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First published online 19 March 2008
doi: 10.1242/dev.014043
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1 Institut für Molekularbiologie, Medizinische Hochschule Hannover,
Carl-Neuberg-Strasse 1, 30625 Hannover, Germany.
2 HNO Klinik, Universitätsklinikum Eppendorf, Universität Hamburg,
Martinistrasse 52, 20246 Hamburg, Germany.
3 Zentrum für Molekulare Neurobiologie Hamburg (ZMNH), Universität
Hamburg, Martinistrasse 52, 20246 Hamburg, Germany.
* Author for correspondence (e-mail: kispert.andreas{at}mh-hannover.de)
Accepted 28 February 2008
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SUMMARY |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Key words: Inner ear, Otic fibrocytes, Otic mesenchyme, Stria vascularis, Deafness, Mouse
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INTRODUCTION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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;
Teubner et al., 2003;
Boettger et al., 2003;
Delprat et al., 2005), and
noise-induced, as well as age-related, hearing deficits are initiated by
changes in fibrocyte physiology
(Hequembourg and Liberman,
2001; Hirose and Liberman,
2003).
Otic fibrocytes represent a heterogeneous population of cells with special
structural and molecular adaptations according to their location and
physiological properties (Spicer and
Schulte, 1991). Fibrocytes are found in the spiral limbus at the
proximal site of the cochlea, and in the spiral ligament in the cochlear
lateral wall, where five subgroups can be distinguished (see
Fig. 1A). Type I fibrocytes
underlie the stria vascularis, a specialized non-sensory epithelial thickening
of the lateral wall, type II fibrocytes are situated under the spiral
prominence, type III fibrocytes line, as a thin layer, the otic capsule, type
IV fibrocytes are located lateral to the basilar membrane and anchor it to the
lateral wall (Henson and Henson,
1988), and type V fibrocytes reside above the stria vascularis.
Fibrocytes of subtypes I, II and V are highly interconnected, and form a
mesenchymal gap junction network. This and an independent epithelial network
couple non-sensory supporting cells of the Organ of Corti with basal and
intermediate cells of the stria vascularis (for a review, see
Kikuchi et al., 2000). Basal
cells form a multi-layered epithelial barrier that separates the extracellular
spaces of the stria vascularis and the spiral ligament. Neural crest-derived
intermediate cells form a discontinuous layer between basal cells and marginal
cells that constitutes an epithelial barrier facing the endolymph in the
cochlear duct (see Fig. 1B for
a scheme of the cellular structure of the stria) (for reviews, see
Forge and Wright, 2002;
Raphael and Altschuler, 2003).
The mesenchymal gap junction network plays a central role in ionic
homeostasis. In fact, recycling of K+-ions through this network is
pivotal for cochlear physiology. Strial marginal cells actively transport
K+-ions into the endolymph to maintain a very high concentration in
this compartment. A voltage gradient between the negative potential inside the
sensory hair cells and the positive endocochlear potential (EP) in the
endolymph, together with the concentration gradient in the same direction,
drives the influx of K+-ions through apical mechano-sensitive
channels and, thus causes the depolarization of hair cells. After secretion by
hair cells and re-uptake by supporting cells, K+-ions are thought
to travel through the epithelial and mesenchymal gap junction networks back to
the stria vascularis (for reviews, see
Kikuchi et al., 2000;
Wangemann, 2002).
Despite the importance of otic fibrocytes for the physiology and pathology
of hearing, little insight has been gained into the genetic circuits
regulating fibrocyte development. Mice mutant for the transcription factor
gene Pou3f4 (also known as Brn4) show ultrastructural
alterations in fibrocyte morphology and exhibit a reduced EP and profound
deafness (Minowa et al., 1999;
Phippard et al., 1999). In
mice mutant for otospiralin (Otos), a gene encoding a small
extracellular matrix (ECM) protein of unknown function, fibrocytes type II and
IV are degenerated (Delprat et al.,
2005). Similar to Pou3f4, the precise role of
Otos in fibrocyte differentiation is unknown.
This report defines a critical role in the development of otic fibrocytes
for Tbx18, a member of the evolutionary conserved family of T-box
transcription factors (Naiche et al.,
2005). Mice carrying a null allele of Tbx18 die shortly
after birth with severe malformations of the vertebral column and the rib cage
(Bussen et al., 2004),
prominent hydroureternephrosis (Airik et
al., 2006) and defective caval veins
(Christoffels et al., 2006),
defects that have been traced to crucial functions of the gene in somite
patterning, differentiation of the ureteric mesenchyme and myocardialization
of caval veins, respectively. Here, we uncover an additional requirement for
Tbx18 in the development of the inner ear. We show that adult
Tbx18-deficient mice with rescued lethality display profound
deafness, and we analyze the electro-physiological, histological and molecular
changes that underlie this phenotype. We correlate the expression of
Tbx18 in otic mesenchyme with defects in fibrocyte composition and
stria vascularis integrity, and characterize the etiology of the defects.
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MATERIALS AND METHODS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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;
Airik et al., 2006). The
generation of a GFP allele of Tbx18 (Tbx18GFP)
that was interchangeably used with the lacZ allele will be described
elsewhere. All mouse lines were maintained on an NMRI outbred background.
Embryos for Tbx18 expression analysis were derived from matings of
NMRI wild-type mice. Tbx18-/- embryos were obtained from
matings of Tbx18 heterozygotes. Mice compound heterozygous for a
Tbx18 mutant allele and the msd::Tbx18 transgene were mated
to derive viable adult double homozygous mice. Wild-type or heterozygous
littermates were used as controls for mutant embryos and mice. Genomic DNA
prepared from yolk sac or tail biopsies was used for genotyping by PCR. For
timed pregnancies, vaginal plugs were checked in the morning after mating,
noon was taken as embryonic day (E) 0.5. Embryos were dissected in
phosphate-buffered saline (PBS) and fixed in 4% paraformaldehyde (PFA) in
PBS.
Histological analyses
Embryos were embedded in paraffin wax and sectioned to 5 µm. Inner ears
were dissected from adult temporal bones and fixed in Bouin's fixative for 48
hours, decalcified in 0.5 M EDTA/PBS for 48 hours, dehydrated, paraffin wax
embedded and sectioned to 5 µm. Sections were stained with Hematoxylin and
Eosin or Picro-Sirius Red (F3BA, Sigma-Aldrich, USA). Histochemistry for
β-galactosidase activity was carried out on cryosections as described
(Lobe et al., 1999).
Ultrastructural analysis
For the preparation of ultra-thin sections, cochleae from anaesthetized
3-week-old Tbx18KO and control animals (one each) were quickly
removed and perfused through the round window with 3% glutaraldehyde in
cacodylat buffer, and then left in the fixative overnight. Fixed cochleae were
decalcified in 10% EDTA (pH 7.3) at 4°C for 3 days, cut in two, post-fixed
in 2% osmium tetroxide for 30 minutes, dehydrated in a graded series of
ethanol solutions and embedded in Epon. Ultra-thin sections (60 nm) were
stained with uranyl acetate and lead citrate and examined with a Zeiss 902
electron microscope.
Immunohistochemistry and immunofluorescence
Adult inner ears were fixed in 4% PFA overnight, decalcified in 0.5 M
EDTA/PBS, pH 8, for 48 hours, embedded in paraffin wax and cut to 5 µm.
Alternatively, decalcified cochleae were immersed in 30% sucrose/PBS for 12
hours, embedded in tissue-freezing medium (Leica, Germany), and cryosectioned
to 5 µm. For the detection of antigens, the following primary antibodies
and dilutions were used. Polyclonal rabbit antisera against Aqp1 (1:200,
Alomone Labs), Barttin (1:100, gift from Friedhelm Hildebrandt)
(Birkenhager et al., 2001),
E-cadherin (1:200, gift from Rolf Kemler)
(Vestweber and Kemler, 1984),
Glut1 (1:200, Dianova), Kcc3 (1:500, gift from Thomas Jentsch)
(Boettger et al., 2003), Kcnq1
(1:250, gift from Thomas Jentsch) (Dedek
and Waldegger, 2001), Kir4.1 (1:200, Alomone Labs), laminin
(1:100, Sigma) and otospiralin (1:250, gift from Christian Hamel)
(Delprat et al., 2002);
polyclonal guinea pig-anti-connexin26 (1:200, gift from Johanna Brandner)
(Brandner et al., 2004); and
monoclonal mouse antibodies against Atp1a1 (Na-K-ATPase subunit 1a, clone a6F,
1:500, developed by Douglas M. Farmbrough and obtained from the Developmental
Studies Hybridoma Bank, University of Iowa) and Cldn11 (clone 37E3, 1:500,
gift from Alexander Gow) (Gow et al.,
2004). Fluorophore-coupled secondary antibodies were purchased
from Dianova, Germany (Rhodamine-Red-X-conjugated donkey-anti-mouse,
Rhodamine-Red-X-conjugated goat-anti-rabbit, Cy3-conjugated goat-anti-guinea
pig), Invitrogen, USA (Alexa-488 conjugated donkey-anti-rabbit) and Santa Cruz
Biotechnology, USA (FITC-conjugated goat-anti-mouse, FITC-conjugated
goat-anti-rabbit), and used at a dilution of 1:200. Non-fluorescent staining
was performed using kits from Vector Laboratories (Mouse-on-Mouse peroxidase
kit, Vectastain ABC peroxidase kit (Rabbit IgG), DAB substrate kit). Labeling
with the primary antibody was performed at 4°C overnight after antigen
retrieval (2 mM EDTA in 0.01 M Tris-HCl, pH9, at 80°C, 1 hour) and
blocking in 3% BSA, 3% normal goat serum for 30 minutes in PBST. For
monoclonal mouse antibodies an additional IgG blocking step was performed
using the Mouse-on-Mouse Kit (Vector Laboratories).
In situ hybridization analysis
In situ hybridization analysis on 10 µm sagittal sections of E18.5 heads
was performed following a standard procedure with digoxigenin-labeled
antisense riboprobes (Moorman et al.,
2001). Details of probes used are available upon request.
Documentation
Sections were photographed using a Leica DM5000 microscope with a Leica
DFC300FX digital camera. Laser scanning microscopy was performed using a Leica
TCS SP2 microscope. All images were processed in Adobe Photoshop CS.
Hearing assessment
Auditory brainstem responses were measured as described previously
(Boettger et al., 2002). In
cases where no auditory response could be recorded at the highest obtainable
level of 137 dB peak equivalent sound pressure level, the hearing threshold
was set to this value for statistical analysis. Measurement of the
endocochlear potential (EP) followed published procedures
(Boettger et al., 2003).
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RESULTS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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) and expression of Pou3f4, whose expression marks
the entire early periotic mesenchyme
(Phippard et al., 1998)
(Fig. 2). Inner ear expression
of Tbx18 was first detected in ventral aspects of the mesenchyme
surrounding the otic vesicle at embryonic day (E) 11.5
(Fig. 2A). Tbx18
expression partially overlapped Sox9 and Pou3f4 expression,
whose domains, however, were clearly more expanded at this stage
(Fig. 2B,C). Expression of
Tbx18 and Sox9 started to become mutually exclusive at
E12.5. Tbx18 expression was confined to the inner zone of the
periotic mesenchyme in the whole inner ear, including the future cochlea and
the vestibular portion (Fig.
2D,G; data not shown), whereas Sox9 expression was
restricted to the outer zone, which undergoes chondrogenic differentiation to
form the otic capsule from E13.5 onwards
(Fig. 2E,H). Expression of
Pou3f4 was found throughout the periotic mesenchyme, overlapping
Tbx18 and Sox9 expression until E13.5
(Fig. 2F,I). From E15.5 until
E18.5, Tbx18 expression was restricted to prospective otic fibrocytes
of the spiral limbus and the spiral ligament
(Fig. 2J,M). Expression was
upregulated in condensing mesenchyme underlying the stria vascularis at E18.5
(Fig. 2P). Sox9
expression was downregulated in the otic capsule after E15.5, but was found in
condensing mesenchymal cells at E18.5 (Fig.
2K,N,Q). Pou3f4 and Tbx18 expression domains
overlapped from E15.5 to E18.5 (Fig.
2L,O,R). Tbx18 expression was not detected at postnatal
day (P) 21, when inner ear development is completed (data not shown). Hence,
Tbx18 expression was restricted to an inner zone of otic mesenchyme
fated to differentiate into otic fibrocytes, throughout inner ear
development.
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).
To evaluate the functional significance of Tbx18 expression in otic
mesenchyme, we sought to overcome the perinatal lethality of
Tbx18-deficient mice by re-introducing somitic expression of
Tbx18. We used transgenic msd::Tbx18 mice that express
Tbx18 throughout the presomitic and somitic mesoderm, but not in the
inner ear (Bussen et al.,
2004) (see Fig. S1 in the supplementary material), to generate
mice compound mutant for a Tbx18 loss-of-function allele and the
msd::Tbx18 transgene. Mice double homozygous for Tbx18 and
msd::Tbx18 were born in the expected Mendelian ratio. They were
smaller than their littermates and exhibited a general impaired mobility,
explainable by skeletal defects. They survived for 3-4 months before they died
from hydronephrotic lesions. Although vestibular function seemed normal in
these mice, acoustic Preyer reflexes were absent, indicating a severe hearing
deficit. We examined auditory function by measuring auditory brainstem
responses (ABRs) to clicks, with an upper frequency limit of 5.5 kHz, shortly
after the onset of hearing (at P14) in three- and twelve-week-old
Tbx18/Tbx18,msd::Tbx18/msd::Tbx18 (Tbx18KO) mice and
Tbx18/+,msd::Tbx18/+ (control) littermates. Tbx18KO mice
showed an ABR with clicks of about 130 dB peak equivalent sound pressure level
(pe SPL) at both time-points; the average increase was >75 dB above the
hearing threshold of control animals (Fig.
3A). Thus, Tbx18KO mice had a pronounced hearing loss at
three weeks of age.
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Lateral wall hypoplasia in Tbx18KO mice
Histological analysis of three-week-old Tbx18KO inner ears did not
reveal any obvious changes in cochlear shape, but an overall reduction in size
was apparent (Fig. 4B), and the
spiral ganglion was not completely surrounded by bone tissue of the modiolus
(Fig. 4D). The lateral wall,
including stria vascularis and otic fibrocytes, of the spiral ligament was
severely hypoplastic, the spiral prominence was variably reduced (white arrow
in Fig. 4F,J). Suprastrial
(type V) fibrocytes were absent, and type IV fibrocytes lateral to the basilar
membrane were partially replaced by bone tissue (arrow and arrowhead in
Fig. 4F,J). The number of cells
underlying the stria vascularis was drastically diminished and the spiral
prominence was different from that of controls, indicating additional defects
in type I and II fibrocytes (Fig.
4D,F). The stria vascularis of Tbx18KO mice was variably
reduced, with a thin stripe of flat marginal cells extending into Reissner's
membrane (Fig. 4J).
Ultrastructural analysis of the stria vascularis architecture at 3 weeks of
age revealed presence of marginal, intermediate and basal cells, but their
densely packed membranous infoldings were severely reduced in Tbx18KO
animals (Fig. 4L). Phenotypic
changes in the lateral wall gradually increased in severity towards the basal
end of the cochlea duct. The Organ of Corti appeared normal at this stage
(Fig. 4H). At 12 weeks of age,
defects of the lateral wall had increased in severity. The stria vascularis
was flattened to a thin layer of simple squamous epithelial cells (see Fig. S2
in the supplementary material).
Hence, the cyto-architecture of the lateral wall, including the fibrocytes of the spiral ligament and the epithelial barrier cells of the stria vascularis, is severely affected by the loss of Tbx18 function in development.
Improper differentiation of otic fibrocytes in Tbx18KO mice
Expression of Tbx18 in otic fibrocytes, disruption of the EP and
histological changes in the lateral wall of Tbx18-deficient mice
suggested a role for Tbx18 in the differentiation of otic fibrocytes.
To explore this possibility in more detail, we analyzed the distribution of
proteins defining fibrocyte subtypes by immunohistochemistry.
Otospiralin (Otos), an ECM protein of unknown function, is expressed in all
otic fibrocytes (Delprat et al.,
2002). In Tbx18KO mice, Otos was present throughout the
spiral ligament, albeit with decreased expression, particularly in the region
(of type IV fibrocytes) beneath the basilar membrane (arrow in
Fig. 5B). The gap junction
protein connexin 26 (Cx26; also known as Gjb2 - Mouse Genome Informatics)
marks type I fibrocytes that underlie the stria vascularis
(Xia et al., 1999). Cx26
staining was absent in Tbx18KO spiral ligament fibrocytes
(Fig. 5D). Expression of
Atp1a1, the alpha1 polypeptide of the
Na+/K+-transporting ATPase, is confined to marginal
cells of the stria vascularis and to type II, IV and V fibrocytes of the
spiral ligament (Xia et al.,
1999). In Tbx18KO mice, the Atp1a1 expression domain was
unchanged but the level of expression in spiral ligament fibrocytes appeared
reduced (Fig. 5F). Aquaporin 1
(Aqp1) expression, which is normally restricted to bone lining fibrocytes type
III (Li and Verkman, 2001)
(arrowheads in Fig. 5G), was
lost in the mutant (Fig. 5H).
Kcc3 (also known as Slc12a6 - Mouse Genome Informatics) represents a
potassium-chloride co-transporter whose expression is found in type I, III and
V fibrocytes (Boettger et al.,
2003). In the mutant, expression of Kcc3 at strongly reduced
levels was homogenous in the spiral ligament
(Fig. 5J). Together,
histological and immunohistochemical analyses revealed that terminal
differentiation of fibrocytes into clearly distinct subpopulations was
severely disturbed in the spiral ligament of Tbx18KO mice
(Fig. 5K).
Loss of basal cells in the stria vascularis of Tbx18KO mice
To evaluate the histological changes in the Tbx18KO stria
vascularis more carefully, we analyzed the localization of marker proteins by
immunofluorescence. The potassium channel Kcnq1 is confined to the apical
surface of strial marginal cells, whereas the chloride channel subunit Barttin
is localized baso-laterally (Estevez et
al., 2001). Subcellular localization of Kcnq1 and Barttin was
unchanged in Tbx18KO mice (Fig.
6B,D), suggesting the presence of marginal cells with normal
apicobasal polarity. However, the area of Barttin staining was reduced and
exhibited an irregular shape, possibly indicating an improper formation of
baso-lateral projections (inset in Fig.
6D). Expression of Kir4.1 (also known as Kcnj10 - Mouse Genome
Informatics), an inwardly-rectifying potassium channel of intermediate cells
(Ando and Takeuchi, 1999), was
reduced in the Tbx18KO stria vascularis
(Fig. 6F), indicating that the
differentiation of intermediate cells, including the formation of membranous
infoldings (inset in Fig. 6F)
was severely affected. The glucose transporter Glut1 (also known as Slc2a1 -
Mouse Genome Informatics) exhibits strong expression in basal and endothelial
cells of the stria vascularis (Ito et al.,
1993). In the mutant, Glut1 expression was found only in
endothelial cells of the few remaining strial vessels
(Fig. 6H). The absence of
expression of the basal cell-specific protein claudin 11 (Cldn11)
(Gow et al., 2004;
Kitajiri et al., 2004) in
Tbx18KO mice (Fig. 6J)
confirmed the dramatic reduction of the basal cell layer.
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; Kikuchi and Hilding,
1966). In addition to the basal lamina of strial vessels, laminin
staining detected the presence of a basal lamina underlying marginal cells in
Tbx18KO mice (Fig.
6L), which is in agreement with the observed reduction of
baso-lateral surface projections of marginal cells. Thus, the cellular
architecture of the stria vascularis in Tbx18KO is severely
disturbed, with a massive decrease of basal cell number, and a vast reduction
of membraneous projections of intermediate and marginal cells
(Fig. 6M).
Loss of mesenchymal condensations underneath the stria vascularis in Tbx18-/- spiral ligaments
The occurrence of strial defects upon loss of Tbx18 in otic
mesenchyme suggested a cellular or trophic contribution of the otic mesenchyme
to strial development. To distinguish these possibilities, we analyzed
Tbx18-/- inner ears at E18.5, when stria vascularis
maturation is initiated (Kiernan et al.,
2002; Xia et al.,
1999). Histological examination revealed condensation of
mesenchymal cells underneath the forming stria in the wild type, whereas in
Tbx18-/- mice cells underneath the stria remained loosely
organized at this stage (arrow in Fig.
7B). During our studies, we established that these mesenchymal
condensates are characterized by the expression of Sox9, E-cadherin
(also known as Cdh1 - Mouse Genome Informatics) and Cx26 in
the wild type (Fig. 7C,E,G).
Expression of all three genes was lost in the Tbx18-deficient spiral
ligament, although the epithelial expression domains of these genes appeared
unaffected (Fig. 7D,F,H).
Further markers were used to assess cytodifferentiation of the stria
vascularis. Expression of Bsnd, which encodes Barttin, in prospective
marginal cells (Birkenhager et al.,
2001) was detected in the mutant, suggesting proper temporal
regulation of marginal cell differentiation
(Fig. 7J). Dct (also
known as Trp2) is a marker for prospective intermediate cells
(Steel et al., 1992).
Dct expression in the mutant was indistinguishable from in the wild
type, demonstrating normal homing of future intermediate cells
(Fig. 7L). We did not detect
changes in the BrdU incorporation of cells of the spiral ligament at E18.5 and
E12.5 (n=3 each, data not shown), which suggests that proliferation
defects are not a causative agent.
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Disturbed boundary formation between the otic capsule and otic fibrocytes
Tbx18 expression shows an early restriction to the inner ring of
otic mesenchyme that will give rise to otic fibrocytes. We wondered whether
the observed defects in otic fibrocyte differentiation might be caused, or at
least be affected, by a mis-patterning of the otic mesenchyme, indicating an
early role for Tbx18 in compartmentalization of this tissue. Improper radial
patterning of the otic mesenchyme should reflect in misallocation of cells to
the inner and outer compartment, i.e. to otic fibrocytes and the otic capsule,
and/or in improper boundary formation. Histological analysis of E18.5 inner
ears revealed a local thickening of the otic capsule adjacent to the spiral
ligament of Tbx18-/- mice (arrow in
Fig. 8E), and an altered
appearance of otic fibrocyte precursors in the spiral ligament of the basal
coil (Fig. 8E). Fibrocytes
appeared highly condensed with a preferential parallel orientation to the
border of the otic capsule. Collagen staining with Picro-Sirius Red showed a
distinct boundary between the fiber systems of the bony otic capsule and the
spiral ligament in the wild type at this stage (arrowhead in
Fig. 8B). By contrast, in
Tbx18-/- mice, the collagen fiber network appeared to be
continuous between the two regions (Fig.
8F). Following the indications of severe histological changes at
the interface between otic capsule and otic fibrocytes, we analyzed expression
of periostin (Postn), a gene expressed in certain types of fibrous
connective tissue, such as the bone-lining periosteum
(Horiuchi et al., 1999).
Postn expression was restricted to cells lining the outer border of
the otic capsule and to a small domain in the proximobasal part of the spiral
ligament in the wild type at E18.5 (Fig.
8C). In Tbx18-/- inner ears, Postn
was expressed in a distal to proximal gradient throughout the spiral ligament
(Fig. 8G). By contrast,
expression of Coch, which we found to be restricted to a proximal
region of the otic mesenchyme underlying the epithelium of the lateral wall in
the wild type at this stage (Fig.
8D), was absent in the mutant
(Fig. 8H). Expression of
Pou3f4 and Otos, which was found throughout the otic
mesenchyme in the wild type, was unchanged in Tbx18-/-
mice at E18.5 (data not shown). Together, these findings suggest that in the
Tbx18-/- inner ear, the outer compartment of the otic
capsule has expanded at the expense of the inner compartment. Mesenchymal
cells of the inner compartment have acquired some basal characteristics of
otic fibrocytes, but seem to differentiate into periosteum-like connective
tissue rather than into distinct fibrocyte subtypes.
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DISCUSSION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Tbx18 is necessary for otic fibrocyte differentiation
To our knowledge, Tbx18 is the earliest molecular marker that
shows radially restricted expression in the periotic mesenchyme, demonstrating
that molecular subdivision into an inner and outer compartment occurs shortly
after mesenchymal aggregation around the otic vesicle. Although we did not
formally address the fate of the Tbx18-positive otic mesenchyme by
genetic lineage tracing experiments, it is likely that Tbx18
expression from at least E12.5 onwards marks prospective otic fibrocytes.
Evidence derives from the mutually exclusive expression domains of
Tbx18 and the chondrogenic marker gene Sox9, which in turn
becomes quickly restricted to the outer ring of mesenchyme destined to form
the otic capsule. In addition, short-time lineage tracing experiments with a
lacZ reporter in the Tbx18 locus revealed an early
restriction of β-galactosidase activity to the inner compartment of
mesenchymal cells.
The spatial restriction of Tbx18 expression in the developing inner ear is compatible with a role of this transcriptional regulator either in patterning of the periotic mesenchyme in capsule versus fibrocyte compartments, or in specification/differentiation of otic fibrocytes. Dramatic changes in subtype composition of lateral wall fibrocytes in the Tbx18-deficient inner ear support a function of Tbx18 in fibrocyte differentiation. However, for several reasons, we suggest that defects in fibrocyte differentiation are subordinate to a primary requirement of Tbx18 in patterning or compartmentalization of the otic mesenchyme. First, loss of type I, III and V, and reduction of type IV fibrocytes shows that populations residing in close proximity to the bony capsule are predominantly affected by the loss of Tbx18, although the expression of Tbx18 appears homogenous. More importantly, failure to restrict cells that normally express Tbx18 to the inner compartment in the mutant after E12.5, local loss of a distinct capsule-fibrocyte boundary at E18.5 coupled with histological alterations of fibrocytes, ectopic expression of Postn, and an absence of Cx26 and Coch expression at E18.5 indicate a disturbance of radial patterning of the periotic mesenchyme and a failure to maintain the boundary between mesenchymal compartments destined to give rise to otic capsule and otic fibrocytes. The acquisition of a fate of periosteum-like connective tissue may hamper further differentiation of fibrocytes into the different subtypes.
The failure to restrict cells that normally express Tbx18 to the
inner compartment of the otic mesenchyme may have one of several molecular
explanations. First, Tbx18-/- cells no longer recognize a
repulsive signal emanating from the outer mesenchymal compartment to restrict
their migration or intermingling with these cells. Second,
Tbx18-mutant cells have lost selective adhesiveness. Alternatively,
the loss of an inhibitory signal from the Tbx18-mutant cells leads to
ectopic induction of Tbx18 in the outer ring of mesenchymal cells.
Notably, Tbx18 has also been implicated in the maintenance of
compartment boundaries in somites and in the ureteric mesenchyme. In
Tbx18-/- somites, posterior somite characteristics expand
anteriorly (Bussen et al.,
2004), whereas in the metanephric field ureteric mesenchymal cells
fail to coalesce onto the ureteric epithelium
(Airik et al., 2006). It
remains to be explored whether these phenotypes can be rationalized by the
disruption of a common molecular program.
To date, a requirement for the proper differentiation of otic fibrocytes
has only been demonstrated for Pou3f4
(Minowa et al., 1999;
Phippard et al., 1999).
Similar to Pou3f4-/- mice, differentiation of fibrocytes
is severely compromised in Tbx18-deficient mice. However, reduction
but not loss of Pou3f4 expression and more severe phenotypic changes
in Tbx18-/- animals suggests that there is not a simple
epistatic relationship between the two transcription factor genes. More
likely, the two genes act in parallel genetic circuits regulating patterning
and differentiation of otic mesenchyme.
Requirement for Tbx18 in fibrocyte differentiation reveals multiple steps in stria vascularis formation
Despite the pivotal role of the stria vascularis in auditory function,
little is known about the developmental processes and the underlying molecular
pathways involved in its formation. Our study provides molecular evidence that
the basal cell layer of the stria vascularis forms by aggregation from, and
subsequent mesenchymal-epithelial transition (MET) of, adjacent otic
fibrocytes.
Evidence is provided by the co-regulation of Sox9, Cx26 and
E-cadherin in condensing mesenchymal cells beneath the stria vascularis.
Sox9, a gene encoding an HMG-type transcription factor, has been
implicated in the regulation of mesenchymal cell condensation in various
contexts (Bi et al., 2001;
Akiyama et al., 2004), thereby
supporting a similar role in this setting. A direct regulation of
Sox9 by Tbx18 is possible. However, it is more likely that the loss
of Sox9 expression is secondary to a prior differentiation defect of
the otic mesenchyme. Intriguingly, Cx26 expression in this domain
precedes the onset of K+-cycling through the gap junction network
of the spiral ligament (Sadanaga et al., 1995;
Yamasaki et al., 2000). Hence,
the expression of Cx26 might indicate a role in compartmentalization
of cells by gap junction-mediated cell-cell communication (for a review, see
Levin, 2007) (see also
Ackert et al., 2001). Finally,
E-cadherin expression has been implicated in a variety of tissue remodeling
processes. The forced expression of E-cadherin in fibroblasts is sufficient to
drive them into epithelial cells, whereas the loss of E-cadherin in epithelial
cells has been linked to mesenchymal transitions (for a review, see
Thiery and Sleeman, 2006) (see
also Vanderburg and Hay,
1996). The absence of E-cadherin expression in condensing
mesenchymal cells beneath the stria vascularis in the
Tbx18-/- inner ear strongly supports a functional role of
E-cadherin in MET as a prerequisite for basal cell layer formation.
The almost complete loss of basal cells in Tbx18-/-
mice offered the opportunity to study the role of these cells for marginal and
intermediate cell differentiation and maturation. Intermediate cell
projections were reduced in the Tbx18-/- stria vascularis
at three weeks of age, although precursor cells were established normally at
E18.5. This favors the idea that expansion, maintenance and/or differentiation
of intermediate cells depends on the presence of an intact basal cell layer.
Marginal cells showed a mature phenotype with the formation of basolateral
processes and expression of the
Na+/K+-ATPase-
1-isoform
(Erichsen et al., 1996). The
collapse of Reissner's membrane that has been reported for certain mutants
with severely impaired endolymphatic K+-secretion was not apparent
in Tbx18-/- mice, suggesting that marginal cells became
functional (Delpire et al.,
1999; Vetter et al.,
1996). We noted, however, that the extension of basolateral
processes of marginal cells and interdigitation with intermediate cells was
severely compromised, showing that parts of the differentiation program of
marginal cells depend on basal and intermediate cells. We propose that basal
cell-mediated degradation of the basal lamina is a prerequisite for these
processes to occur.
Tbx18 and deafness
The measurement of ABRs revealed profound deafness in
Tbx18-deficient mice. At three weeks of age, i.e. shortly after the
onset of hearing, the EP was completely abolished. Several of our findings
suggest that the defect of otic fibrocyte differentiation in Tbx18KO
mice structurally and functionally interferes with the establishment of a
normal EP, both at the level of its strial generation and with the recycling
of K+-ions by the mesenchymal gap junction network.
First, it has recently been demonstrated that expression of the potassium
inwardly-rectifying channel Kir4.1 in strial intermediate cells is required
for EP generation in the mouse (Marcus et
al., 2002). Second, functional integrity of the basal cell layer
of the stria vascularis is necessary to establish a distinct intrastrial
compartment that, in turn, is required for generation of the EP. Previous
studies using Cldn11 mutant mice showed that loss of the tight
junction barrier in basal cells causes a strong decrease of the EP
(Gow et al., 2004;
Kitajiri et al., 2004). Third,
fibrocytes of the spiral ligament have been implicated in the uptake and
transport of perilymphatic K+-ions (for a review, see
Kikuchi et al., 2000). Fourth,
mice mutant for connexin 30, which exhibits overlapping cochlear expression
and forms heteromeric gap junction channels with connexin 26, do not establish
a normal EP, illustrating the importance of the spiral ligament gap junction
network (Forge et al., 2003;
Teubner et al., 2003). Hence,
the lack of Kir4.1-positive intermediate cells and the almost complete absence
of Cldn11-expressing basal cells in the stria vascularis, the failure in
terminal fibrocyte differentiation and the absence of Cx26 expression in the
spiral ligament collectively explain the complete abrogation of the EP in
Tbx18-deficient mice.
In conclusion, we have shown that lack of the T-box transcription factor Tbx18 in otic mesenchyme leads to changes in the compartmentalization and differentiation of otic fibrocytes, and to subsequent defects in stria vascularis formation. Cooperatively, these defects result in a failure to generate a normal EP, dramatically demonstrating the importance of the mesenchymal cells of the lateral wall for auditory function.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/135/9/1725/DC1
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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