1 National Institute on Deafness and other Communication Disorders, 5 Research
Court, Rockville, MD 20850, USA
2 Division of Biology, 139-74, California Institute of Technology, Pasadena, CA
91125, USA
* Author for correspondence (e-mail: wud{at}nidcd.nih.gov)
Accepted 18 February 2005
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SUMMARY |
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Key words: Inner ear, Axis, Axial specification, Induction, Hindbrain, Rhombomere, Sonic hedgehog, SHH, Chicken
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Introduction |
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The vertebrate inner ears are paraxial structures that originate as
thickenings of the ectoderm, known as otic placodes, that form adjacent to
rhombomeres 5 (R5) and 6 of the hindbrain. Normal development of the inner ear
is dependent on the positional information provided by surrounding tissues,
including the neural tube (Kiernan et al.,
2002). Indeed, secreted signaling molecules, especially FGF family
members, expressed in the hindbrain and paraxial mesoderm are implicated in
otic placode induction (Ladher et al.,
2000
; Maroon et al.,
2002
; Phillips et al.,
2001
; Wright and Mansour,
2003a
). The otic placode, once committed, begins to invaginate to
form the otocyst, which undergoes elaborate morphogenesis to develop into a
structurally complex inner ear (Kiernan et
al., 2002
; Torres and
Giraldez, 1998
).
An important step during early inner ear development is acquisition of
axial identities from the surrounding tissues, which in turn influence the
positional information and development of all inner ear components
(Fekete and Wu, 2002). It is
not clear when otic tissue acquires its axial identity, and the timing of this
specification appears to vary across different species. In salamanders, the AP
axis of the inner ear appears to be specified during or shortly after otic
placode induction (Harrison,
1936
). In chicken, the AP axis appears to be specified later in
development (Wu et al., 1998
).
Nevertheless, results from both species indicate that the AP axis is specified
before the DV axis, suggesting that axial specification occurs in multiple
stages (Harrison, 1936
;
Wu et al., 1998
). The
mechanisms involved in acquiring axial identity, however, remain elusive in
both species.
The role of hindbrain in inner ear development has been well established
(Kiernan et al., 2002).
Hindbrain mutants with defects in the region of R5 are often associated with
inner ear malformations. Therefore, it is possible that the hindbrain confers
AP axial identity to the inner ear. Nevertheless, it is not clear from the
typical hindbrain mutants such as Hoxa1/ and
kreisler whether their inner ear defects reflect a failure in AP patterning
(Gavalas et al., 1998
;
Ruben, 1973
). Furthermore, as
the border between R5 and R6 corresponds to the AP midline of the otocyst, it
has been postulated that unique signals from each rhombomere may provide AP
axial information required for inner ear patterning
(Brigande et al., 2000
).
Although the role of the hindbrain in AP patterning of the inner ear is not
clear, studies from inner ear analysis of Shh knockout mice have
implicated the hindbrain in DV patterning of the inner ear
(Riccomagno et al., 2002). The
absence of ventral inner ear structures in
Shh/ mutants prompted the proposal that SHH
secreted from the ventral midline (floor plate and notochord) is required to
pattern the ventral axis of the inner ear
(Riccomagno et al., 2002
),
similar to its role in the specification of ventral somites
(Lassar and Munsterberg,
1996
). However, a more recent study shows that immunoreactivity of
SHH, as well as low levels of its mRNA (using RT-PCR) are detected in the
mouse otocyst, unlike the situation in the somites
(Liu et al., 2002
). Thus, an
issue remains of which source of SHH, the ventral midline or the otic tissue
itself, is more important for patterning the ventral axis of the inner ear.
Furthermore, as formation of multiple organs are disrupted in
Shh/ mutants, including the notochord
(Chiang et al., 1996
), it is
possible that the inner ear phenotype observed in
Shh/ mutant is compounded by the loss of
other structures that are also required for ear development.
In this study, we have tested the roles of hindbrain in axial specification of the inner ear in ovo. We demonstrate that the hindbrain is nonessential for AP but crucial for DV patterning of the inner ear. Furthermore, we show that SHH secreted from either the floor plate or notochord is required for ventral patterning of the inner ear.
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Materials and methods |
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Hindbrain rotations
Embryos at embryonic day 1.5 (E1.5) equivalent to 10-13 somite stages (ss)
or Hamburger Hamilton stage 10-11 (HH 10-11) were used
(Hamburger and Hamilton,
1951). A segment of the neural tube including the notochord
between R4 and R7 was surgically separated from its surrounding tissues using
a microsurgical blade. The separated tissue was rotated either vertically (DV
rotation) or horizontally (AP rotation) in ovo. The operated embryos were
further incubated and subsequently harvested at E2.5-E3 for gene expression
analysis using whole-mount in situ hybridization, or harvested at E7 for
anatomical analysis using the paint fill technique
(Bissonnette and Fekete,
1996
).
Ablation of the midline structures
To ablate the ventral midline structures from 10-13 ss embryos, the neural
tube and the notochord between R4-R7 were removed by making horizontal slits
at R3-R4 and R7-R8 junctions, as well as longitudinal slits along both sides
of the neural tube between R4 to R7. The dissected tissues were transferred to
a Petri dish containing PBS, and the ventral region of the neural tube,
including the notochord, was severed from the neural tube. The remaining
dorsal part of the neural tube was returned to the embryo. To ablate the
notochord alone, the isolated neural tube and notochord were transferred to a
Petri dish containing 25% (v/v) dispase (Roche) in PBS, and the tissue was
triturated through a narrow pipette tip until the notochord was separated from
the neural tube. Only the neural tube was returned to the original embryo. To
ablate the neural tube alone, a micro-surgical blade was carefully inserted
between a partially freed neural tube segment and the underlying notochord in
ovo, and the neural tube was then dislodged using a back and forth motion of
the surgical blade.
Otic tissue transplantation
To reverse the AP axis of the otic tissue, a right otic cup of the host
embryo was replaced with a left otic cup from an age-matched donor embryo at
11-16 ss (Wu et al., 1998). To
guide the orientation and tracking of the transplanted tissue in host embryos,
0.05% CM-DiI (Molecular Probes) in 300 mM sucrose solution was injected into
the anterior region of the left otic cup before transplantation. The
transplanted otic tissues were monitored using a fluorescent microscope 24
hours after surgery, and embryos with appropriately transplanted otic tissues
were further incubated and harvested as indicated.
Hybridoma cell implantation
Hybridoma cells secreting anti-SHH antibody (5E1) and anti-GAG antibody
(3C2; Developmental Studies Hybridoma Bank, University of Iowa, IA) were
labeled with 0.05% CM-DiI for 10 minutes at 37°C, washed three times with
PBS, and resuspended in 300 mM sucrose solution containing 0.1% Fast Green
(Sigma-Aldrich). The labeled cells were implanted either underneath the neural
tube of E1.5 embryos or into the mesenchyme beneath the otocyst of E2.5
embryos using a pulled glass micropipette. The implanted cells were monitored
using a fluorescent microscope.
Whole-mount in situ hybridization
Whole-mount in situ hybridizations were performed as described
(Wu and Oh, 1996). Riboprobes
for Lfng (Laufer et al.,
1997
), NeuroD
(Roztocil et al., 1997
),
EphrinA4 (EphA4) (Patel
et al., 1996
), Hoxb1
(Guthrie et al., 1992
),
Shh (Echelard et al.,
1993
), Six1 (Heanue
et al., 1999
) and Gbx2
(Niss and Leutz, 1998
) were
prepared as previously described. A 562 bp of 3'-untranslated region
(nucleotides 1183-1745) of chicken Otx2 (GenBank: AJ489221) was used
to generate an antisense probe for Otx2.
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Results |
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SHH from the ventral midline structures is required for inner ear patterning
To confirm that SHH is indeed the key molecule from the midline structures
required for ventral inner ear patterning, we specifically inhibited its
function in vivo using a function-blocking antibody. Hybridoma cells secreting
anti-SHH antibody (5E1) (Ericson et al.,
1996; Wechsler-Reya and Scott,
1999
) or control anti-viral gag antibody (3C2)
(Stoker and Bissell, 1987
)
were injected underneath the hindbrain region between R4-R7 at E1.5 (10-13 ss,
HH 10-11; Fig. 5A). To
facilitate visualization of hybridoma cells, they were pre-labeled with DiI
before implantation and assessed 1 day after surgery
(Fig. 5B,C). By E7, the inner
ears from 5E1-implanted embryos show a complete absence of ventral structures
including the basilar papilla and saccule
(Fig. 5D; n=10),
whereas embryos implanted with control hybridoma cells have no obvious
malformations (data not shown). This inner ear phenotype elicited by 5E1 cells
closely resembles embryos from which ventral midline structures are ablated
(compare Fig. 4F with
5D). These results demonstrate
that SHH expressed by the notochord or floor plate is indeed important for
ventral patterning of the inner ear.
|
Signals from the hindbrain play a major role in the DV specification of the inner ear
Next, we tested the importance of hindbrain signaling in establishing the
DV axis of the inner ear. As SHH expressed from the ventral midline is
critical for the ventral inner ear patterning (Figs
4,
5), we investigated whether the
ventral neural tube and the notochord are sufficient to confer ventral fates
in dorsal otic tissues. This was accomplished by surgically rotating the
neural tube between R4-R7 (including the notochord) along its horizontal axis
between 10-13 ss (Fig. 6A), an
age well before DV axis is specified in the inner ear
(Wu et al., 1998). Presumably,
such an operation would allow dorsal otic tissues to receive ventral signals
from the rotated hindbrain in the presence of other potential dorsalizing
signals from tissues such as the ectoderm and mesenchyme. The success of
neural tube rotation was verified by examining the changes in gene expression
in the neural tube 24 to 36 hours after surgery (E2.5-E3). In embryos in which
the neural tube is rotated dorsoventrally, Shh expression is observed
dorsally, closest to the dorsal (Fig.
6C,E) rather than the ventral region of the otocyst
(Fig. 6B,D). Likewise, genes
that are normally expressed in the dorsal neural tube such as Bmp5
and Msx1 are observed ventrally (data not shown). However, the
original DV identity of the rotated neural tube is no longer maintained 48
hours after surgery, as evidenced by the loss of Shh expression in
the dorsal region of the neural tube (data not shown). Therefore, the DV axial
specification of the inner ears in the neural tube-rotated embryos was
assessed prior to E3.5.
|
We further assessed the dorsal fates of these inner ears using the
expression pattern of Gbx2, which is normally associated with the
dorsomedial region of the otocyst (Fig.
7A,D) (Hidalgo-Sanchez et al.,
2000). Consistent with the expression patterns of ventrally
expressed genes, Gbx2 expression in the dorsal otic tissues is
abolished in the operated embryos, indicating the loss of dorsal fates
(Fig. 7B,E; n=14).
However, Gbx2 expression is not induced in the ventral otocyst of
embryos with dorsoventrally rotated neural tube. Presumably, this is due to a
considerable increase in distance between the original dorsal neural tube
tissue and the otic epithelium after rotation
(Fig. 7D,E; vertical bar).
Interestingly, in some specimens, ectopic otocysts are present ventrally due
to the inadvertent translocation of a part of the otic epithelium during
neural tube rotation and Gbx2 expression is induced in these ectopic
ventrally positioned otocysts (Fig.
7C,F; arrows; n=9). This suggests that intrinsic signals
in the dorsal neural tube are sufficient to confer dorsal otic fates, despite
potential ventral signals from other surrounding tissues. Taken together,
these experiments demonstrate the importance of the hindbrain in establishing
the DV axis of the inner ear.
|
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Discussion |
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Roles of the hindbrain in AP axial specification of the inner ear
In all vertebrates, the inner ear develops in approximately the same
position along the body axis, adjacent to R4-R6. Cooperative signals emanating
from adjacent tissues including the hindbrain, mesoderm and endoderm are
likely to contribute to the location of the ear
(Baker and Bronner-Fraser,
2001). In particular, signals from the hindbrain have been
implicated not only in induction but also in the later morphogenesis of the
inner ear (Kiernan et al.,
2002
; Torres and Giraldez,
1998
; Whitfield et al.,
2002
). For example, hindbrain mutants such as
Hoxa1/ and kreisler that are missing R5,
display severe inner ear malformations
(Gavalas et al., 1998
;
Ruben, 1973
). However, despite
the malformations, the AP axial identity of the inner ears appears to be
unaffected (Gavalas et al.,
1998
) (D. Choo, personal communication). Our results indicate that
the hindbrain has no major function in conferring AP axial identity to the
otic tissue. Reversing the AP orientation of the hindbrain of R4 to R7 prior
to axial specification of the ear had no adverse effect on inner ear
development even when larger regions of the hindbrain were rotated (data not
shown). Taken together, these observations demonstrate that despite the
important role of rhombomeres in inner ear development, they are nonessential
for conferring AP axial information to the inner ear. Thus far, tissue(s)
responsible for conveying AP axial identity to the inner ear remains
unknown.
SHH signaling from the ventral midline is required for inner ear patterning
Analyses of Shh-knockout mice demonstrate that SHH is required for
ventral patterning of the inner ear
(Riccomagno et al., 2002).
However, the source of SHH, whether from the ventral midline or the otic
tissue itself, was not clear (Liu et al.,
2002
). Using microsurgical ablation and localized inhibition with
function-blocking antibodies in chicken, we demonstrate that SHH from the
ventral midline is the primary signal for inner ear patterning in chicken,
with either the floor plate or the notochord being sufficient to mediate this
function. These conclusions are similar to those drawn in zebrafish
(Hammond et al., 2003
).
Consistent with this idea, inner ear morphology is normal in
Gli2-null mice that lack the floor plate
(Ding et al., 1998
;
Matise et al., 1998
) (D.
Epstein and D.K.W., unpublished). The sufficiency of SHH from floor plate or
notochord in conferring ventral inner ear fates is similar to their role in
induction of ventral properties in somites
(Brand-Saberi et al., 1993
;
Pourquie et al., 1993
).
Although we demonstrate that SHH from the midline is the primary source for
establishing ventral inner ear patterning, these results do not preclude the
possibility that SHH expressed within the otic tissues
(Liu et al., 2002
) and
cochlear-vestibular ganglion may play a later role in inner ear
development.
In zebrafish, Hedgehog signaling is required for AP patterning of
the inner ear (Hammond et al.,
2003), yet AP patterning in mouse
Shh/ inner ears appears normal
(Riccomagno et al., 2002
).
Similarly, we do not observe any AP patterning defects in chicken inner ears
in which the SHH signaling is disrupted. This suggests that the mechanisms for
axial patterning in zebrafish are different from those of mice and chicken.
Furthermore, in chicken embryos where the SHH signaling is disrupted, the
dorsal region of the inner ear is normal. These results are also different
from those observed in Shh/ mice, which
include dorsal inner ear defects
(Riccomagno et al., 2002
). As
all axes of the Shh/ mutants are correctly
established initially, except for the ventral axis, we attribute these dorsal
phenotypes observed in Shh mutants as secondary defects, possibly
owing to disorganization of the neural tube
(Riccomagno et al., 2002
).
Possible mechanism of SHH function on inner ear patterning
How does SHH expressed in the ventral midline exert its effect on inner ear
tissues? One possibility is that it travels from the midline and acts on otic
tissues directly. Alternatively, it may mediate its effect indirectly by
activating genes in the mesoderm, which in turn induce ventral inner ear
development. There is evidence to support both scenarios. Both
Brn4/Pou3f4 and Tbx1 expression levels in the mesoderm
surrounding the inner ear have been shown to be regulated by SHH
(Riccomagno et al., 2002).
Although the specific role of mesodermal Tbx1 in inner ear
development is unclear, the lack of Brn4/Pou3f4 in the mesoderm
results in cochlear defects in mice
(Phippard et al., 1999
;
Raft et al., 2004
;
Vitelli et al., 2003
).
However, SHH has been shown to be capable of traveling considerable distances
to exert its function in other systems
(Goetz et al., 2002
). Several
lines of evidence favor the idea of direct action of SHH on otic tissue.
First, genes known to directly respond to SHH signaling, such as
Patched and Gli1, are expressed in the otic epithelium of
both mice and chicken (Ozaki et al.,
2004
; Riccomagno et al.,
2002
) (J.B., unpublished). Second, the expression of these
SHH-responsive genes is upregulated when Shh is ectopically expressed
in otic tissue in mice (Riccomagno et al.,
2002
) or when SHH-soaked beads are implanted to the developing
otocysts in chicken (J.B., unpublished). Third, locally blocking SHH signaling
beneath the otocyst at a later stage is sufficient to block ventral inner ear
patterning (Fig. 5), further
supporting the idea that SHH acts directly on the otic epithelium. These late
implantation results also suggest that SHH action on otic tissue is required
continuously. This is consistent with previous studies showing that
specification of the DV axis, which is dependent on SHH, occurs well after
otocyst formation (Wu et al.,
1998
).
SHH traveling from a source to a target area sets up a concentration
gradient that is highest at the source and gradually decreases with the
distance away (Goetz et al.,
2002). This concentration gradient of SHH is thought to be an
important mechanism in other systems for conveying unique positional
information to cells and tissues located various distances from a source
(Goetz et al., 2002
). For
example, it has been shown in the neural tube that different types of neurons
are induced in response to a graded SHH concentration
(Ericson et al., 1997
;
Ericson et al., 1996
).
Similarly, a SHH concentration gradient has been shown to be important for
correct digit patterning during limb development
(Yang et al., 1997
). SHH may
play a similar role in patterning the fine structure of the cochlea. The
vertebrate cochlea is a tonotopically organized structure, such that each
region of the cochlea is sensitive to a specific range of sound frequencies,
with the base of the cochlea most sensitive to high frequency sounds and the
apex to low frequency sounds (Davis,
2003
). This tonotopic organization is reflected by a gradient of
morphological and physiological differences along the cochlea
(Davis, 2003
). Since SHH
function is crucial for the formation of the cochlea, an intriguing
possibility is that a concentration gradient of SHH protein could be
established during initiation of the cochlear outgrowth and may underlie the
structural bases for the tonotopic organization that develops later.
Signals from the hindbrain play a major role in the DV axial specification of the inner ear
The present results show that rotating the DV axis of the neural tube,
including the notochord is sufficient to convert presumably dorsal otic tissue
to ventral fates, resulting in downregulation of dorsally expressed genes such
as Gbx2 and upregulation of ventral specific genes such as Lfng,
NeuroD, Six1 and Otx2 in dorsal otic tissues (Figs
6,
7). Similar results were
obtained when the neural tube was rotated without the notochord (see Fig. S1
in the supplementary material), suggesting that the ventral neural tube alone
is sufficient to confer the ventral inner ear fate, overriding any potential
dorsalizing signals from surrounding ectodermal or mesodermal tissues.
Concomitantly, most of the ventrally expressed genes, except Otx2 are
downregulated in the ventral otic tissue after DV rotation of the hindbrain
(Fig. 6). It is not yet clear
if ventral Otx2 expression is sustained in the absence of SHH
signaling, although in mice ventral Otx2 expression is dependent on
Shh (Riccomagno et al.,
2002).
We did not observe upregulation of Gbx2 in the ventral otocyst
after DV axial rotation of the neural tube. We speculate that this is not
because the dorsal neural tube is incapable of providing signals to confer
dorsal fate to ventral otic tissue, but rather reflects the considerable
increase in distance between the two tissues after rotation. This speculation
is supported by the observation that Gbx2 is upregulated in
ectopically located otic tissues adjacent to the rotated dorsal neural tube.
Taken together, these results suggest that even though the ectopic otic
tissues are in a ventral environment, signals from the dorsal neural tube are
still sufficient to upregulate a dorsally expressed gene in a ventrally
located position. What might be the dorsalizing signals? In the neural tube
(Mehler et al., 1997;
Muroyama et al., 2002
),
secreted signaling molecules from the dorsal neural tube such as BMPs and WNTs
are sufficient to confer dorsal fates in the neural tube as well as their
adjacent somites (Fan et al.,
1997
; Marcelle et al.,
1997
). Interestingly, preliminary data suggest that WNT signaling
from the dorsal neural tube might play an essential role in specification of
the dorsal fate of the inner ear in mice (D. Epstein, personal
communication).
Recently, Six1 has been implicated in the ventral patterning of
the inner ear in mice. Lack of Six1 causes apoptosis of ventral otic
tissues and a ventral expansion of dorsal-specific genes
(Ozaki et al., 2004;
Zheng et al., 2003
). However,
the two signaling pathways that regulate ventral inner ear patterning,
Shh and Six1, appear independent. Six1 expression
in the otic tissue is not affected in Shh-null mutants, and the
expression of SHH target genes in the otic epithelium are normal in
Six1-null mutants (Ozaki et al.,
2004
). Six1 expression in the otic epithelium is
regulated by Eya1 (Ozaki et al.,
2004
), although little is known about how Eya1 expression
is regulated in the inner ear. Interestingly, our data show that Six1
expression in the otocyst is shifted after DV neural tube rotation, suggesting
that the Six1 expression in the otic tissue is also controlled by
signal(s) from the neural tube.
In summary, our data show that there is a temporal difference between
placode induction and axial specification in the chicken inner ear, suggesting
that there may be distinct regulatory mechanisms for these two processes.
Although multiple tissues appear to be capable of inducing placode formation,
the specification of DV axis is mainly conferred by the hindbrain. Even though
signals from specific segments of the hindbrain (R4-R6) participate in placode
induction (Wright and Mansour,
2003b) and development of the inner ear at later stages
(Kiernan et al., 2002
), our
data show that they do not play a role in conferring AP axial identity to the
inner ear.
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ACKNOWLEDGMENTS |
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Footnotes |
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Supplementary material for this article is available at http://dev.biologists.org/cgi/content/full/132/9/2115/DC1
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