). By
contrast, its expression in the hindbrain was relatively normal in
Six1-/- embryos (Fig.
6A,B). Fgf10, another member of the Fgf superfamily, is
expressed in the otic placode and vesicle and by E10.5, its expression became
concentrated to a broad region in the ventral half, a small patch in the
posterodorsal wall and in the neuroblast cells and gVIII
(Fig. 6C) (Pirvola et al.,
2000). The expression of both Fgf3 and Fgf10 in the
ventrolateral wall of the otic vesicle and in the neuroblasts and gVIII
overlaps and both genes are suggested to function through their receptor
Fgfr2 IIIb (Pauley et al.,
2003
). When both are knocked out, the otic vesicle fails to form
(Wright and Mansour, 2003
). In
Six1-/- embryos, Fgf10 expression was unaffected
in the otic placode (data not shown). However by E10.5, residual
Fgf10 expression was only observed in the ventromedial wall of
Six1-/- otic vesicle (arrow,
Fig. 6D). The expression of
both Fgf3 and Fgf10 was undetectable in the gVIII in
Six1-/- embryos, further confirming the absence of this
structure in Six1 homozygotes. Bmp4, a member of the
Tgfß superfamily, is expressed in a broad region of the lateral otic
vesicle at E9.5 and by E10.5, its expression is restricted to two patches, one
in the dorsal and the other in the lateral region of the otic vesicle which
mark the sensory anlagen of the cristae
(Fig. 6E)
(Wu and Oh, 1996
). In
Six1-/- embryos, Bmp4 was expressed in the otic
vesicle at E9.5 but its expression significantly diminished at E10.5 with the
dorsal expression domain disappeared and the lateral domain weakened greatly
(arrowhead and arrow, Fig. 6F).
Alteration of Fgf3, Fgf10 and Bmp4 expression in
Six1-/- embryos could indicate that Six1
regulates the transcription of these genes or that Six1 is required
for the specification of the cells that express these genes or both.
Nonetheless, these results indicate that Six1 is required for the
normal expression of Fgf3, Fgf10 and Bmp4 in the otic
vesicle.

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Fig. 6. Loss of Six1 function alters the expression of Fgf3, Fgf10,
Bmp4, Nkx5.1 and Gata3 in the otic vesicle. (A-D,G-J) Transverse
sections; (E,F) Horizontal sections. (A,B) Fgf3 begins to be
expressed in the ventrolateral region of the otic vesicle (ov) at E9.5 and its
expression was undetectable in Six1-/- embryos. Note
Fgf3 expression in the hindbrain (hb) is relatively normal in
Six1-/- embryos. (C,D) Fgf10 is normally
expressed in a broad region of ventral otic vesicle and the gVIII in wild-type
embryos at E10.5; however, residual expression of Fgf10 was only
detected in the ventromedial otic vesicle in Six1-/-
embryos (arrow). (E,F) Bmp4 is normally observed in two specific
spots, one dorsal and one ventrolateral in the otic vesicle of wild-type
embryos at E10.5. In Six1-/- embryos, the dorsal
expression spot (arrowhead) was undetectable and the ventrolateral spot became
very weak (arrow). (G,H) Nkx5.1 is expressed in the dorsolateral
region of the otic vesicle in wild-type embryos at E10.5. In
Six1-/- embryos, however, Nkx5.1 expression is
excluded from the dorsolateral region (arrow) and its expression domain
shifted or expanded ventrally, including the ventral-most region. (I,J)
Gata3 is expressed strongly dorsolaterally and weakly ventromedially
in the wild-type embryos at E10.5. Similarly, in Six1-/-
embryos it is not expressed in the dorsolateral region (arrow) and its
expression domain shifted ventrally. By contrast, its expression in the
ventromedial region of the otic vesicle was unaffected in
Six1-/- embryos. Scale bars: 100 µm.
|
|
Loss of Six1 function alters the expression profile of
Nkx5.1 and Gata3 in the otic vesicle
During mouse inner ear development, the semicircular canals form from the
dorsal region of the otic vesicle, the vestibular epithelia form from the
middle region and the auditory epithelia form from the ventral region of the
otic vesicle, respectively (Li et al.,
1978
). Because only severely affected semicircular canal-like
structure was observed in some Six1 homozygotes at E12.5, we further
analyzed additional markers that are well characterized and localized along
the dorsoventral axis of the otic vesicle. Nkx5.1 is expressed early
in the otic placode and its expression is restricted to the dorsolateral otic
vesicle, which will give rise to the vestibular apparatus of the inner ear
(Fig. 6G)
(Hadrys et al., 1998
;
Wang et al., 1998
).
Inactivation of Nkx5.1 leads to agenesis of the semicircular canals
and circling behavior (Hadrys et al.,
1998
). In Six1-/- embryos, Nkx5.1
expression was normal in the otic placode at E8.5 (data not shown). However in
the otic vesicle, Nkx5.1 expression was excluded from the
dorsolateral region at E10.5 (arrow, Fig.
6H) and its expression shifted or expanded ventrally, including
the ventral-most wall (Fig.
6G,H). Gata3 is normally expressed throughout the otic
placode and by E10.5 its expression is restricted to two regions in the otic
vesicle, strongly in the dorsolateral region and weakly in the ventromedial
region (Fig. 6I)
(Lawoko-Kerali et al., 2002
).
In Six1-/- embryos, no significant difference of
Gata3 expression was detected by E9.5 (data not shown). However
similar to Nkx5.1, Gata3 expression in the dorsolateral region was
shifted ventrally in Six1-/- otic vesicle at E10.5
(Fig. 6I,J). By contrast, its
expression in the medial region was unaffected in Six1-/-
otic vesicle (Fig. 6I,J). The
failure to express Nkx5.1 and Gata3 in the dorsolateral
region of the otic vesicle lacking Six1 is unlikely to be due only to
abnormal cell death, as apoptotic cells were detected throughout the lateral
region at E9.5 (Fig. 3J). Although it is unclear whether the ventrolateral region of
Six1-/- otic vesicle expressing Nkx5.1 and
Gata3 will give rise to the vestibule-like structure observed in some
Six1-/- embryos at E12.5, these data indicate that
Six1 regulates the establishment of regional specification of the
otic vesicle.
Six1 and Eya1 expression in otic vesicle is
Pax2 independent
Because the Pax2 mutant inner ear phenotype is less severe than
that seen in Eya1-/- or Six1-/- mice
(Torres et al., 1996
), it is
unclear whether Pax genes function in the same genetic pathway with
Eya1 and Six1. To further clarify their genetic
relationships during early inner ear development, we next examined the
expression of Eya1 and Six1 in Pax2-/-
embryos. Surprisingly, the expression of both Eya1 and Six1
was unaffected in the otic vesicle and its derivative gVIII of
Pax2-/- embryos (Fig.
7A-D and data not shown), indicating that Six1 and
Eya1 expression in the otic epithelium does not require Pax2
function. Taken together, our results suggest that Pax2 may function
independently or in parallel with Eya1 and Six1 during early
mammalian otic development.

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Fig. 7. Six1 and Eya1 expression in the otic vesicle is
Pax2 independent. (A-D) Eya1 (A,B) and Six1 (C,D)
are expressed in the ventral otic vesicle (ov) and its derivative gVIII of
wild-type embryos at E10.5 and their expression was unaffected in these
structures in Pax2-/- embryos. Scale bars: 100 µm.
|
|
Eya1 and Six1 interact during mammalian inner ear
morphogenesis
As Eya1 and Six1 genes function in the same genetic
pathway in early otic development and both gene products physically interact
in vitro and in cultured cells (Buller et
al., 2001
), we further tested whether these two genes interact in
a molecular pathway during mammalian inner ear morphogenesis by examining the
inner ear gross structure of
Six1+/-/Eya1+/- compound heterozygotes
using latex paintfilling (Table
1 and Fig. 8). At
E16.5, the membranous labyrinth developed to its mature shape and the cochlea
reached 1.75 turns (Morsli et al.,
1998
). The inner ear phenotypes in each single or double
heterozygous mice were variable (Table
1). Each single heterozygote alone on 129 background revealed
malformed endolymphatic duct and sac and
18% of
Six1+/- or Eya1+/- ears exhibited
slightly shortened cochlea (arrowhead and arrow,
Fig. 8B; data not shown). Some
Six1+/- ears also revealed a small or mis-shaped saccule
(asterisk, Fig. 8B). By
contrast, 19 out of 40 (47.5%) Eya1 and Six1 compound
heterozygous ears revealed more severely affected cochlea
(Table 1). The severe phenotype
showed a coiled but abnormal cochlea, which only completed less than 1 turn at
E16.5 with abnormally shaped distal tips (insets,
Fig. 8C,D). In addition, seven
out of 40 (17.5%) Eya1/Six1 double heterozygous ears showed a missing
posterior ampulla and a truncation or complete absence of the posterior
semicircular canal (asterisk, Fig.
8D). This defect was not seen in each single heterozygote on 129
background (Table 1). No
enhancement of the defects of the endolymphatic duct and sac and the saccule
was observed in the compound heterozygous inner ears
(Table 1). Similar observation
was obtained in C57BL/6J background (data not shown). In summary, these data
suggest that Eya1 and Six1 genetically interact during inner
ear morphogenesis and this interaction is crucial for the normal morphogenesis
of the cochlea and the posterior ampulla.
We next analyzed early otic development in Eya1/Six1 double
homozygous embryos (Fig. 8E-H).
At E9.5, the otic vesicle in
Eya1+/-/Six1+/- double heterozygotes
was well formed at rhombomere 5 (r5) level and showed a restricted expression
of Six1lacZ (Fig.
8E). In the double homozygotes, the otic vesicles appeared to be
formed in the correct place at r5 level but were severely hypoplastic
(Fig. 8F-H). Because
Six1lacZ expression in the otic vesicle is Eya1
dependent (Fig. 5A,B), the otic
vesicle of Eya1/Six1 double homozygous embryos lacked the
Six1lacZ expression (arrows,
Fig. 8F-H). Although the neural
tube of the hindbrain was significantly reduced in size in
Eya1-/-/Six1-/- embryos, it appeared
to be patterned correctly and Six1lacZ expression was
ectopically turned on in r2, r4 and r6
(Fig. 8F-H). These data further
indicate that both Eya1 and Six1 are not required for the
initiation of otic placode morphogenesis to form the otic vesicle, but are
required for the normal growth of the otic vesicle.
 |
DISCUSSION
|
---|
Six1 regulates normal growth of the otic epithelium
Inner ear development begins with the induction of the otic placode. Once
the otic placode initiates to form the otic vesicle, extensive morphogenetic
processes and cellular events, such as differentiation, proliferation and
apoptosis, take place to ensure the formation of the highly organized
structures of the adult inner ear. Six1 expression is turned on in
the invaginating otic placode at around E8.75 and in the absence of
Six1, the otic vesicle formed without an apparent morphological
alteration at E9.5. Thus, Six1 is unlikely to be involved directly in
either the induction of the otic placode or the initiation of the otic placode
morphogenesis to form the otic vesicle. This conclusion was further
strengthened by the fact that the otic vesicle also formed in the correct
position in Eya1/Six1 double homozygous embryos. Taken together,
these findings demonstrate that both Eya1 and Six1 are not
required for the initiation of mammalian inner ear organogenesis.
Our studies clearly demonstrate that Six1 controls the inner ear
morphogenesis by regulating the programmed cell death and proliferative growth
of the otic epithelium, directly or indirectly. Secreted diffusable factors
are proposed to play roles in the growth regulation of the inner ear. Of
particular interest, retinoic acid (RA) and the growth factor Bmp4 have been
shown to influence regional patterning and specification of the inner ear,
particularly for the vestibular structures
(Chang et al., 1999
;
Dupe et al., 1999
;
Gerlach et al., 2000
;
Niederreither et al., 2000
;
Pasqualetti et al., 2001
;
Merlo et al., 2002
).
Interestingly, the inner ears of Six1-/- embryos bear some
similarities to the phenotype displayed by mice exposed to excess all-trans RA
(at-RA) or isotretinoin (13cis-RA), or mice lacking Raldh2,
a gene that catalyzes RA formation (Burk
and Willhite, 1992
;
Niederreither et al., 2000
).
In the otic vesicle of both Six1-/- and
Raldh2-/- embryos, Nkx5.1 expression is expanded
ventrally (Niederreither et al.,
2000
). RA is also able to rescue the Hoxa1-/-
inner ear phenotype, including vestibular malformation and a lack of cochlear
duct outgrowth (Pasqualetti et al.,
2001
). Recently, Bmp4 has been suggested to function together with
RA through the same pathway or intersecting pathways, as RA represses
Bmp4 transcription in otocyst cells
(Thompson et al., 2003
).
However, it is unclear how exactly the RA and Bmp4 signaling controls the
patterning of the inner ear. In the present study, we found that the
maintenance of Bmp4 expression in the otic vesicle requires
Six1 function. Therefore, it is possible that Six1 regulates
normal growth of the otic vesicle through the RA-Bmp4-signaling pathway. This
could explain why the morphogenetic defects in Six1-/-
mice are not restricted to the ventral inner ear but extend to the dorsal
inner ear where Six1 is not expressed. Thus, the disruption of Six1
exerts some indirect, nonautomous effects on developing inner ear structures.
Such long-distance influence may also be regulated by the expression of
Six1 in the periotic mesenchyme. It will be interesting to test
whether RA can rescue the Six1-/- inner ear phenotype.
The role of Six1 in the specification of neuroblast
cells
As the otic placode invaginates, a population of otic epithelial cells near
the center of the otic cup and ventral otic vesicle emigrates into the
underlying mesoderm. These cells are neuroblasts for the vestibuloacoustic
ganglion (gVIII) and they are the first cell lineage specified within the otic
epithelium before leaving the otic epithelium. The basic helix-loop-helix
(bHLH) transcription factors neurogenin 1 (Ngn1) and Neurod1 have been shown
to be essential for the formation of gVIII
(Ma et al., 1998
;
Liu et al., 2000
;
Kim et al., 2001
).
Ngn1 was proposed to play a role for the determination of neuroblast
precursor fate (Ma et al.,
1998
), while Neurod1 is required for the delamination of
neuroblasts and for their survival during differentiation process
(Liu et al., 2000
;
Kim et al., 2001
). Recently,
the transcription coactivator Eya1 has been shown to be required for the
formation of the gVIII, as this structure failed to form in
Eya1-/- mice (Xu et
al., 1999a
). The zinc-finger protein Gata3 is expressed in the
neuroblasts and also plays a role in the formation of gVIII
(Karis et al., 2001
). Growth
factors Fgf3 and Fgf10 and their receptor Fgfr2 IIIb also play a role in the
formation of the gVIII, as mutations in each of these genes led to severe
hypomorphic development of the gVIII
(Mansour et al., 1993
; Pirvola
et al., 2000). However, the molecular mechanisms controlling the specification
of neuroblast cell fate are currently not well understood. In the present
work, we found that Six1 is expressed in the ventral otic epithelium
within which the neuroblast precursors are specified and in the gVIII. In the
absence of Six1, the gVIII failed to form, similar to that observed
in Eya1-/- embryos. Thus, Six1 is likely to play
a direct role in the determination of neuroblast cell fate and this cell
lineage may not be specified in the absence of Six1. This hypothesis
was further supported by loss of specific marker expression, including
Fgf3 and Fgf10 in the neuroblasts and gVIII of
Six1-/- embryos (Fig.
6 and data not shown).
As both Eya1 and Six1 are required for the activation of
Fgf3 expression in the otic epithelium and both proteins physically
interact in vitro and in cultured cells
(Buller et al., 2001
), it is
possible that Fgf3 is a common downstream target for both
Eya1 and Six1. Based on these observations, we propose that
both Eya1 and Six1 control the initial selection of
neuroblast precursors by regulating the expression of Ngn1, Neurod1
and Fgf3, directly or indirectly. This hypothesis will be
strengthened if Ngn1 and Neurod1 expression in the otic
epithelium and neuroblasts also requires both Eya1 and Six1
function. Expression studies of Ngn1 and Neurod1 in both
Eya1 and Six1 mutants are under way in our laboratory.
The role of Six1 in the specification of sensory
regions
The commitment of the otocyst to form prospective vestibular and auditory
sensory areas is controlled by patterning genes. However, it is unclear
exactly how these genes are involved in this complicated process. Our studies
show that Six1 is expressed in the middle and ventral otic vesicle
and all middle and ventral derivatives failed to form in the absence of
Six1. To analyze the molecular defects in Six1-/-
otic vesicle, we examined the expression of ventromedial otic markers,
including Pax2, Pax8, the zinc-finger gene Sall1 and
dachshund 1 (Dach1), in Six1-/- embryos and
failed to detect obvious changes in their expression between wild-type and
Six1-/- embryos. In addition, Gata3 expression in
the ventromedial region was also unaffected in Six1-/-
embryos. Further studies are required to establish the molecular mechanisms by
which Six1 acts to regulate the patterning of this region in the
inner ear.
Interestingly, in addition to the loss of ventral cell fates, the absence
of Six1 impacts the positioning of dorsolateral markers, including
Nkx5.1 and Gata3. Although Nkx5.1 appears to be
necessary for the correct expression of Bmp4 in the otocyst and for
regional control of apoptosis and Bmp4 was suggested to have a role
in the specification of sensory organ formation
(Oh et al., 1996
;
Morsli et al., 1998
;
Cole et al., 2000
;
Merlo et al., 2002
), it is
unclear how these genes function together to control the morphogenesis of the
sensory system. We found that Six1 is expressed in all sensory
regions of the inner ear and Six1-/- mice lacked all
sensory organ formation. The expression domain of Bmp4, which marks
the sensory anlage of the posterior crista was lost and its expression level
in the other sensory anlagen was also largely reduced in
Six1-/- embryos. Coincidentally, Eya1 and
Six1 interaction critically affects the morphogenesis of the
posterior ampulla and Bmp4 expression was also lost in
Eya1-/- embryos at E10.5 (data not shown). Therefore, it
is likely that both Eya1 and Six1 regulate the expression of
Bmp4 the dosage of which is crucial for the morphogenesis of the
sensory organs, particularly for the posterior ampulla. Nonetheless, our
results indicate that Six1 is probably an early regulator for the
specification of all sensory organs of the inner ear.
The regulatory relationship between Pax, Eya and Six genes during
mammalian inner ear morphogenesis
In the ear, both Eya1 and Six1 are co-expressed during
mammalian auditory system development and their mutant mice had similar
defects in all three parts of the ear (Xu
et al., 1999a
). Our studies have clearly demonstrated that the
Drosophila Eya-Six regulatory cassette is evolutionarily conserved
during mammalian inner ear development.
In Drosophila eye imaginal disc, the fly Pax6 gene
ey has been shown to function upstream of both eya and
so (Halder et al.,
1998
). In mammalian inner ear, Pax2 expression overlaps
with Eya1 and Six1 in the medial otic vesicle and the inner
ear phenotype in Pax2-/- mice is less severe than that
seen in Eya1-/- or Six1-/- mice
(Torres et al., 1996
).
Pax8, a paralog of Pax2, is also expressed in the otic
placode (Pfeffer et al.,
1998
). Although Pax8 mutants do not exhibit an otic
phenotype (Mansouri et al.,
1998
), Pax2 and Pax8 may function redundantly
during early otic morphogenesis with Pax2 alone executing later
functions. This could explain why the Pax2 mutant phenotype appears
to occur slightly later. If Pax2 and Pax8 function redundantly in early otic
development and a crucial threshold of Pax2/Pax8 protein expression in otic
epithelium regulates Eya1 and Six1 expression, Eya1
and Six1 expression should be reduced or lost in Pax2/Pax8
double homozygotes. We are currently testing this hypothesis by generating
Pax2/Pax8 compound mutants in C3H/He background, as
Pax2/Pax8 compound heterozygous females in either 129 or C57BL/6J
strain had a blind-ending vagina, similar to the recent observation by
Bouchard et al. (Bouchard et al.,
2002
). Alternatively, Pax2 and Pax8 could
function in parallel or independently of Eya1 and Six1 in
early otic morphogenesis, as Eya1 and Six1 expression was
unaffected in Pax2-/- otic vesicle. Evidence obtained from
the analysis of the cochlea phenotype in Eya1/Pax2 compound
heterozygous mice suggests that Eya1 and Pax2 may interact
during cochlear development, because the cochlea phenotype is enhanced in
Eya1/Pax2 compound heterozygotes than in each single heterozygote
(data not shown). It should also be noted that our recent studies indicate
that the genetic and regulatory relationship between Pax, Eya and Six genes
varies between different organs during mammalian development
(Xu et al., 2003
). Probably,
the Pax, Eya and Six genes function in the same or parallel pathway but with
different combinations of regulatory relations in different organs. Detailed
examination of inner ears in Pax2/Pax8, Pax2/Eya1, Pax2/Six1 or
Eya1/Six1/Pax2 compound knockouts will enhance our understanding on
the possible molecular and genetic interactions between these transcription
factors during early mammalian inner ear morphogenesis.
 |
ACKNOWLEDGMENTS
|
---|
We thank P. Gruss for the Pax2 and Pax8 mutant mice, B.
Fritzsch and G. Carlson for helpful comments, and G. Sajithlal and W. Provance
for technical assistance. Photomicroscopy and image analysis was made possible
by equipment purchased with a grant from the M.J. Murdock Charitable Trust.
This work was supported by NIH RO1 DC05824, NCRR P20RR15583, Oberkotter
foundation and Evan Drammis Research Foundation (all to P.-X.X.).
 |
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