1 Division of Biology, Center for Molecular Medicine, Jichi Medical School,
Tochigi 329-0498, Japan
2 Department of Molecular Neurobiology, Institute of Development, Aging and
Cancer, Tohoku University, Sendai 980-8575, Japan
3 Division of Transgenic Technology, Center for Animal Resources and
Development, Kumamoto University, Kumamoto 860-0811, Japan
4 Department of Otolaryngology, Tokyo Medical and Dental University, Tokyo
113-8519, Japan
5 Division of Nephrology, Department of Internal Medicine, Jichi Medical School,
Tochigi 329-0498, Japan
6 Division of Cell Biology, Center for Experimental Medicine, Institute of
Medical Science, University of Tokyo, Tokyo 108-8639, Japan
* Author for correspondence (e-mail: kkawakam{at}jichi.ac.jp)
Accepted 24 October 2003
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SUMMARY |
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Key words: Six1, Otic vesicle, Inner ear, Pattern formation, Cell proliferation, Shh, Mouse
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Introduction |
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Six1 is expressed in otic vesicles, nasal epithelia, branchial
arches/pouches, nephrogenic cords, somites and a limited set of ganglia
(Oliver et al., 1995b).
However, it is unknown whether or how Six1 is involved in the
development of the inner ear, nose, branchial arch/pouch-derived organs,
kidney, ganglia and skeletal muscles. To address this question, we generated
and analyzed the organ development of Six1-deficient mice. The inner
ear, nose, thymus, kidney and skeletal muscles are severely affected in
Six1-deficient mice, suggesting crucial roles for Six1 in
the development of these organs. Among these phenotypes, the defects in inner
ear development in the mutant mice are intriguing because inner ears develop
elaborate structures with precise disposition and orientation in normal
embryogenesis. They are derived from the otic vesicle by successive
transformation and compartmentalization, but it is poorly understood how the
patterning of the otic vesicle is established and what are the key factors for
such complex processes. Thus, this paper focused on the analysis of inner ear
development and identified the essential roles of Six1 in otic
vesicle patterning.
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Materials and methods |
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In this study, Shh mutant mice with a targeted deletion of exon 2
of the gene were also used (Chiang et al.,
1996) (kindly supplied by C. Chiang and C. C. Hui).
Mice were kept under specific pathogen-free conditions in environmentally controlled clean rooms at the Center for Experimental Medicine, Jichi Medical School, and at the Laboratory Animal Research Center, Institute of Medical Science, University of Tokyo. All mice used in this study were sacrificed by cervical translocation or anesthetization with diethyl ether. The experiments were conducted according to the institutional ethical guidelines for animal experiments and safety guidelines for gene manipulation experiments.
Histological examinations
Embryos and neonates were fixed in 10% formalin or 4% PFA in PBS, embedded
in paraffin wax and then cut into 5-µm thick serial sections. De-waxed
sections were stained with hematoxylin and eosin as described previously
(Ozaki et al., 2001). Alcian
Blue/Alizarin Red staining of neonatal skeletons was performed as described
previously (Wallin et al.,
1994
).
RNA in situ hybridization
In situ hybridization was performed using digoxygenin (DIG)-labeled
antisense riboprobes as described previously
(Xu and Wilkinson, 1998).
Eya1 riboprobe was synthesized from a 528 bp HindIII
fragment of pHM6Eya1 (Ohto et al.,
1999
) subcloned into pBluescript KS(+). Six4 riboprobe
was synthesized from a 630 bp PstI fragment (ntd 1545-2175 of
Six4 SM type cDNA) subcloned into pBluescript KS(+). The following
cDNAs were also used for in situ hybridization probes: Six1
(Oliver et al., 1995b
),
Otx1 and Otx2 (Matsuo et
al., 1995
), Fgf3
(Wilkinson et al., 1988
),
Lfng (Morsli et al.,
1998
), Dlx5 (Miyama
et al., 1999
), Dach1
(Caubit et al., 1999
),
Dach2 (Davis et al.,
2001
), Pax2
(Nishinakamura et al., 2001
),
Bmp4 (a kind gift from N. Ueno), Hmx3
(Wang et al., 1998
),
Shh (Urase et al.,
1996
), Ptch (Goodrich
et al., 1996
), Gli1
(Hui et al., 1994
),
Wnt2b (Riccomagno et al.,
2002
).
TUNEL analysis
For terminal deoxynucleotidyl transferase-mediated dUDP nick-end labeling
(TUNEL), embryos were fixed in 4% PFA in PBS, embedded in OCT compound, and
frozen and sectioned into serial cryosections. Apoptotic cells were detected
with the In Situ Cell Death Detection Kit, POD (Roche Diagnostics Mannheim,
Germany). Briefly, fragmented DNA in apoptotic cells was end-labeled with
fluorescein and the labeled DNA was detected with anti-fluorescein antibody
conjugated with peroxidase and a chromogenic substrate.
BrdU incorporation
Pregnant female mice of gestation day 10.5 and 11.5 were intraperitoneally
injected with 100 mg 5-bromo-2'-deoxyuridine (BrdU) per kg body weight.
Embryos were collected 1.5 hours later and processed for preparation of
8-µm thick paraffin sections as described above. De-waxed serial sections
crossing otic vesicles were treated with 2 N HCl/0.5% Triton X-100 in PBS for
30 minutes at room temperature and rinsed with 0.1 M borate buffer (pH 8.5),
followed by incubation in 0.6% H2O2 in PBS for 30
minutes at room temperature. Subsequently, the sections were incubated
overnight in peroxidase-labeled anti-BrdU (Roche) at 4°C. After washing,
sections were stained in 0.4 mg/ml diaminobenzidine, 0.68 mg/ml imidazole,
0.01% H2O2, and 50 mM Tris-HCl (pH 7.4).
Paint-fill analysis
Paint-fill was performed as described previously
(Bissonnette and Fekete, 1996).
In brief, embryos were fixed in Bodian's fixative, dehydrated through graded
ethanol solutions, then cleared in methyl salicylate and injected into the
lumen of the membranous labyrinth with white paint diluted 1 to 100 in methyl
salicylate.
ABR threshold measurements
The auditory evoked response was recorded with stainless steel needle
electrodes inserted subcutaneously into the vertex (active), left and right of
the retro-auricular regions (inactive) and the opposite thigh (ground). The
stimulus sound in peak equivalent sound pressure level (peSPL) of a tone pips
of 0.1 millisecond slopes, 1 millisecond duration, 70 millisecond repeat
interval with 5.6, 8.0, 12.0, 18.0, 24.0, 32.0 kHz frequencies was given by
free field in an electrically shielded room. A tweeter (PT-RIII, Pioneer) was
placed 10 cm in front of the external acoustic foramen. The stimulus sound
pressure was corrected by a Bruel & Kjaer-type 2636 noise meter. A
microcomputer (ER-2104, GE Marquet) was used to analyze the response. Auditory
thresholds were obtained for each stimulus by varying at 10 dB steps up and
down to identify the lowest level at which an auditory brain response (ABR)
pattern could be recognized. These experiments were conducted in five
wild-type and six heterozygous mice at 5 to 6 weeks of age.
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Results |
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Homozygous mutants were born at Mendelian frequency and showed few body movements but were apnoeic and died immediately after birth. They had micrognathia, and the eyelids were sometimes open (data not shown). No Six1 mRNA was detected in homozygotes (Fig. 1C), confirming that the entire coding region of Six1 was replaced by EGFP gene in this mutant. In the following analyses, we used neonates and embryos from F1 heterozygous matings.
Defects in ears, nose, thymus, kidneys and skeletal muscles of Six1-deficient neonates
Dissection analyses and hematoxylin and eosin (H-E) staining of sections of
the neonates revealed defects in the ears, nose, thymus, kidneys and skeletal
muscles in the Six1-deficient mice. In the inner ear, the dorsalmost
parts of semicircular canals and common crus remained as a common fused space.
The endolymphatic sac was present but was irregularly larger in size than that
of wild-type littermates (Fig.
2A,B) The enlargement was confirmed by comparing the diameter of
the paint-filled endolymphatic sacs of the Six1-deficient and the
wild-type embryos (data not shown). The expansion of the expression domain of
Wnt2b, an expression marker for the endolymphatic sac and duct, also
supports the enlargement of the endolymphatic sac
(Fig. 2C,E). Other parts of the
inner ear were completely absent, including the cochlea, vestibule and
accompanying vestibulo-acoustic ganglia
(Fig. 2G,H, data not shown).
These structural defects were also demonstrated by paint-fill analyses
(Fig. 2Q,R,R'). Because
Six1 expression was evident in the branchial arch and periotic
mesenchymes (Fig. 1C, Fig. 4C), we examined the
middle ear defects in the Six1-deficient neonates and found
malformations of the malleus and the incus and the absence of the stapes
(Fig. 2I,J). In the nose,
Six1-deficient mice manifested a hollowed nasal region with traces of
nasal bleeding (data not shown). A pair of mere simple, rounded nostrils was
present with no nasal epithelium, by contrast to the well-branched cavities
with thick layers of nasal epithelia in the wild-type littermates
(Fig. 3A,B). Both nasal
cavities did not connect with the oral cavity or the nasopharynx, and the
vomeronasal organs were absent in Six1-deficient mice (data not
shown). The surrounding ossified region was abnormally enlarged
(Fig. 3B), as observed in the
inner ear (Fig. 2B,H).
Six1-deficient mice also lacked a thymus
(Fig. 3C,D). Kidneys were
severely affected to variable degrees (Fig.
3E,F). Small kidneys with normal structure were found in mild
cases (data not shown), while both kidneys were absent in extreme cases,
although the ureters were always formed but were occasionally shorter
(Fig. 3F). We also found
markedly reduced skeletal muscle mass of the trunk, limbs, diaphragm and
tongue (Fig. 3G,H, data not
shown). The thymus, kidney, ear, nose and skeletal muscle defects are
consistent with the Six1-deficient mice with different targeting
strategy (Laclef et al.,
2003a; Laclef et al.,
2003b
; Xu et al.,
2003
). These affected organs correlated well with the expression
sites of Six1 during development, such as otic vesicles, nasal pits,
branchial arches/pouches, nephrogenic cords and somites
(Oliver et al., 1995b
). These
results indicate that Six1 is required for the formation of the ear,
nose, thymus, kidneys and skeletal muscles.
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In summary, the development of the inner ear was defective at mid-gestation around E10.5-12.5.
Expression of Six1 in the developing inner ear
To gain insight into the function of Six1 during inner ear
development, we first examined the expression pattern of Six1 by in
situ hybridization in the wild type (Fig.
4A-F) and GFP fluorescence in heterozygous embryos
(Fig. 4G-I). Six1 mRNA
was first detected in the otic placode and the surrounding surface ectoderm at
E8.5 (Fig. 4A). Six1
expression became prominent at the invaginating otic pit and the nascent otic
vesicle at E9.5 (Fig. 4B,C),
consistent with previous observations
(Oliver et al., 1995b).
Notably, the expression level was considerably lower in the dorsalmost region
than in the other region of the otic vesicle
(Fig. 4C). At E10.5,
Six1 expression was limited to the ventral half of the otic vesicle
(Fig. 4D,G). Subsequently, the
expression domain of Six1 became gradually restricted to the cochlear
region at E11.5 (Fig. 4E) and
E12.5 (Fig. 4F,H). At later
stages, Six1 transcripts were detected exclusively in the cochlea at
E14.5 (Fig. 4I), and the
expression of Six1 in the cochlear duct persisted in the neonate
(data not shown).
Six1 is required for correct patterning of the otic vesicle
The morphological defects in Six1-deficient mice were not
restricted to the cochlea but extended to all regions of the inner ear except
the dorsal extremity of the semicircular canals
(Fig. 2). The missing ventral
structures of the mutant mouse inner ear appeared to be related to the
expression domain of Six1 in the ventral otic vesicle at E9.5-10.5
(Fig. 4C,D). The absence of
cochlea and vestibule and the enlargement of the endolymphatic sac prompted us
to examine the following three possibilities: that the specification along the
dorsoventral axis within the otic vesicle is altered in
Six1-deficient embryos, that the cells within the ventral region of
the Six1-deficient otic vesicle undergo enhanced apoptotic cell
death, and that the cells within the ventral region of the
Six1-deficient otic vesicle proliferate at a lower rate than those of
the wild type. We assessed the first possibility by comparing the expression
pattern of genes differentially expressed within the otic vesicle at
E9.5-10.5. The ventralmost cells of the otic vesicle are marked by the
co-expression of Otx1 and Otx2
(Morsli et al., 1999).
Otx1 and Otx2 were not expressed in the
Six1-deficient otic vesicle, by contrast to the wild type, although
an ectopic faint expression of Otx1 was reproducibly detected in the
dorsalmost region (Fig. 5A-D).
Lunatic Fringe (Lfng), a component of the Notch signaling
pathway, is known as a molecular marker for inner ear sensory structures
(Morsli et al., 1998
).
Lfng was expressed in the rostroventral region in the wild type
(Fig. 5E), but no such
expression was noted in the Six1-deficient otic vesicle
(Fig. 5F). Fgf3, which
is required for normal morphogenesis of the inner ear
(Mansour et al., 1993
), was
expressed in the rostroventral region of the wild-type otic vesicle as
Lfng (Fig. 5G), while
the expression of Fgf3 was absent in the Six1-deficient otic
vesicle (Fig. 5H). Bmp4 is an early marker for the superior, lateral and posterior
cristae. It was expressed in the restricted regions of the otic vesicle in the
wild type (Fig. 5I), but no
such expression was noted in Six1-deficient mice
(Fig. 5J).
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We also examined the expression pattern of Pax2, Eya1 and Six4 to clarify whether the expression of these genes is dependent on Six1. These genes are components of the Pax-Six-Eya-Dach gene network and are co-expressed in the otic vesicle. Pax2 was expressed in the medial side of the otic vesicle of the wild-type and Six1-deficient embryos (Fig. 5S,T). Eya1 expression in the ventral side of the wild-type otic vesicle was maintained in the Six1-deficient otic vesicle (Fig. 5U,V). Six4 was expressed in the ventral side of the otic vesicle in wild-type embryos, and this expression pattern was almost the same in the Six1-deficient embryo (Fig. 5W,X). However, the most abundantly expressed regions of Eya1 and Six4 appeared slightly shifted from the ventromedial (wild-type) to the ventrolateral (Six1-deficient) side of the otic vesicle. These results suggest that the expression of Pax2, Eya1 and Six4 in the otic vesicle is not dependent on Six1.
In conclusion, loss of Six1 expression leads to marked changes in the expression domains of many genes in the otic vesicle, suggesting that the first possibility listed above is the case: i.e. the specification along the dorsoventral axis within the otic vesicle is altered in Six1-deficient embryos. Next, we assessed the second and third possibilities by TUNEL method and BrdU incorporation.
Enhanced apoptosis and reduced cell proliferation in the ventral otic vesicle
We examined whether enhanced apoptotic cell death or reduced cell
proliferation within the ventral region of the otic vesicle contributes to the
inner ear phenotype. TUNEL method was used to detect apoptotic cells in the
otic vesicle at E10.5 and E11.5, just before the extensive morphological
changes. Several apoptotic cells were detected in the wild type, while
enhanced apoptotic cell death was observed in the ventral and medial sides of
the otic vesicles of Six1-deficient embryos at E11.5
(Fig. 6A,B). Statistical
analysis revealed significant augmentation of apoptosis at E10.5 and E11.5
(Fig. 6C). We also examined
BrdU incorporation in the otic vesicle at the same developmental stages. In
the wild type and in the Six1-deficient embryos at E11.5, BrdU
incorporation was abundant in the ventral region of the otic vesicle
(Fig. 6D). By contrast, the
incorporation was profoundly reduced in the ventral side of the otic vesicles
of Six1-deficient embryos at E11.5
(Fig. 6E). A significant
decrease in the number of BrdU-incorporated cells was observed at E11.5 but
not at E10.5 (Fig. 6F). The
reduced cell proliferation observed in Six1-deficient otic vesicles
may be in line with the roles of Six1 in cell cycle control
(Ford et al., 1998). These
results suggest that the lack of ventral structures of the inner ear in the
Six1-deficient mice is partly due to enhanced apoptosis and reduced
cell proliferation, as well as altered patterning of the otic vesicle.
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Discussion |
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Furthermore, our results showed a marked reduction of cell proliferation and enhanced apoptosis in the ventral otic vesicle in Six1-deficient embryos (Fig. 6). This may contribute to the inner ear phenotype lacking most of the ventral structures. Thus, Six1 controls inner ear development by regulating cell death and proliferation as well as by establishing otic vesicle patterning.
Phenotypic similarity of the inner ear compared with Shh-deficient mice
Previous and present studies indicated that specification of the cochlea is
dependent on Shh signaling and that perturbation of otic vesicle pattering in
Shh-deficient mice (Riccomagno et
al., 2002) is similar to that of Six1-deficient mice.
Considering these phenotypic similarities of inner ear formation between
Six1- and Shh-deficient mice, we assumed a genetic
interaction between Six1 and Shh. However, the expression
patterns of Shh, Gli1 and Ptch in Six1-deficient
mice and that of Six1 in Shh-deficient mice
(Chiang et al., 1996
) indicate
that the expressions of Shh, Gli1 and Ptch are not dependent
on Six1, and that the expression of Six1 is not dependent on
the Shh signaling pathway in and around the otic vesicle at E10.5
(Fig. 7). Another possible mode
of genetic interaction is through functional cooperation between Six1 and the
components of Shh signaling cascades. Shh protein is emanated from the
notochord and/or the floor plate, probably giving a gradient of Shh across the
otic vesicle with a high concentration in the ventral side and a low
concentration in the dorsal side. This Shh gradient would enhance putative
collaborative interaction between downstream components of Shh signaling
cascades and Six1 in the ventral otic vesicle. Modulation of the
transactivating function of Six1 by Shh signaling would be one of the
plausible mechanisms. However, we cannot exclude independent actions of Six1
and components of Shh signaling cascades in the otic vesicle. For example,
expression of Pax2 in the medialventral otic vesicle is maintained in
Six1-deficient mice (Fig.
5S,T), but is downregulated in Shh-deficient mice
(Riccomagno et al., 2002
). To
determine whether Six1 and Shh interact genetically, it
would be important to examine the phenotypes of the Six1/Shh
double mutant.
Roles of Six1 in Pax-Six-Eya-Dach gene network
Six genes function as components of the Pax-Six-Eya-Dach gene network in
organ development. In the ventral otic vesicle, Six1 is co-expressed
with Pax2, Pax8, Six4 and Eya1 to control inner ear
development. Outside the otic vesicles, various combinations of Pax, Six, Eya
and Dach genes are co-expressed in the primordia of the organs affected in
Six1-deficient mice: the olfactory placode (Pax6, Six1, Six2,
Six3, Six4, Six6, Eya1, Eya2, Eya4); the thymus (Pax9, Six1, Six4,
Eya1); the metanephros (Pax2, Pax8, Six1, Six2, Six4, Eya1); and
the somite/myotome (Pax3, Six1, Six4, Eya1, Eya2, Eya4, Dach1).
Six1 plays important roles in the development of these organs,
probably through the control of patterning and/or cell proliferation, as
observed in the otic vesicle. Notably, Dach genes are not co-expressed with
Six and Eya genes in the ventral otic vesicle, nose or kidney (Figs
4,
5, data not shown).
Furthermore, Dach expression domains were expanded ventrally in the
Six1-deficient otic vesicle, indicating that Six1 represses
the expression of Dach genes in the ventral otic vesicle. Likewise,
augmentation of Dach expression was observed in the nasal pit of
Six1-deficient embryos (data not shown), indicating that expression
of the Dach gene is repressed by Six1 also in the nasal pit. These
findings are in contrast to Drosophila compound eye formation and
chick myogenesis. In both those cases, Pax, Six and Eya are co-expressed with
Dach, cooperatively to execute the developmental programs. Thus, the
Pax-Six-Eya gene network lacking Dach may demarcate the two placode-derived
sensory organs, the inner ear and the nose, and the kidney from other organs
such as the eye and the skeletal muscles. In addition, hierarchy among Pax,
Six, Eya and Dach genes in the otic vesicle has been revealed in this study.
That is, the expression patterns and levels of Eya1 and Pax2
were not affected but expression domains of Dach1 and Dach2
were expanded ventrally in the Six1-deficient otic vesicle
(Fig. 5). Conversely,
Six1 expression is lost but Pax2 expression is not disturbed
in the Eya1-deficient otic vesicle
(Xu et al., 1999). Thus, in
the otic vesicle, expression of Eya1 and Pax2 is independent
of Six1, expression of Six1 depends on Eya1, and
Six1 controls Dach1 and Dach2 expression. In the
myotome, Six4 expression is not dependent on Six1, as
observed in the otic vesicle (Laclef et
al., 2003a
), but Pax2 expression is dependent on
Six1 in metanephric mesenchyme
(Xu et al., 2003
). The
similarities among these organ primordia in the context of the
Pax-Six-Eya(-Dach) network and the diversity in selecting members from
respective gene hierarchies among them raise interesting issues regarding the
ontogeny of these organs during evolution.
In conclusion, our study identified the essential role of Six1 in the regulation of otic vesicle patterning. Together with mice homozygous for other Pax, Six, Eya and Dach genes, Six1-deficient mice should allow a comprehensive understanding of the roles of the Pax-Six-Eya-Dach gene network in various organogeneses.
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ACKNOWLEDGMENTS |
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