1 Department of Neuroscience, Albert Einstein College of Medicine, 1300 Morris
Park Avenue, Bronx, NY 10461, USA
2 Department of Molecular Genetics, Albert Einstein College of Medicine, 1300
Morris Park Avenue, Bronx, NY 10461, USA
Author for correspondence (e-mail:
morrow{at}aecom.yu.edu)
Accepted 22 December 2003
![]() |
SUMMARY |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: Tbx1, Ngn1, NeuroD, Bmp4, Otx1, Otocyst, Neurogenesis, Patterning, Mouse
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Inner ear sensory organs and VIIIth ganglion neurons arise from the
otocyst, an epithelial vesicle that forms by invagination of an ectodermal
(otic) placode (Kiernan et al.,
2002). A subset of otocyst epithelial cells express the
Atonal-related basic helix-loop-helix (bHLH) genes neurogenin 1
(Ngn1) and NeuroD (Neurod1), delaminate into the
mesenchyme, and coalesce with neural crest-derived glial progenitors to form a
ganglion (Rubel and Fritzsch,
2002
; D'Amico-Martel and
Noden, 1983
). Ngn1 is required for neural progenitor
determination and formation of the VIIIth ganglion
(Ma et al., 1998
;
Ma et al., 2000
), while
NeuroD requires Ngn1 activity for its expression and
controls delamination and growth factor-mediated neuronal survival
(Kim et al., 2001
;
Liu et al., 2000
). Attainment
of inner ear morphology by growth/remodeling of the otocyst follows a major
period of neurogenesis (Adam et al.,
1998
). However, during neurogenic stages, homeobox patterning
genes such as Otx1, Pax2, Dlx5 and Hmx2/3 are regionally
expressed in the otocyst, and their inactivation in mice causes selective loss
or malformation of inner ear sensory organs
(Morsli et al., 1999
;
Torres et al., 1996
;
Merlo et al., 2002
;
Wang et al., 2001
;
Wang et al., 1998
;
Hadrys et al., 1998
). Other
genes expressed during the period of neurogenesis and implicated in early
sensory organ development include bone morphogenetic protein 4 (Bmp4)
and the Notch signaling modulator lunatic fringe (Lfng)
(Morsli et al., 1998
;
Cole et al., 2000
).
Divergence of otocyst-derived cell lineages has been modeled as a series of
binary fate choices (Fekete and Wu,
2002), however, no single factor or pathway has been identified
that differentially influences neural and sensory organ fate specification in
vivo. Fate specification in the otocyst could involve compartmentalization
(Brigande et al., 2000
;
Kiernan et al., 1997
), change
in competence of a pluripotent progenitor field as a function of time
(Fritzsch et al., 2002
;
Cole et al., 2000
), or some
combination of the two mechanisms.
Tbx1 was identified by sequence homology to the DNA binding domain
of Brachyury (T) (Bollag
et al., 1994). Brachyury/
gastrulae generate insufficient mesoderm, and posterior structures are absent
at later embryonic stages (Chesley,
1935
; Yanagisawa et al.,
1981
; Herrmann et al.,
1990
; Wilkinson et al.,
1990
). Other T-box genes such as Drosophila
optomotor-blind and mouse Tbx6 also specify tissue fate and
control posteriorization during development
(Kopp and Duncan, 1997
;
Chapman and Papaioannou,
1998
). T-box gene mutations cause a variety of human congenital
malformations (Bamshad et al.,
1997
; Basson et al.,
1997
; Li et al.,
1997
; Braybrook et al.,
2001
), and TBX1 haploinsufficiency is implicated in the
etiology of Velocardiofacial/DiGeorge syndrome
(Merscher et al., 2001
;
Lindsey et al., 2001). Inner ear and cranial ganglion dysplasias have been
described in case reports of this syndrome
(Adkins and Gussen, 1974
;
Black et al.,1975
; Ohatni and
Schuknecht, 1984), and in mice, Tbx1 null homozygous mutation causes
otocyst hypoplasia and arrest of inner ear morphogenesis
(Vitelli et al., 2003
;
Jerome and Papaioannou, 2001
).
We have previously reported an occurrence of middle and inner ear
abnormalities in adult BAC-transgenic mice expressing TBX1, with the
inner ear phenotype characterized by dysplastic and ectopic/supernumerary
sensory organs (Funke et al.,
2001
). Here, we show that Tbx1 suppresses neural fate
determination in the otocyst epithelium and is required for proper
morphogenesis of inner ear sensory organs.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Immunohistochemistry
Undiluted mAb4D5 (anti-islet1/2) or mAb2H3 (anti-neurofilament, 165kDa)
supernatant (Developmental Studies Hybridoma Bank) was applied to tissue
sections (for 3 hours at room temperature) and detected with a biotinylated
horse anti-mouse IgG conjugate (1:200; Vectalab), avidin-biotin complex
formation (Vectalab) and DAB reaction (Research Genetics). Tissue was prepared
by Carnoy fixation of whole embryos, which were paraffin wax embedded and
serially sectioned at a thickness of 7 µm. Sections were counterstained
with either Eosin or Cresyl Violet. Affinity-purified rabbit anti-MATH1
(Helms and Johnson, 1998) was
diluted 1:50 in PBS/0.2% Triton X-100/1% goat serum, incubated overnight at
4°C on 4% paraformaldehyde-fixed 12 µm serial cryosections, and
detected with a TRITC-conjugated goat anti-rabbit IgG (Jackson
Immunoresearch). Double-label immunofluorescence was performed by sequential
incubation with anti-MATH1 (anti-Atoh1) and mAb2H3, followed by detection with
goat anti-rabbit and goat anti-mouse secondary antibodies conjugated to TRITC
and FITC (Jackson Immunoresearch).
Whole-mount in situ hybridization
Digoxigenin-labeled RNA probes for Tbx1, TBX1
(Funke et al., 2001),
Ngn1 (Ma et al.,
1998
), NeuroD (Lee et
al., 1995
), Bmp4, Lfng
(Morsli et al., 1998
),
Gata3 (George et al.,
1994
), Fgf3 (Riccomagno, 2002), Otx1
(PstI/SacI cDNA fragment), and Pax2 (554 bp coding
fragment cloned by PCR) were prepared by standard methods. Whole-mount
hybridization and anti-digoxigenin immunochemistry were performed according to
the method of Wilkinson (Wilkinson,
1992
), with minor modifications. Following enzymatic color
development, embryos were post-fixed (3.7% formaldehyde in 0.1 M Mops, pH
7.5), dehydrated, and embedded in paraffin wax for serial sectioning at a
thickness of 7 µm. Bmp4-, Lfng- and
Tbx1-hybridized embryos were also sunk in 30% sucrose overnight,
embedded in OCT compound (Tissue Tek) and cryosectioned at a thickness of 30
µm. Some Pax2- and Gata3-hybridized embryos were embedded
in agarose (3.5%, 8% sucrose, PBS) and vibratome sectioned at a thickness of
40 µm.
Quantitative analyses
Transverse 7 µm serial sections were digitized and imported into either
Photoshop7.0 (Adobe) or Openlab3.1.1 (Improvision) at a resolution of
0.32-0.64 µm/pixel. NeuroD and Ngn1 signal occurrence
data were obtained from consecutive serial sections by marking of (perinuclear
ring) hybridization signals and automated counting in Photoshop with an Image
Processing Toolkit plug-in (Reindeer Graphics). Volumetric data were obtained
by integrating traced areas of interest in Openlab3.1.1. Data were tested for
significance by multivariate analyses of variance in SAS version 8.1e.
Three-dimensional reconstruction
Serial section sets were aligned using AutoAligner2 software (Bitplane AG).
Regions of interest were traced in Photoshop7.0 and reconstructed using
Imaris3 software (Bitplane AG).
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
|
|
Complementary expression of Tbx1 and neural bHLH genes
We next compared Tbx1 expression with that of the neural bHLH
genes Ngn1 and NeuroD, which are expressed sequentially
during cranial sensory ganglion development
(Ma et al., 1998).
Ngn1 expression is first detected at the onset of invagination (13-14
somites) in a small number of cells located near the AP midline of the placode
(data not shown). This is followed by expression of NeuroD in the
same region; thus, neural determination is initiated near a Tbx1
expression border (Fig. 1A,D).
Epithelial domains of Ngn1 and NeuroD coincide through E11.5
(Fig. 4A,B; data not shown).
Both expression domains expand into anterolateral, anteroventral and
posteroventral portions of the invaginating placode
(Fig. 1E,H,J) and overlap with
low intensity Tbx1 signal posteriorly
(Fig. 1G,H). By E9.5 a
definitive border of NeuroD expression emerges at the otocyst AP
midline (Fig. 2D), and the
posterolateral region remains Ngn1/NeuroD negative
throughout otocyst stages (Fig.
2D-F, Fig. 4B,
Fig. 5A). Expression mapping at
E9.5 suggests that NeuroD and Tbx1 expression borders are
juxtaposed laterally at the AP midline, but overlap in the posteroventral
otocyst (Fig. S2,
http://dev.biologists.org/supplemental/).
At E9.5, all otocyst regions expressing bHLH genes show
NeuroD-positive cells delaminating from the epithelium (data not
shown).
|
Suppression of neural determination in TBX1 gain-of-function otocysts
To test whether an expanding domain of Tbx1 activity can suppress
otic neurogenesis, Ngn1 and NeuroD expression was analyzed
in a transgenic mouse line carrying tandem copies (eight to ten) of human BAC
DNA (line 316.23) (Funke et al.,
2001). TBX1 and flanking genes (5' and 3' of
TBX1) are expressed in transgenic embryos, suggesting the presence
and activity of local TBX1 regulatory elements. Of the four human
genes present, only TBX1 and PNUTL1 hybridization signals
are detected in the transgenic otocyst
(Funke et al., 2001
).
Transgenic embryos hybridized with a species-specific TBX1 probe show
recapitulation of the endogenous Tbx1 signal together with ectopic
signal in the anterior otic placode and otocyst
(Fig. 1C,
Fig. 2P,Q). TBX1
transgene and endogenous Tbx1 otocyst expression patterns are similar
at later otocyst stages (Fig.
2C,R). By contrast, TBX1 signal onset in transgenic
mesenchyme is delayed (compare Fig. 1C and
B and Fig. 2P and
A), affording a period (E9-9.5) during which any gain-of-function
effects are probably of epithelial origin.
Ngn1 and NeuroD otic epithelial expression is effectively abolished posteriorly and significantly reduced anteriorly in transgenic embryos (Fig. 1F,I,L, Fig. 2M-O, Fig. 5A). The transgene suppresses neurogenesis in Tbx1+/+, Tbx1+/ and Tbx1/ backgrounds (Fig. 1L; data not shown). Transgenic otocysts show differences in neural domain patterning when endogenous gene copy number is varied, but in all instances, NeuroD domain borders are shifted anteriorly in the presence of the transgene (Fig. 2N; Fig. S2, http://dev.biologists.org/supplemental/). At E10, Tg;Tbx1+/ ganglion volume, identified by NeuroD signal, is 39% that of wild type (0.314±0.113 x106 vs. 0.798±0.206 x106 µm3; n=8 ganglia/class; P<0.0005; compare Fig. 2N and E). No qualitative difference with wild type is detected in size of the adjacent facial ganglion rudiment (Fig. 2, asterisks), the neural progenitors of which derive from an epibranchial placode.
Tbx1 loss-of-function causes ectopic neural determination
The generation of a Tbx1 null mutation by gene targeting has been
described (Merscher et al.,
2001). The Tbx1/ otocyst is
hypoplastic (Fig. 2K,L; Fig.
S1P-T,
http://dev.biologists.org/supplemental/),
in accordance with previous accounts
(Vitelli et al., 2003
;
Jerome and Papaioannou, 2001
),
and we find earlier evidence of impaired growth during placode invagination
(Fig. 1K). Posteriorly, the
invaginating placode of Tbx1/ embryos shows
an increase in Ngn1/NeuroD expression over that in wild type
(Fig. 1I,K; data not shown).
The Tbx1/ otocyst has a uniform distribution
of Ngn1/NeuroD expression (laterally and ventrally) along
its entire AP length, and no AP midline border forms
(Fig. 2J,K,L,
Fig. 3I, Fig. 4B,
Fig. 5A). Extreme dorsal and
medial borders of wild-type Ngn1/NeuroD expression, together
with signal intensity gradients for these markers along the medial-lateral
axis, are preserved in Tbx1/ otocysts (Fig.
S1F-J,P-T,
http://dev.biologists.org/supplemental/).
Tbx1+/ otocysts show a stereotyped pattern of transient ectopic neurogenesis, first posteroventromedially and then anterodorsolaterally (Fig. 2G,H,I, Fig. 4A,B). These loci correspond to sites and times of concerted Tbx1 expression onset and bHLH gene downregulation described above, suggesting that neurogenic suppression is delayed in the heterozygous state. Ectopic neurogenesis in heterozygotes correlates with increased accumulations of delaminated NeuroD-positive cells compared to wild type (Fig. S1K-O, http://dev.biologists.org/supplemental/).
The more persistent and widespread ectopic neurogenesis observed in Tbx1 null homozygotes correlates with ectopic delamination (Fig. 5C,E) and duplication of VIIIth ganglion rudiment form about the otocyst AP midline (Fig. 2K,L, Fig. 5B). At E10, Tbx1/ ganglion volume, identified by NeuroD signal, is 1.83-fold that of wild type (1.46±0.367 x106 vs. 0.798±0.206 x106 µm3; n=8 ganglia/class; P<0.001). By E10.5, anti-islet1/2 (mAb4D5) immunochemistry reveals histodifferentiation of the wild-type ganglion into lateral and ventromedial subdivisions (Fig. S3A,B, http://dev.biologists.org/supplemental/), and islet-positive nuclear size and packing density vary between subdivisions (Fig. 5D; Fig. S3C, http://dev.biologists.org/supplemental/). These features are present along the entire AP length of the E10.5 Tbx1/ ganglion rudiment (Fig. 5C). Evidence of ganglion rudiment splitting near the otocyst AP midline is observed by E11.5 (data not shown), and early labyrinth-stage (E13.5) Tbx1/ ears commonly show a secondary compound ganglion apposed to and innervating the epithelial posterior pole (Fig. 8E,F,G; 4/8 ears auditory and vestibular neuronal cytology; remainder vestibular only). No posterior VIIIth ganglia analyzed possessed a central projection. Anterior to the epithelium, all E13.5 Tbx1/ VIIIth ganglia analyzed (8/8) showed auditory and vestibular neuronal cytology and partial retention of auditory and vestibular ganglion morphology; in addition, all anterior ganglia possessed both peripheral and central projections (Fig. 8A-D). Tbx1/ posterior and anterior VIIIth ganglia are necrotic beyond E14.5 and largely absent by E16.5, suggesting an additional requirement for Tbx1 in neuronal survival at late embryonic stages.
|
|
Pax2 and Gata3 expression was assayed to test for effects
of Tbx1 mutation on medial-lateral otocyst patterning. Along this
axis, their otocyst expression domains are reciprocal and partially
overlapping (Lavoko-Kerali et al.,
2002). Pax2 is expressed medially and is required for
genesis of the cochlear duct (Nornes et
al., 1990
; Torres et al.,
1996
). Gata3 hybridization at E10.5 yields a strong
lateral signal (Zheng et al.,
2003
), and the inner ear of
Gata3/ mice is severely dysmorphic
(Karis et al., 2001
). No
change was detected in expression patterning of either Pax2 or
Gata3 in Tbx1/ otocysts between
E9.5 and E10.5. Wild-type features of these genes' expression, such as dorsal
borders and ventral gradients are preserved in the mutant otocyst
(Fig. 6I,J).
Otx1 is expressed in the posteroventrolateral otocyst by E10.25
and loss-of-function results in sensory organ absence (lateral vestibular
canal/ampulla) or dysmorphogenesis (lateral crista, maculae, cochlea); no
neurogenic phenotype pertaining to the ear has been reported
(Fritzsch et al., 2001;
Morsli et al., 1999
;
Acampora et al., 1996
). E10.5
and E11 Tbx1/ otocysts lack Otx1
expression (Fig. 6H) and show
ectopic neurogenesis posteroventrolaterally
(Fig. 2L, Fig. 3I,
Fig. 5A), which persists
through to at least E11.5. Thus, Tbx1 loss-of-function causes
anterior (neurogenic) transformation of a posterior otocyst region that
contributes to sensory organ development.
Tbx1 is required for proper patterning and maintenance of Bmp4 expression
To further explore the effects of Tbx1 activity on sensory organ
patterning, Bmp4 expression was analyzed in
Tbx1/ and Tbx1+/
embryos. Local antagonism of BMP signaling in the developing chick causes
dysmorphogenesis of multiple inner ear sensory organs, with the most severe
and frequent defects related to vestibular canal formation
(Chang et al., 1999;
Gerlach et al., 2000
). In the
E10.5 mouse otocyst, Bmp4 hybridization marks the presumptive
anterior and lateral cristae (anterior stripe) and posterior crista
(Morsli et al., 1998
)
(Fig. 3G). We have localized
the origin of the anterior stripe at E10 to a juxtaposition of Tbx1
and Lfng/neural bHLH gene expression domains
(Fig. 2B,E,
Fig. 3B,C).
Bmp4 otocyst expression in E10 and E10.5 Tbx1/ embryos is mis-patterned. E10 mutant embryos show a diffuse anterodorsolateral signal in place of an anterior stripe, while an extreme dorsolateral band of signal terminating at the otocyst posterior pole is preserved (Fig. 3D). By E10.5, the wild-type anterior stripe and posterior focus form discrete signals (Fig. 3G), but Tbx1/ otocysts of this stage show a pattern similar to that of E10 mutants (Fig. 3H, Fig. 7D,E). The anterior stripe and posterior focus persist in wild-type otocysts through E11, however, Tbx1/ embryos of this stage showed little to no signal in the otocyst epithelium (Fig. 7F). Bmp4 otocyst signal in E10 and E10.5 Tbx1+/ embryos (15/15) was indistinguishable from wild-type (data not shown). However, all E11 heterozygotes analyzed (four embryos) showed a diffuse anterodorsolateral signal bilaterally in place of an anterior stripe (Fig. 7C), suggesting a requirement for proper Tbx1 gene dosage in patterning of Bmp4 otocyst expression.
|
Identifiable vestibular and auditory sensory organs fail to develop in Tbx1/ embryos (Fig. 8E,I). The E13.5 Tbx1/ inner ear is composed of two ventral chambers a lateral tube and medial sac and a dorsal projection, which are continuous and communicate via a narrow duct near the AP midline. Ventral tube and medial sac formation begins with a furrow that runs anteroposteriorly along the ventral wall of the otocyst. The furrow, which is positioned roughly at the otocyst medial-lateral midline and intersects the neurogenic region, deepens and fuses with the dorsal wall of the otocyst (data not shown, but see Fig. 5C). The dorsal projection appears to derive from the endolymphatic projection of the mutant otocyst and exhibits simple cuboidal epithelial morphology typical of the wild-type endolymphatic duct.
By E13.5, the mutant ventral epithelium, previously a site of neurogenesis, is stratified, innervated, and MATH1 positive (Fig. 8C,F,J,K,L). Neurites of the VIIIth ganglion envelop the base of upper layer MATH1-positive cells (Fig. 8H,J,K), and MATH1 immunoreactivity and innervation patterns are similar across the epithelium (Fig. 8K,L; data not shown). These patterns consist of two patches separated by the cleft between lateral tube and medial sac (Fig. 8I), thus segregation of an initially unitary neural/sensory-competent region may be related to morphogenetic movements of the late otocyst. Lateral patch structure is polarized along the AP axis with respect to the number and morphology of innervated, MATH1-positive cells (Fig. 8J,K,L). Architecture of the medial sac patch is similar to that of the posterior lateral tube.
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Tbx1 specifies regional identity and establishes a fate boundary in the otocyst
It is hypothesized that compartment boundaries exist along the three major
axes of the otocyst (Fekete,
1996). At the lateral wall of the otocyst, a putative AP midline
boundary is defined by anterior expression of
NeuroD/Lfng/Fgf3 and posterior expression of
Otx1 (Fekete and Wu,
2002
). Otx1 exhibits particular attributes of an otocyst
selector gene. Its regional expression encompasses primordia of the
non-sensory cochlear duct, lateral canal and lateral crista, and it is
required for proper morphogenesis of these end organs and the
positioning/segregation of the lateral crista and other proximate sensory
epithelia (Morsli et al.,
1999
; Fritzsch et al.,
2001
). However, Otx1 otocyst expression is first detected
at E10.25, which precludes a role for this gene in early otocyst
patterning.
We find that Tbx1 expression is localized posterolaterally in the
invaginating placode and defines an AP midline expression border prior to
otocyst formation. Tbx1 expression is maintained in the
posterolateral otocyst through the time of Otx1 expression onset.
Apart from partial, transient overlap, Tbx1 and NeuroD
expression domains are complementary throughout otocyst stages. TBX1
gain-of-function displaces the NeuroD domain border anteriorly.
Tbx1 loss-of-function eliminates AP midline borders of NeuroD,
Lfng and Fgf3 expression as early as E9.5 and causes ectopic
posterolateral expression of all three genes. By contrast, Otx1
expression is not detected in Tbx1/
otocysts, and the posterolateral otocyst is a site of ectopic neural
progenitor delamination between E9.5 and E11.5. At later stages,
Otx1-dependent sensory organ structures are missing or
indistinguishable by morphological criteria. These results suggest that
Tbx1 patterns sensory organ and neural fate assignment in parallel,
through local control of regional gene expression
(Fig. 9B). Tbx1
appears to establish a midline boundary between an anterior region of combined
neural and sensory epithelial competence and posterior tissue destined to form
sensory organ structural components such as the lateral wall of the cochlear
duct and lateral canal plate (Fig.
9A). Ectopic expression of Ngn1 in the
Tbx1/ posterolateral otocyst further
supports this hypothesis, as Ngn1 activates NeuroD
expression and may mediate the selection of neural progenitors from otic
epithelium through Notch pathway signaling
(Ma et al., 1998).
|
Incomplete symmetry of the Tbx1/ ear
about the AP midline may also result from complex and dynamic changes of
Tbx1 expression border morphology during otocyst growth. At E10, the
dorsal part of the AP midline border deviates anteriorly from the midline. By
E11, it has shifted to a position perpendicular to the AP midline and forms a
dorsal-ventral border. During these stages, a stripe of Bmp4
expression co-localizes with Tbx1 expression at its interface with
the NeuroD/Lfng domain border
(Fig. 9A).
decapentaplegic (dpp), a Drosophila homolog of
Bmp4, is expressed in a narrow stripe of cells adjacent to the
imaginal wing disc AP compartment boundary and encodes a morphogen that
controls growth and patterning of the disc
(Nellen et al., 1996;
Lecuit et al., 1996
). The
existence of a DPP morphogen gradient is implied by expression of its target
genes, one of which is optomotor-blind, a T-box gene that controls
wing disc regional identity (Podos and
Ferguson, 1999
). Interestingly, in the otocyst, we find
high-intensity Tbx1 signal at all sites of Bmp4
expression.
In the mouse, Bmp4 expression marks sites of developing cristae
(Morsli et al., 1998), however
the effects of Bmp4 loss-of-function on inner ear development are
unknown because of early embryonic lethality
(Winnier et al., 1995
).
Perturbation of BMP signaling in the chick ear causes a range of sensory organ
defects, and vestibular canal morphogenesis is most frequently and severely
affected (Chang et al., 1999
;
Gerlach et al., 2000
).
Tbx1/ ears lack vestibular canals and it was
recently reported that Tbx1 is required for otocyst Bmp4
expression (Vitelli et al.,
2003
). Contrary to this result, we find that
Tbx1/ otocysts express Bmp4 but do
not form an anterior stripe and epithelial expression is lost by E11.
Tbx1+/ otocysts show normal patterning of
Bmp4 through E10.5 and a loss of the anterior stripe by E11. Thus,
Bmp4 patterning appears sensitive to Tbx1 gene dose in a
stage-specific manner. These results, together with the observation of
high-intensity Tbx1 signal at all sites of Bmp4 expression,
suggest that a complex regulatory interaction between Tbx1 and
Bmp4 is localized to a fate boundary in the anterodorsolateral
otocyst (Fig. 9A,B). Studies of
comparative expression and epistatic relationships between Tbx1 and
other regulators of Bmp4 patterning and vestibular canal
morphogenesis, such as Hmx2 (Wang
et al., 2001
), Hmx3
(Wang et al., 1998
) and
Dlx5 (Merlo et al.,
2002
) may elucidate cooperative interactions among these
genes.
Tbx1 suppresses neurogenesis at some prospective sensory epithelial territories
Evidence of a common progenitor for VIIIth ganglion neurons and
mechanosensory cells has been obtained by clonal analyses in chick
(Satoh and Fekete, 2003).
Furthermore, expression overlap of Lfng and neural fate markers in
both chick and mouse leads to the suggestion that neural progenitors and
utricular and saccular maculae derive from a common anterior otocyst region
(Fekete and Wu, 2002
;
Cole et al., 2000
). In the
wild-type anterior otocyst, we find overlapping Ngn1, NeuroD, and
Lfng expression that is complementary to the Tbx1 domain
(Fig. 9A). Neural bHLH gene
expression persists in this region through E11.5, the latest stage assayed for
these markers. Tbx1 loss-of-function has little to no effect on
neurogenic activity in this region and does not preclude the subsequent
development of anteroventral sensory epithelium. Thus,
Tbx1-independent pathways probably control neural and sensory
epithelial fate assignment at this otocyst region.
The Lfng-positive posteroventral otocyst is the presumptive anlage
of the organ of Corti (Fekete and Wu,
2002) and initially, this region is Tbx1-negative. We
show that transient wild-type expression of Ngn1 and NeuroD,
together with delamination, precedes the local onset of Tbx1
expression in this region (see Fig.
4A). Regression of posteroventral neurogenesis is delayed in
Tbx1 heterozygotes, and neurogenesis persists in this region through
E11.5 in Tbx1/ otocysts. Conversely,
TBX1 gain of function effectively eliminates posteroventral
neurogenesis. Interestingly, Tbx1 heterozygotes at E11 show delayed
regression of neurogenesis at the anterodorsolateral otocyst (see
Fig. 2I) and loss of a
definitive Bmp4 anterior stripe. These phenotypes are observed toward
the end of a period (E9.75-E11) during which Tbx1 expression expands
into the anterodorsolateral otocyst. Together these results suggest that at
some otocyst regions, Tbx1 regulates the developmental timing by
which neural and sensory epithelial competent states are expressed.
Functionally, this differs from the effect of Tbx1 activity at the
posterolateral otocyst, where neural competence is fully suppressed at all
times and sensory organ structural epithelium is formed.
Conclusion
In this study we have characterized the differential effects of
Tbx1 mutation on neurogenesis and inner ear sensory organ
development. Tbx1 specifies regional identity in the otocyst and is
required for the positioning of a fate boundary. Our data support the
hypothesis of a relationship between neural and sensory epithelial competence
in the otocyst. Furthermore, absence of Tbx1 causes expression of
neural competence in a portion of the otocyst associated with formation of
sensory organ structural epithelia. Taken together, our results suggest that
Tbx1 regulates otocyst gene expression locally but affects inner ear
growth and morphogenesis in a global manner. Tbx1 may therefore
function as an otocyst selector gene in its control of neurogenesis and
sensory organ development. Studies aimed at dissecting the contributions of
epithelial and mesenchymal Tbx1 activity to various aspects of inner
ear development using tissue-specific gene inactivation strategies are
currently in progress.
In Xenopus gastrula ectoderm, BMP signaling suppresses neural
competence and induces epidermal fate
(Weinstein and Hemmati-Brivanlou,
1999). In the mouse otocyst, the specific effects of Bmp4
on neural and sensory organ development and its range of action are unknown.
Nevertheless, co-localization of Bmp4 expression and robust ectopic
neurogenesis at the Tbx1/ otocyst posterior
pole through E10.5 provides a preliminary clue that Tbx1-mediated
neural suppression may proceed independently of Bmp4 activity.
Finally, the homeobox genes Eya1 and Six1
(Xu et al., 1999
;
Zheng et al., 2003
) may
function reciprocally to Tbx1 with respect to neurogenesis, as both
are expressed at regions of neural progenitor determination and are required
for VIIIth ganglion rudiment formation.
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
Footnotes |
---|
* Present address: Department of Cell and Molecular Biology, House Ear
Institute, 2100 W 3rd Street, Los Angeles, CA 90057, USA
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Acampora, D., Mazan, S., Avvantaggiato, V., Barone, P., Tuorto, F., Lallemand, Y., Brulet, P. and Simeone, A. (1996). Epilepsy and brain abnormalities in mice lacking the Otx1 gene. Nat. Genet. 14,218 -222.[Medline]
Adam, J., Myat, A., le Roux, I., Eddison, M., Henrique, D.,
Ish-Horowicz, D. and Lewis, J. (1998). Cell fate
choices and the expression of Notch, Delta, and Serrate homologues in the
chick inner ear: parallels with Drosophila sense-organ development.
Development 125,4645
-4654.
Adkins, W. Y. and Gussen, R. (1974). Temporal bone findings in the third and fourth pharyngeal pouch (DiGeorge) syndrome. Arch. Otolaryngol. 100,206 -208.[CrossRef]
Bamshad, M., Lin, R. C., Law, D. J., Watkins, W. C., Krakowiak, P. A., Moore, M. E., Franceschini, P., Lala, R., Holmes, L. B., Gebuhr, T. C. et al. (1997). Mutation in human TBX3 alter limb, apocrine and genital development in ulnar-mammary syndrome. Nat. Genet. 16,311 -315.[Medline]
Basson, C. T., Bachinsky, D. R., Lin, R. C., Levi, T., Elkins, J. A., Soults, J., Grayzel, D., Kroumpouzou, E., Traill, T. A., Leblanc-Straceski, J. et al. (1997). Mutations in human TBX5 cause limb and cardiac malformation in Holt-Oram syndrome. Nat. Genet. 15,30 -35.[Medline]
Bermingham, N. A., Hassan, B. A., Price, S. D., Vollrath, M. A.,
Nissim, B.-A., Eatock, R. A., Bellen, H. J., Lysakowski, A. and Zoghbi,
H. Y. (1999). Math1: An essential gene for the generation of
inner ear hair cells. Science
284,1837
-1841.
Black, F. O., Spanier, S. S. and Kohut, R. I. (1975). Aural abnormalities in partial DiGeorge syndrome. Arch. Otolaryngol. 101,129 -134.[Abstract]
Bollag, R. J., Siegfried, Z., Cebra-Thomas, J., Garvey, N., Davidson, E. M. and Silver, L. M. (1994). An ancient family of embryonically expressed mouse genes sharing a conserved protein motif with the T locus. Nat. Genet. 7, 383-389.[Medline]
Braybrook, C., Doudney, K., Marcano, A. C., Arnason, A., Bjornsson, A., Patton, M. A., Goodfellow, P. J., Moore, G. E. and Stanier, P. (2001). The T-box transcription factor gene TBX22 is mutated in X-linked cleft palate and ankyloglossia. Nat. Genet. 29,179 -183.[CrossRef][Medline]
Brigande, J. V., Kiernan, A. E., Gao, X., Iten, L. E. and
Fekete, D. M. (2000). Molecular genetics of pattern formation
in the inner ear: Do compartment boundaries play a role? Proc.
Natl. Acad. Sci. USA 97,11700
-11706.
Chang, W., Nunes, F., de Jesus-Escobar, J. M., Harland, R. M. and Wu, D. K. (1999). Ectopic noggin blocks sensory and nonsensory organ morphogenesis in the chicken inner ear. Dev. Biol. 216,369 -381.[CrossRef][Medline]
Chapman, D. L., Garvey, N., Hancock, S., Alexiou, M., Agulnik, S. I., Gibson-Brown, J. J., Cebra-Thomas, J., Bollag, R. J., Silver, L. M. and Papaioannou, V. E. (1996). Expression of the T-box family genes, Tbx1-Tbx5, during early mouse development. Dev. Dyn. 206,379 -390.[CrossRef][Medline]
Chapman, D. L. and Papaioannou, V. E. (1998). Three neural tubes in mouse embryos with mutations in the T-box gene Tbx6. Nature 391,695 -697.[CrossRef][Medline]
Chen, P., Johnson, J. E., Zoghbi, H. Y. and Segil, N. (2002). The role of Math1 in inner ear development: uncoupling the establishment of the sensory primordium from hair cell fate determination. Development 129,2495 -2505.[Medline]
Chesley, P. (1935). Development of the short-tailed mutation in the house mouse. J. Exp. Zool. 29,437 -438.
Cole, L. K., le Roux, I., Nunes, F., Laufer, E., Lewis, J. and Wu, D. K. (2000). Sensory organ generation in the chicken inner ear: contributions of Bone Morphogenetic Protein 4, Serrate1, and Lunatic Fringe. J. Comp. Neurol. 424,509 -520.[CrossRef][Medline]
D'Amico-Martel, A. and Noden, D. M. (1983). Contributions of placodal and neural crest cells to avian cranial peripheral ganglia. Am. J. Anat. 166,445 -468.[Medline]
Dahmann, C. and Basler, K. (1999). Compartment boundaries at the edge of development. Trends Genet. 15,320 -326.[CrossRef][Medline]
Day, S. J. and Lawrence, P. A. (2000).
Measuring dimensions: the regulation of size and shape.
Development 127,2977
-2987.
Fekete, D. M. (1996). Cell fate specification in the inner ear. Curr. Opin. Neurobiol. 6, 533-541.[CrossRef][Medline]
Fekete, D. M. and Wu, D. K. (2002). Revisiting cell fate specification in the inner ear. Curr. Opin. Neurobiol. 12,35 -42.[CrossRef][Medline]
Fritzsch, B., Beisel, K. W., Jones, K., Farinas, I., Maklad, A., Lee, J. and Reichardt, L. F. (2002). Development and evolution of inner ear sensory epithelia and their innervation. J. Neurobiol. 53,143 -156.[CrossRef][Medline]
Fritzsch, B., Signore, M. and Simeone, A. (2001). Otx1 null mutant mice show partial segregation of sensory epithelia comparable to lamprey ears. Dev. Genes Evol. 211,388 -396.[CrossRef][Medline]
Funke, B., Epstein, J. A., Kochilas, L. K., Lu, M. M., Pandita,
R. K., Liao, J., Bauerndistel, R., Schuler, T., Schorle, H., Brown, M.
C. et al. (2001). Mice overexpressing genes from the 22q11
region deleted in velo-cardio-facial syndrome/DiGeorge syndrome have middle
and inner ear defects. Hum. Mol. Genet.
10,2549
-2556.
Garcia-Bellido, A., Ripoll, P. and Morata, G. (1973). Developmental compartmentalization of the wing disk of Drosophila. Nature 245,251 -253.
George, K. M., Leonard, M. W., Roth, M. E., Lieuw, K. H.,
Kioussis, D., Grosveld, F. and Engel, J. D. (1994).
Embryonic expression and cloning of the murine GATA-3 gene.
Development 120,2673
-2686.
Gerlach, L. M., Hutson, M. R., Germiller, J. A., Nguyen-Luu, D.,
Victor, J. C. and Barald, K. F. (2000). Addition of
the BMP4 antagonist, noggin, disrupts avian inner ear development.
Development 127,45
-54.
Goldberg, M. E. and Hudspeth, A. J. (2000). The vestibular system. In Principles of Neural Science (ed. E. R. Kandel, J. H. Schwartz and T. M. Jessell), pp.801 -815. New York: McGraw-Hill.
Hadrys, T., Braun, T., Rinkwitz-Brandt, S., Arnold, H. H. and
Bober, E. (1998). Nkx5-1 controls semicircular canal
formation in the mouse inner ear. Development
125, 33-39.
Hammond, K. L., Loynes, H. E., Folarin, A. A., Smith, J. and
Whitfield, T. T. (2003). Hedgehog signaling is
required for correct anteroposterior patterning of the zebrafish otic vesicle.
Development 130,1403
-1417.
Helms, A. W. and Johnson, J. E. (1998).
Progenitors of dorsal commissural interneurons are defined by MATH1
expression. Development
125,919
-928.
Harrison, R. G. (1936). Relations of symmetry in the developing ear of Amblystoma punctatum. Proc. Natl. Acad. Sci. USA 22,238 -247.
Herrmann, B. G., Labeit, S., Pustka, A., King, T. R. and Lehrach, H. (1990). Cloning of the T gene required in mesoderm formation in the mouse. Nature 343,617 -622.[CrossRef][Medline]
Hudspeth, A. J. (2000). Hearing. In Principles of Neural Science (ed. E. R. Kandel, J. H. Schwartz and T. M. Jessell), pp. 801-815. New York: McGraw-Hill.
Irvine, K. D. and Wieschaus, E. (1994). fringe, a boundary-specific signaling molecule, mediates interactions between dorsal and ventral cells during Drosophila wing development. Cell 79,595 -606.[Medline]
Jerome, L. A. and Papaioannou, V. E. (2001). DiGeorge syndrome phenotype in mice mutant for the T-box gene, Tbx1. Nat. Genet. 27,286 -291.[CrossRef][Medline]
Karis, A., Pata, I., Hikke van Doorninck, J., Grosveld, F., de Zeeuw, C. I., de Caprona, D. and Fritzsch, B. (2001). Transcription factor GATA-3 alters pathway selection of olivocochlear neurons and affects morphogenesis of the inner ear. J. Comp. Neurol. 429,615 -630.[CrossRef][Medline]
Kawamoto, K., Ishimoto, S.-I., Minoda, R., Brough, D. E. and
Raphael, Y. (2003). Math1 gene transfer generates new
cochlear hair cells in mature guinea pigs in vivo. J.
Neurosci. 23,4395
-4400.
Kiernan, A. E., Nunes, F., Wu, D. K. and Fekete, D. M. (1997). The expression domain of two related homeobox genes defines a compartment in the chicken inner ear that may be involved in semicircular canal formation. Dev. Biol. 191,215 -229.[CrossRef][Medline]
Kiernan, A. E., Steele, K. P. and Fekete, D. M. (2002). Development of the mouse inner ear. In Mouse development (ed. J. Rossant and P. Tam), pp.539 -566. San Diego: Academic Press.
Kim, J., Irvine, K. D. and Carroll, S. B. (1995). Cell recognition, signal induction, and symmetrical gene activation at the dorsal-ventral boundary of the developing Drosophila wing. Cell 82,795 -802.[Medline]
Kim, W.-Y., Fritzsch, B., Serls, A., Bakel, L. A., Huang, E. J.,
Reichardt, L. F., Barth, D. S. and Lee, J. E. (2001).
NeuroD-null mice are deaf due to a severe loss of the inner ear sensory
neurons during development. Development
128,417
-426.
Kopp, A. and Duncan, I. (1997). Control of cell
fate and polarity in the adult abdominal segments of Drosophila by
optomotor-blind. Development
124,3715
-3726.
Lavoko-Kerali, G., Rivolta, M. N. and Holley, M. (2002). Expression of the transcription factors GATA3 and Pax2 during development of the mammalian inner ear. J. Comp. Neurol. 442,378 -391.[Medline]
Lawrence, P. A. and Struhl, G. (1996). Morphogens, compartments, and pattern: Lessons from Drosophila? Cell 85,951 -961.[Medline]
Lecuit, T., Brook, W. J., Ng, M., Calleja, M., Sun, H. and Cohen, S. M. (1996). Two distinct mechanisms for long-range patterning by Decapentaplegic in the Drosophila wing. Nature 381,387 -393.[CrossRef][Medline]
Lee, J. E., Hollenberg, S. M., Snider, L., Turner, D. L., Lipnick, N. and Wientraub, H. (1995). Conversion of Xenopus ectoderm into neurons by NeuroD, a basic helix-loop-helix protein. Science 268,836 -844.[Medline]
Li, Q. Y., Newbury-Ecob, R. A., Terrett, J. A., Wilson, D. I., Curtis, A. R., Yi, C. H., Gebuhr, T., Bullen, P. J., Robson, S. C., Strachan, T. et al. (1997). Holt-Oram syndrome is caused by mutation in TBX5, a member of the Brachyury (T) gene family. Nat. Genet. 15,21 -29.[Medline]
Lindsay, E. A., Vitelli, F., Su, H., Morishima, M., Huynh, T., Pramparo, T., Jurecic, V., Ogunrinu, G., Sutherland, H. F., Scambler, P. J. et al. (2001). Tbx1 haploinsufficiency in the DiGeorge syndrome region causes aortic arch defects in mice. Nature 410,97 -101.[CrossRef][Medline]
Liu, M., Pereira, F. A., Price, S. D., Chu, M.-J., Shope, C.,
Himes, D., Eatock, R. A., Brownell, W. E., Lysakowski, A. and Tsai,
M.-J. (2000). Essential role of BETA2/NeuroD1 in development
of the vestibular and auditory systems. Genes Dev.
14,2839
-2854.
Ma, Q., Anderson, D. J. and Fritzsch, B. (2000). Neurogenin1 null mutant ears develop fewer, morphologically normal hair cells in smaller sensory epithelia devoid of innervation. JARO 1,129 -143.[Medline]
Ma, Q., Chen, Z., del Barco Barantes, I., de la Ponpa, J. L. and Anderson, D. J. (1998). neurogenin1 is essential for the determination of neuronal precursors for proximal cranial sensory ganglia. Neuron 20,469 -482.[Medline]
Mansour, S. L., Goddard, J. M. and Capecchi, M. R.
(1993). Mice homozygous for a targeted disruption of the
proto-oncogene int-2 have developmental defects in the tail and inner ear.
Development 117,13
-28.
McKay, I., Lewis, J. and Lumsden, A. (1996). The role of FGF-3 in early inner ear development: an analysis in normal and kreisler mutant mice. Dev. Biol. 174,370 -378.[CrossRef][Medline]
Merlo, G. R., Paleari, L., Mantero, S., Zerega, B., Adamska, M., Rinkwitz, S., Bober, E. and Levi, G. (2002). The Dlx5 homeobox gene is essential for vestibular morphogenesis in the mouse embryo through a BMP4-mediated pathway. Dev. Biol. 248,157 -169.[CrossRef][Medline]
Merscher, S., Funke, B., Epstein, J. A., Heyer, J., Anne, P., Lu, M. M., Xavier, R., Demay, M. B., Russell, R. G., Factor, S. et al. (2001). TBX1 is responsible for cardiovascular defects in velo-cardio-facial/DiGeorge syndrome. Cell 104,619 -629.[Medline]
Milan, M. and Cohen, S. M. (2003). A
re-evaluation of the contributions of Apterous and Notch to the dorsoventral
lineage restriction boundary in the Drosophila wing.
Development 130,553
-562.
Morsli, H., Choo, D., Ryan, A., Johnson, R. and Wu, D. K.
(1998). Development of the mouse inner ear and origin of its
sensory organs. J. Neurosci.
18,3327
-3335.
Morsli, H., Tuorto, F., Choo, D., Pia Postiglione, M., Simeone,
A. and Wu, D. K. (1999). Otx1 and Otx2 activities are
required for the normal development of the mouse inner ear.
Development 126,2335
-2343.
Nellen, D., Burke, R., Struhl, G. and Basler, K. (1996). Direct and long-range action of a DPP morphogen gradient. Cell 85,357 -368.[Medline]
Nornes, H. O., Dressler, G. R., Knapik, E. W., Deutsch, U. and Gruss, P. (1990). Spatially and temporally restricted expression of Pax2 during murine neurogenesis. Development 109,797 -809.[Abstract]
Ohtani, I. and Schuknecht, H. F. (1984). Temporal bone pathology in DiGeorge's syndrome. Ann. Otol. Rhinol. Laryngol. 93,220 -224.[Medline]
Pauley, S., Wright, T. J., Pirvola, U., Ornitz, D., Beisel, K. W. and Fritzsch, B. (2003). Expression and function of FGF10 in mammalian inner ear development. Dev. Dyn. 227,203 -215.[CrossRef][Medline]
Pirvola, U., Spencer-Dene, B., Xing-Qun, L., Kettunen, P.,
Thesleff, I., Fritzsch, B., Dickson, C. and Ylikoski, J.
(2000). FGF/FGFR-2(IIIb) signaling is essential for inner ear
morphogenesis. J. Neurosci.
20,6125
-6134.
Pirvola, U., Ylikoski, J., Trokovic, R., Hebert, J., McConnell, S. and Partanen, J. (2002). FGFR1 is required for the development of the auditory sensory epithelium. Neuron 35,671 -680.[Medline]
Podos, S. D. and Ferguson, E. L. (1999). Morphogen gradients: new insights from DPP. Trends Genet. 15,396 -402.[CrossRef][Medline]
Riccomagno, M. M., Martinu, L., Mulheisen, M., Wu, D. K. and
Epstein, D. J. (2002). Specification of the mammalian
cochlea is dependent on Sonic hedgehog. Genes Dev.
16,2365
-2378.
Rubel, E. W. and Fritzsch, B. (2002). Auditory system development: primary auditory neurons and their targets. Annu. Rev. Neurosci. 25,51 -101.[CrossRef][Medline]
Satoh, T. and Fekete, D. M. (2003). Mechanosensory epithelial cells and ganglion cells are clonally related. Assoc. Res. Otolaryngol. 23 (Abstr. No. 1776).
Shailam, R., Lanford, P. J., Dolinsky, C. M., Norton, C. R., Gridley, T. and Kelly, M. W. (1999). Expression of proneural and neurogenic genes in the embryonic mammalian vestibular system. J. Neurocytol. 28,809 -819.[CrossRef][Medline]
Sher, A. E. (1971). The embryonic and postnatal development of the inner ear of the mouse. Acta Otolaryngologica, Suppl. 285,5 -77.
Theiler, K. (1989). The House Mouse: Atlas of Embryonic Development. New York: Springer.
Torres, M., Gomez-Pardo, E. and Gruss, P.
(1996). Pax2 contributes to inner ear patterning and optic nerve
trajectory. Development
122,3381
-3391.
Vitelli, F., Viola, A., Morishima, M., Pramparo, T., Baldini, A.
and Lindsay, E. A. (2003). TBX1 is required for inner
ear morphogenesis. Hum. Mol. Genet.
12,2041
-2048.
Wang, W., Chan, E. K., Baron, S., van de Water, T. R. and Lufkin, T. (2001). Hmx2 homeobox gene control of murine vestibular morphogenesis. Development 128,5017 -5029.[Medline]
Wang, W., van de Water, T. R. and Lufkin, T.
(1998). Inner ear and maternal reproductive defects in mice
lacking the Hmx3 homeobox gene. Development
125,621
-634.
Weinstein, D. C. and Hemmati-Brivanlou, A. (1999). Neural induction. Annu. Rev. Cell Dev. Biol 15,411 -433.[CrossRef][Medline]
Wilkinson, D. G. (1992). In Situ Hybridization: A Practical Approach. Oxford: IRL Press.
Wilkinson, D. G., Bhatt, S. and Herrmann, B. G. (1990). Expression pattern of the mouse T gene required in mesoderm formation in the mouse. Nature 343,617 -622.[CrossRef][Medline]
Winnier, G., Blessing, M., Labosky, P. A. and Hogan, B. L. M. (1995). Bone morphogenetic protein-4 is required for mesoderm formation and patterning in the mouse. Genes Dev. 9,2105 -2116.[Abstract]
Wu, D. K., Nunes, F. D. and Choo, D. (1998).
Axial specification for sensory organs versus non-sensory structures of the
chicken inner ear. Development
125, 11-20.
Xu, P.-X., Adams, J., Peters, H., Brown, M. C., Heaney, S. and Maas, R. (1999). Eya1-deficient mice lack ears and kidneys and show abnormal apoptosis of organ primordia. Nat. Genet. 23,113 -117.[CrossRef][Medline]
Yanagisawa, K. O., Fujimoto, H. and Urushihara, H. (1981). Effects of the Brachyury (T) mutation on morphogenetic movement in the mouse embryo. Dev. Biol. 87,242 -248.[Medline]
Zecca, M., Basler, K. and Struhl, G. (1996). Direct and long-range action of a wingless morphogen gradient. Cell 87,833 -844.[Medline]
Zheng, J. L. and Gao, W.-Q. (2000). Overexpression of Math1 induces robust production of extra hair cells in postnatal rat inner ears. Nat. Neurosci. 3, 580-586.[CrossRef][Medline]
Zheng, W., Huang, L., Zhu-Bo, W., Silvius, D., Tang, B. and Xu,
P.-X. (2003). The role of Six1 in mammalian auditory system
development. Development
130,3989
-4000.
Related articles in Development: