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First published online 1 February 2006
doi: 10.1242/dev.02267
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Gonda Department of Cell and Molecular Biology, House Ear Institute, 2100 West 3rd Street, Los Angeles, CA 90057, USA.
* Author for correspondence (e-mail: agroves{at}hei.org)
Accepted 28 December 2005
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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 placode, Fgf, Competence, Induction, Chick
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INTRODUCTION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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; Streit,
2004). Several lines of evidence support the idea that
craniofacial placodes may derive from a common, `pre-placodal' field during
early development. First, morphological studies in a variety of vertebrates
suggest that some, or sometimes all placodes can derive from a single
epithelial thickening on the head (Braun,
1996; Knouff,
1935; Miyake et al.,
1997; O'Rahilly and
Müller, 1985; Platt,
1896; van Oostrom and
Verwoerd, 1972; Verwoerd and
van Oostrom, 1979). Second, embryological experiments in which
cranial ectoderm adjacent to the anterior neural plate was rotated along the
anteroposterior axis demonstrated that nasal, lens and otic placodes are not
determined in the young embryo, and that presumptive or `pre-placodal' tissue
is competent to generate a variety of cranial placodes if transplanted at an
appropriately early age (Jacobson,
1963c; Jacobson,
1966). Third, fate-mapping experiments in chick and zebrafish show
that cells destined to give rise to different cranial placodes are
intermingled in young embryos, suggesting that different placodes arise from
common ectodermal domains (Bhattacharyya et
al., 2004; Kozlowski et al.,
1997; Streit,
2002; Whitlock and
Westerfield, 2000). Finally, the expression of a variety of genes
in a border region around the neural plate from which cranial placodes derive
has provided molecular evidence for the existence of a genetically distinct
pre-placodal domain (reviewed by Groves,
2005; Streit,
2004).
The functional significance of a genetically distinct pre-placodal identity
is not clear at present. For example, it is not known whether the competence
to form cranial placodes is contingent upon the assumption of pre-placodal
identity, or whether naïve ectoderm can differentiate into particular
placodal derivatives without first expressing pre-placodal genes. It has been
suggested that placode induction proceeds in a series of stages, in which an
initial induction is required to instill some form of common placodal identity
in cranial ectoderm, followed by subsequent local inductive interactions which
single out individual placodes (Baker and
Bronner-Fraser, 2001; Streit,
2004). The advent of molecular markers has now rendered this
question experimentally tractable, and in the present study we have used the
induction of the otic placode as a test of this sequential model.
The otic placode forms adjacent to the posterior half of the hindbrain, and
will ultimately give rise to the entire inner ear and the vestibulo-acoustic
ganglion (Barald and Kelley,
2004; Brown et al.,
2003; Riley and Phillips,
2003; Torres and Giraldez,
1998; Whitfield et al.,
2002). Studies in a variety of vertebrates demonstrate the primacy
of Fgf signaling in the induction of the otic placode. Several Fgf family
members are expressed in tissues adjacent to the presumptive otic placode in
different species, such as Fgf3, Fgf4, Fgf8, Fgf10 and Fgf19
(Karabagli et al., 2002;
Kil et al., 2005;
Ladher et al., 2000;
Mahmood et al., 1995;
Maves et al., 2002;
Shamim and Mason, 1999;
Wright et al., 2003). Some of
these family members are necessary for placode induction; for example,
Fgf3 and Fgf8, and Fgf3 and Fgf10 are
necessary for placode induction in zebrafish and mouse respectively
(Leger and Brand, 2002;
Liu et al., 2003b;
Maroon et al., 2002;
Phillips et al., 2001;
Solomon et al., 2004;
Wright and Mansour, 2003).
Mis-expression of Fgf family members can also induce ectopic placodes in
amphibians and chick (Lombardo et al.,
1998; Vendrell et al.,
2000), although it has yet to be determined whether this is a
direct action of Fgf on presumptive placodal ectoderm, as opposed to an
indirect upregulation of other inducing signals. Fgf19 has also been proposed
to induce otic markers in co-operation with Wnt8c in chick embryos
(Ladher et al., 2000).
To determine the relationship between the existence of a pre-placodal domain and the induction of the otic placode by Fgf family members, we undertook an analysis of Fgf-mediated induction of the otic placode in chick embryos. In the present study, we show that Fgf signaling is both necessary and sufficient for the induction of some, but not all markers of the otic placode, and that Fgf acts directly on presumptive placodal ectoderm to induce placodal markers. In a series of grafting experiments, we demonstrated that the competence of embryonic ectoderm to express otic placode markers in response to Fgf signaling correlates with the expression of genes that define the pre-placodal region. This work thus provides the first functional evidence for a sequential, two-step model of placode induction, and suggests a molecular basis for the competence of ectoderm to give rise to the otic placode.
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MATERIALS AND METHODS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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).
Explant cultures
Head chunks comprising the prospective otic ectoderm were dissected from
the 0- to four-somite stage (HH stage 6 to stage 8) and five- to eight-somite
stage embryos (HH stage 9) in Ringer's solution using 30-gauge hypodermic
needles. Tissues were stored in complete medium on ice (10% fetal bovine serum
in L15 medium) until required, then rinsed three times in Ringer's before
transplanting into collagen gels. To isolate ectodermal tissue from 0- to
four-somite stage chicks, embryos were treated with 0.1 mg/ml dispase in
DMEM/F12 medium for 15 minutes on ice and 5 minutes at 37°C. The embryos
were allowed to recover for at least 10 minutes on ice in complete medium (10%
fetal bovine serum in L15 medium) and were then transferred to Ringer's
solution. Using a 30-gauge hypodermic needle, ectodermal tissue was isolated
either from prospective otic ectoderm, prospective trigeminal ectoderm or
lateral tissue from the level of prospective trigeminal ectoderm. The
dissected tissues were kept in complete medium on ice until required. Tissues
were rinsed three times in Ringer's solution prior to transfer into collagen
gels. Embryonic anterior epiblast was isolated from HH stage 3+ to 4 quail and
chick embryos by transferring yolks to PBS and cutting the yolk
circumferentially slightly above the equator, peeling the embryo from the yolk
with forceps, and peeling the blastoderm off the vitelline membrane.
Blastoderms were pooled in Ringer's solution containing 5 mM EDTA for 5
minutes at room temperature to enable easier dissection. An area comprising
the anterior 25% of the region between Hensen's node and the anterior extent
of the zona pellucida was isolated with 30-gauge hypodermic needles, and the
epiblast freed from underlying tissue. Epiblasts were stored in Ringer's
solution on ice until required, and then cut to appropriately sized pieces
immediately before transfer.
Quail-chick grafts
Chick embryos were cultured using the filter paper carrier method developed
by Chapman and colleagues (Chapman et al.,
2001). Eggs were cracked into a glass Petri dish and the thick
albumen covering the blastoderm removed using a piece of folded tissue paper.
A filter paper with a central aperture of 
0.5 inches was placed on top of
the blastoderm. The vitelline membrane was cut around the paper and the embryo
and filter paper carefully lifted from the yolk using forceps and pooled in
Ringer's solution. The blastoderm was peeled off the vitelline membrane which
was placed on a 35 mm agar albumen plate
(Chapman et al., 2001) still
attached to the filter paper. The area opaca was cut off the embryo and the
embryo placed back on the vitelline membrane, dorsal side upwards. The graft
site was prepared using a pulled glass needle. The graft was transferred from
donor (quail) to host (chick) with a glass mouth pipette, and the graft
positioned dorsal side upwards using a blunt pulled glass needle. The lid was
placed on the agar albumen plate and immediately incubated at 37°C in a
covered 150 mm Petri dish with moistened tissue paper lining the base. Some
embryos were sacrificed after 4, 8 or 24 hours and fixed overnight at 4°C
in 4% paraformaldehyde. Other embryos were transferred to Ringer's solution
after 2 or 8 hours, and the graft removed with 30 gauge hypodermic needles and
cultured in collagen gels for 12 hours. Grafts placed into chick hosts for up
to 8 hours could be removed by mechanical dissection with little or no
contamination of mesodermal or neural tissue, obviating the need for enzymatic
treatment prior to dissection.
Collagen gel cultures
Collagen matrix gels were prepared as previously described
(Groves and Bronner-Fraser,
2000). Briefly, 900 µl of collagen solution, 100 µl of
10xMEM were combined. A few drops of 7.5% sodium bicarbonate were added
to adjust the pH to 7.5. For head chunk cultures, 10 µl drops of the
prepared collagen solution were plated and allowed to set. DMEM-BS medium (1
ml) [a modification of the chemically defined medium of Bottenstein and Sato
(Bottenstein and Sato, 1979;
Wolswijk and Noble, 1989] was
added with either SU5402 or DMSO vehicle. Slits were cut in the gel, and the
heads slipped in. Cultures were grown at 37°C and 5% CO2 for 24
hours. Cultures were fixed in 4% paraformaldehyde overnight at 4°C. For
ectoderm explants, 5 µl drops of the prepared collagen solution were plated
and allowed to set. When set, 1 ml of DMEM-BS medium was added together with
50 ng/ml or 100 ng/ml Fgfs. The following Fgfs were used: Fgf1, Fgf2, Fgf4,
Fgf5, Fgf6, Fgf7, Fgf8, Fgf9, Fgf10, Fgf16, Fgf17, Fgf18 and Fgf19. Explants
were collected with a mouth pipette and placed in the collagen matrix. Each
gel contained 6-10 explants. Cultures were grown at 37°C and 5%
CO2 for 4, 6, 7.5, 21 or 24 hours and fixed in 4% paraformaldehyde
overnight at 4°C. Explants were processed for immunocytochemistry or
dissected free of the gel for in situ hybridization.
Immunocytochemistry
Fixed embryos or fixed gels containing the cultured heads were equilibrated
in PBS containing 30% sucrose and mounted and frozen in OCT. Sections (12
µm) were collected on Superfrost Plus slides (Fisher) and stored at
-20°C. Slides were washed twice with PBS containing 0.1% Triton X-100 and
blocked in PBS containing 0.1% Triton X-100 and 5% goat serum for 2-3 hours.
Primary antibodies were diluted in blocking buffer and applied overnight at
4°C. The slides were washed twice in PBS/0.1% Triton X-100 and secondary
antibodies were diluted in blocking solution and applied for 45 minutes at
room temperature. Slides were washed twice in PBS/0.1% Triton X-100 and
incubated in DAPI solution for 10 minutes, then washed in PBS before being
mounted in Fluoromount G (Southern Biotechnology). The following antibodies
were used in this study: a rabbit polyclonal antibody to Pax2 (Zymed; 1:500);
a rabbit polyclonal antibody to Epha4 (a gift from Elena Pasquale; 1:1000); a
rabbit polyclonal antibody to pan-Distalless proteins (a gift from Grace
Boekhoff-Falk and Jhumku Kohtz, 1:1000)
(Panganiban et al., 1997); and
Pax3 and QCPN IgG1 monoclonal antibodies (DSHB, University of Iowa, USA; 1:1).
For immunocytochemistry against Dlx3, the slides containing the explants were
blocked in PBS containing 10% donkey serum and incubated in 1:1500 Dlx3 goat
polyclonal antibody (a gift from Sujata Bhattacharyya) overnight at 4°C.
Secondary antibodies (Molecular Probes) were diluted in blocking buffer and
applied for 2 hours at room temperature. Slides were washed four times in PBS
between each incubation.
In situ hybridization
We performed whole-mount in situ hybridization and cRNA probe synthesis
according to the protocol of Stern (Stern,
1998) with modifications as described by Kil et al.
(Kil et al., 2005).
Whole-mount in situ hybridization and QCPN antibody staining was performed as
described previously (Stern,
1998). The probes used in this study were obtained from the
following sources: chick Pax2 (Domingos Henrique), Sip1
(Claudio Stern), Bmp7 (Brian Huston), Krox20 (David
Wilkinson), Wnt8c (Jane Dodd), Sax1 and tailless (Kate
Storey), Brachyury (Susan Mackem), Eya2 (Guillermo Oliver), and
Zic2 (Cliff Tabin).
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RESULTS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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;
Ladher et al., 2005;
Represa et al., 1991;
Vendrell et al., 2000). To
determine whether Fgf signaling is necessary for otic placode induction, we
isolated chunks of embryonic head tissue containing presumptive otic placode
ectoderm, neural tube, mesoderm and endoderm from a region corresponding to
the hindbrain level of 0- to eight-somite stage (ss) embryos. We cultured the
head chunks in a chemically defined medium together with SU5402, an inhibitor
of Fgf receptors (Mohammadi et al.,
1997) and examined the expression of otic placode markers in the
cultures after 24 hours by antibody staining or in situ hybridization
(Fig. 1A). Previous work from
our laboratory showed that the otic placode becomes specified with respect to
the otic placode marker Pax2 from the 4 ss onwards
(Groves and Bronner-Fraser,
2000). Unspecified 0-4 ss explants treated with DMSO vehicle alone
expressed Pax2, Epha4, Bmp7 and Dlx3
(Table 1). However, blocking
Fgf signaling with SU5402 resulted in the loss or great reduction of Pax2 and
Epha4 expression in the otic ectoderm (Fig.
1B). Interestingly, expression of Bmp7 or Dlx3 was
unaffected by SU5402 (Table 1).
These results suggest that Fgf signaling is necessary for the induction of
some, but not all otic placode markers. To test if Fgf signaling was required
for the maintenance of otic placode markers after their specification, we
isolated head chunks from older embryos (5-8 ss), in which the otic placode is
already specified (Groves and
Bronner-Fraser, 2000). In these older embryos, SU5402 treatment
failed to block the expression of all otic placode markers tested
(Table 1;
Fig. 1B), suggesting that
although Fgf signaling is necessary for the induction of some otic placode
markers, it is not required for their maintenance.
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;
Lombardo et al., 1998;
Vendrell et al., 2000).
However, as these studies were performed in living embryos, it was not clear
whether Fgf signaling alone was sufficient to induce some or all otic placode
markers, or whether it was acting directly or indirectly. We therefore
determined if Fgf signaling was sufficient to induce otic markers in the
absence of other tissues. Presumptive ectoderm from the trigeminal level of
0-4 ss embryos was cultured for 24 hours in chemically defined medium
containing a variety of different Fgf family members
(Fig. 2A). Trigeminal level
ectoderm (taken from adjacent to the presumptive midbrain)
(Baker et al., 1999) was used
in these experiments as it is highly competent to express otic placode markers
(Groves and Bronner-Fraser,
2000), but does not normally do so when cultured in vitro. Pax2 was induced in 0-4 ss presumptive trigeminal ectoderm in the presence of between 5 and 50 ng/ml Fgf2 (Fig. 2A). To determine the time course of the Fgf response in ectoderm, we examined Pax2 expression in Fgf2-treated presumptive trigeminal ectoderm exposed to Fgf2 for 4, 6, 7.5 or 21 hours (Fig. 2B). No Pax2 expression was found after 4 hours (n=4). Pax2 expression appeared as early as 6 hours, after which time 60.6% of explants were positive for Pax2 (n=33). The number of positive explants increased with time of exposure: 91% after 7.5 hours (n=11) and 100% after 21 hours (n=8). Explants cultured for 6 hours were then tested for the expression of other otic markers. Most of the explants expressed Epha4 (75%), whereas only a few expressed Dlx3 (30%) and none expressed Bmp7. These data along with those obtained after a 24 hour incubation (Table 2; Fig. 2C) suggest that Fgf2 is sufficient to induce and maintain Pax2, Epha4 and Dlx3 but not Bmp7. To test whether other Fgf family members were also sufficient to induce the otic placode, we screened for Pax2 expression in explants treated with Fgf1, Fgf4, Fgf5, Fgf6, Fgf7, Fgf8, Fgf9, Fgf10, Fgf16, Fgf17, Fgf18 and Fgf19. All Fgf family members tested apart from Fgf2 failed to induce Pax2 expression at 50 ng/ml, although Fgf1 and Fgf4 were able to induce Pax2 in 40% (n=10) and 37.5% (n=48) of explants at a concentration of 100 ng/ml. As Fgf2 has been reported to activate isoforms of all four Fgf receptors and gave robust induction of several otic markers, we used it in all subsequent assays in our study.
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; Ladher et al.,
2000; Ladher et al.,
2005; Woo and Fraser,
1998; Wright and Mansour,
2003). To test this possibility, we assayed for the expression of
neural and mesodermal markers in our Fgf2-treated cultures. We first examined
the expression of the pan-neural markers Sip1 and Zic2. Sip1
is first expressed in the chick neural plate at stage 4+ and remains
throughout the neural plate at least until the 10 ss
(Sheng et al., 2003) and
Zic2 is expressed throughout the neural plate at similar stages (K.M.
and A.K.G., unpublished). We did not observe expression of either gene in
presumptive trigeminal explants treated with Fgf2 after 3, 6 or 24 hours
(Fig. 3;
Table 3). We also examined a
panel of regionally expressed neural genes in our Fgf2-treated explants.
Tailless is expressed in the forebrain (Yu
et al., 1994), Krox20 in rhombomeres 3 and 5
(Nieto et al., 1995),
Wnt8c in rhombomere 4 and the caudal neural plate
(Hume and Dodd, 1993;
Kil et al., 2005), and
Sax1 in the caudal neural plate
(Spann et al., 1994). None of
these regionally expressed markers was expressed in Fgf2-treated explants at
any time point examined (Table
3; Fig. 3).
Finally, none of our Fgf2-treated explants expressed the brachyury gene (a
marker of axial and primitive streak mesoderm)
(Liu et al., 2003a) at any
time points examined (Fig. 3).
Together, these results suggest that Fgf2 is sufficient to induce several otic
placode markers in competent ectoderm without first inducing neural or
mesodermal tissue.
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;
Jacobson, 1963a;
Jacobson, 1963b;
Kaan, 1926;
Yntema, 1933). In birds, young
quail embryonic ectoderm can express otic placode markers, form an otocyst and
generate vestibulo-acoustic neurons when grafted adjacent to the posterior
hindbrain (Groves and Bronner-Fraser,
2000). To test whether different populations of cranial ectoderm
were equally competent to respond to Fgf2 in vitro, we isolated unspecified
otic and trigeminal level ectoderm from 0-4 ss embryos, and anterior epiblast
from HH stage 4 embryos and cultured the ectoderm explants in the presence of
50 ng/ml Fgf2 (Fig. 4A).
Although both unspecified trigeminal and otic ectoderm expressed Pax2 in
response to Fgf2 (73.8% and 80% explants, respectively; n=84 and 45),
no anterior epiblast explants expressed Pax2 in the presence of Fgf2 (0%,
n=40; Fig. 4B,C). A
small number of explants of presumptive otic and presumptive trigeminal
ectoderm expressed Pax2 in the absence of Fgf2. This is probably due to some
of the otic explants being already specified at the time of isolation
(Groves and Bronner-Fraser,
2000), and possible contamination of some trigeminal explants with
otic tissue or Pax2-expressing midbrain tissue. As both the trigeminal and
otic level ectoderm explants were taken from within the domain of pre-placodal
gene expression lying adjacent to the neural plate
(McLarren et al., 2003;
Streit, 2002), we tested
whether ectoderm from this pre-placodal region was uniquely competent to
respond to Fgf2. To do this, we again isolated presumptive trigeminal level
ectoderm from stage 0-4 ss embryos, but this time took the ectoderm from a
position lateral to the neural plate, outside the pre-placodal region defined
by genes such as Dlx5, Dlx6, Eya2 and Six1
(McLarren et al., 2003;
Streit, 2002). No explants
taken from regions lateral to presumptive trigeminal ectoderm expressed Pax2
following Fgf2 treatment (0/13; Fig.
4B,C).
The above results show that two populations of ectoderm taken from within
the pre-placodal domain (presumptive otic and trigeminal) were competent to
express otic markers in response to Fgf2, whereas two populations of ectoderm
from outside the pre-placodal domain (anterior epiblast and lateral trigeminal
level ectoderm) were not competent to respond to Fgf2. These results were
surprising, as we had previously shown that all four populations of ectoderm
were competent to express otic placode markers when grafted adjacent to the
posterior hindbrain in vivo (Groves and
Bronner-Fraser, 2000) (A.K.G., unpublished). One explanation for
these results is that both presumptive otic and trigeminal ectoderm fall
within a region in which pre-placodal genes (such as Dlx5, Dlx6, Eya2
and Six1) are expressed, whereas lateral ectoderm and anterior
epiblast do not (Fig. 4D). We
hypothesized that naïve ectoderm such as anterior epiblast must first
assume a pre-placodal cell identity in order for it to be competent to respond
subsequently to Fgf2 by expressing otic placode markers. To test this, we
grafted anterior epiblast tissue from stage 4 quail embryos adjacent to the
neural plate of 0-4 ss chick hosts at the level of the presumptive trigeminal
ganglion. This region lies within the pre-placodal region as defined by the
expression of genes such as Dlx5, Dlx6, Eya2 and Six1
(McLarren et al., 2003;
Streit, 2002). We fixed the
grafted embryos after either 4 or 8 hours and examined the QCPN-expressing
quail grafts for expression of Eya2 by in situ hybridization, or Dlx
genes by immunostaining with a pan Distalless antibody (pan-Dll)
(Panganiban et al., 1997).
Stage 4 anterior epiblast tissue did not express Eya2 or pan-Dll at
the time of isolation (data not shown). When embryos were examined 4 hours
after receiving grafts, only a small number of grafts expressed pre-placodal
markers (pan-Dll: 20%, n=10; Eya2: 8.7%, n=23;
Table 4;
Fig. 5). However, after 8
hours, the majority of quail grafts expressed both markers
(Table 4;
Fig. 5). Grafts of epiblast to
the trigeminal region will express the trigeminal marker Pax3 after 24 hours
(Baker et al., 1999); however,
none of the grafts expressed Pax3 protein at 4 or 8 hours after grafting
(Table 4 and data not shown),
suggesting that more than 8 hours is required to induce this marker. In
contrast to grafts made into the pre-placodal region, grafts placed lateral to
the pre-placodal region at the level of the trigeminal ganglion did not label
with pan-Dll or probes to Eya2 after either 4 or 8 hours
(Fig. 5). We also grafted
epiblast to the most anterior region of the pre-placodal region (at the level
of the future nasal and lens placodes; Fig.
6A) and also adjacent to the neural plate posterior the
pre-placodal region (in the trunk at the level of somites 5-7;
Fig. 6A). After 8 hours, grafts
adjacent to the anterior neural plate expressed both Eya2 and labeled
with pan-Dll, but grafts to the trunk did not
(Fig. 6A). We have previously
shown that young trunk ectoderm (0-6 ss) is competent to give rise to the otic
placode after grafting, but that older ectoderm (7 ss and older) is not
(Groves and Bronner-Fraser,
2000). To see whether this differential response correlates with
the ability to upregulate pre-placodal genes, we grafted old (7-9 ss) or young
(0-2 ss) trunk ectoderm to the level of the presumptive otic placode, and
examined the expression of Eya2 and labeling with pan-Dll after 8
hours (Fig. 6B). Young trunk
ectoderm expressed both markers after grafting (pan-Dll: 62%, n=26;
Eya2: 71%, n=7). Older trunk ectoderm grafts did not express Eya2
(0%, n=15), and fewer old grafts expressed pan-Dll (30%,
n=20).
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DISCUSSION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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;
McLarren et al., 2003).
However, the significance of the pre-placodal region is still uncertain; for
example, it is not clear whether the assumption of a pre-placodal cell state
is necessary for the subsequent differentiation of particular placodes, and
some authors have questioned the assumption that a pre-placodal state exists
(Begbie and Graham, 2001).
Moreover, the function of genes expressed in the pre-placodal region is only
starting to be understood. For example, overexpression of Six1
expands the pre-placodal region in Xenopus embryos, whereas loss of
function expands neural crest and epidermal domains at the expense of
pre-placodal tissue (Brugmann et al.,
2004). Similar results have also been reported with a
Xenopus Iroquois homologue, Xiro1
(Glavic et al., 2004). In this
study, we have shown that competence of chick cranial ectoderm to respond to
otic placode-inducing signals correlates with the pre-placodal cell state.
This suggests that induction of cranial placodes occurs in two steps - an
initial step inducing pre-placodal cell identity adjacent to the anterior
neural plate, and a subsequent set of steps in which particular placodes are
induced by local signals.
The role of Fgf signaling in otic placode induction
In the present study we show that Fgf signaling is both necessary and
sufficient for the induction of some, but significantly not all, markers of
the otic placode. Fgf signaling was both necessary and sufficient for the
induction of Pax2 and Epha4, sufficient but not necessary for the induction of
Dlx3 and neither necessary nor sufficient for the induction of Bmp7.
This suggests that some otic placode-specific genes such as Dlx3 may be
induced by redundant factors in addition to Fgfs, and that other genes such as
Bmp7 are induced entirely independently of Fgf signaling. Other
studies have also suggested that not all otic markers require Fgf signaling
for their induction. For example, one study in zebrafish suggested that Fgf
signaling is not necessary for the induction of the otic marker Pax8
(Maroon et al., 2002),
although a second study performed in an apparently similar fashion came to
opposite conclusions (Leger and Brand,
2002). Other zebrafish studies also demonstrated that foxi1,
dlx3b, dlx4 and sox9b can be induced in the absence of
Fgf3 and Fgf8 (Liu et
al., 2003b; Solomon et al.,
2004), and that Fgf signaling is not sufficient to induce
foxi1 (Phillips et al.,
2004). These data suggest that at least two signaling pathways -
both Fgf-dependent and Fgf-independent - converge to induce the inner ear.
Our data suggest that the inducing action of Fgf2 on unspecified ectoderm
is likely to be direct, rather than indirect. First, the induction of Pax2
protein by Fgf2 is reasonably rapid, being observed after 6 hours. Second, we
were unable to detect expression of the early mesodermal marker
brachyury, or the pan-neural markers Sip1 and Zic2
in our explants after 3, 6 or 24 hours exposure to Fgf2. We were also unable
to detect regional markers of the nervous system in our explants. Together,
these results suggest that Fgf2 is capable of inducing otic placode markers
without first inducing either neuronal or mesodermal tissue, both of which
have been suggested to have otic placode-inducing potential
(Groves, 2005). It is formally
possible that Fgf2 is acting exclusively on ectoderm to induce a second
inducing molecule without inducing neural or mesodermal tissue. Experiments to
demonstrate cell-intrinsic otic placode induction by Fgfs using activated Fgf
receptors, and to establish the Fgf signaling pathways involved in this
induction are currently in progress.
The significance of pre-placodal cell identity in otic placode induction
Our results show that ectoderm taken from a region adjacent to the border
of the anterior neural plate is qualitatively different in its response to
Fgf2 than ectoderm taken from more lateral regions. Since this region of
Fgf-responsive ectoderm is characterized by the expression of a battery of
`pre-placodal' genes, we sought to demonstrate a correlation between Fgf
responsiveness and pre-placodal cell identity. We find that a number of
pre-placodal genes such as Dlx genes and Eya2 are
upregulated in anterior epiblast tissue between 4-8 hours after grafting to
the pre-placodal region at the level of the otic, trigeminal or lens/nasal
placodes. Fgf responsiveness correlates with the upregulation of these
pre-placodal genes, as epiblast grafted into the pre-placodal region for 8
hours and then treated with Fgf2 expresses otic markers, while epiblast
grafted into the pre-placodal region for only 2 hours does not. Our results
suggest that adoption of a pre-placodal cell state is a necessary prerequisite
to respond to otic placode-inducing signals.
We have shown previously that many regions of cranial ectoderm, and
epiblast from the gastrulating chick embryo are competent to form an otic
placode when grafted adjacent to the posterior hindbrain
(Groves and Bronner-Fraser,
2000). However, our present results suggest that some populations
of competent ectoderm - those that lie within the pre-placodal region - can
express otic placode markers in the presence of Fgf2, while other populations
of competent ectoderm - those do not express pre-placodal region genes -
cannot respond to Fgf2. In order to reconcile these results, we propose a
two-step, sequential model of otic placode induction. In the first step,
naïve ectoderm is induced to form the pre-placodal region. Recent work by
Streit and colleagues suggests that these signals are derived from cranial
mesoderm progenitors and the neural plate, and include both Wnt and BMP
antagonists, together with activation of the Fgf pathway
(Litsiou et al., 2005). In a
second step, cells within the pre-placodal region are induced to form
particular placodes by local inducing signals, which in the case of the otic
placode include Fgfs. Under this two step model, competence to form an otic
placode (see Gallagher et al.,
1996; Groves and
Bronner-Fraser, 2000) is resolved into two sequential
competencies. Naïve ectoderm from many regions of the embryo is competent
to respond to pre-placodal inducing signals (such as those described in
Litsiou et al., 2005) by
upregulating pre-placodal genes, but is not competent to express otic markers
in the presence of Fgf2. Naïve ectoderm only becomes competent to respond
to otic placode-inducing signals such as Fgfs when it has first adopted a
pre-placodal identity. Our model predicts that the induction of other cranial
sensory placodes will also occur in a sequential fashion, with induction of
pre-placodal genes being followed by induction of a particular placode in
response to local signals. We have no evidence at present to suggest that our
model can be generalized to other placodes, although the identification of
candidate inducing tissues or molecules for other placodes
(Baker et al., 1999;
Begbie et al., 1999) suggests
tests of this hypothesis.
At present, we do not know which genes in chick are necessary or sufficient
to confer competence on naive ectoderm to respond to pre-placodal region
inducing signals, or which pre-placodal region genes are necessary or
sufficient to confer Fgf responsiveness on pre-placodal region ectoderm.
Although Six1 is necessary for placode development and upregulation
of other pre-placodal genes in Xenopus
(Brugmann et al., 2004), no
single transcription factor can confer pre-placodal properties on naïve
ectoderm in chick embryos (Andrea Streit, personal communication). In
zebrafish, the dlx3b gene is expressed in the pre-placodal region
(and later upregulated in the otic placode) and appears necessary for the
induction of Pax2a in the presence of Fgf signaling
(Liu et al., 2003b;
Solomon and Fritz, 2002).
Dlx5 and Dlx6 are expressed in chick and mice in a similar
pattern to Dlx3b in zebrafish
(Brown et al., 2005;
McLarren et al., 2003;
Ohyama and Groves, 2004), but
perturbation of these genes in chick and mice does not appear to affect
induction of the otic placode (McLarren et
al., 2003; Robledo et al.,
2002). It is also possible that additional competence factors
expressed in a restricted region of the pre-placodal region may be required to
induce particular placodes. For example, the zebrafish foxi1 gene is
expressed initially in a domain broader than the otic placode and is necessary
for the induction of Pax8 in the presence of Fgf signaling
(Hans et al., 2004;
Nissen et al., 2003;
Solomon et al., 2003), but is
not itself induced by Fgf signals. Although loss-of-function experiments can
reveal candidate competence factors, further gain-of-function experiments are
required to establish whether any of the pre-placodal genes or foxi1
are sufficient to confer competence on ectoderm to respond to placode inducing
signals such as Fgfs.
The work of Grainger and colleagues has identified a stage in lens
induction which they refer to as `lens-forming bias'
(Grainger et al., 1997;
Henry and Grainger, 1987)
(reviewed by Grainger, 1992;
Grainger, 1996). This concept
arose from the observation that presumptive lens ectoderm from embryos of
progressively older ages shows an increasing response to the very weak
lens-inducing activity of the optic vesicle
(Grainger et al., 1988;
Henry and Grainger, 1987).
Expression of the transcription factors Otx2 and Pax6
correlate with the presence of lens-forming bias in ectoderm
(Zygar et al., 1998), although
this ectoderm probably contains progenitors of both lens and olfactory
placodes (Bhattacharyya et al.,
2004), both of which require Pax6 for their correct development
(Grindley et al., 1995;
Quinn et al., 1996). As the
molecular basis for lens-forming bias is presently unknown, it is not clear if
`bias' sensu Grainger is a different property from the pre-placodal state that
we now show correlates with Fgf responsiveness. Pre-placodal ectoderm is
characterized by the expression of genes such as Six1, Six4, Eya2, Dlx5,
Dlx6 and Ern1 in a region surrounding the entire anterior neural
plate, whereas lens-forming bias correlates with expression of Pax6
and Otx2, which are expressed in the most anterior border of the
neural plate or the neural plate proper, respectively
(Bhattacharyya et al., 2004;
Chapman et al., 2002;
Fernandez-Garre et al., 2002).
Lens-forming bias can be observed in presumptive lens ectoderm from the neural
plate stage onwards, but is only observed in ectoderm surrounding the
presumptive lens ectoderm at neural tube stages
(Grainger et al., 1997). By
contrast, pre-placodal markers such as Six1 are already expressed at
the border of the neural plate shortly after gastrulation in amphibians and
birds (Brugmann et al., 2004;
Litsiou et al., 2005;
McLarren et al., 2003;
Pandur and Moody, 2000;
Streit, 2002). Finally, we
show that the upregulation of pre-placodal genes correlates with
Fgf-unresponsive ectoderm becoming responsive, whereas the acquisition of bias
appears to represent a more graded response to lens-inducing signals
(Grainger et al., 1997;
Henry and Grainger, 1987). A
more detailed molecular description of biased lens ectoderm and pre-placodal
ectoderm is required to understand the differences and similarities between
these two precursor populations.
The most important point to be stressed from our work is how the concept of
competence to form a particular tissue can change in the light of new
molecular data. Our sequential model of otic placode induction suggests that
competence to respond to pre-placodal gene inducing signals is both
molecularly and functionally distinct from the competence to express otic
placode genes in response to Fgf2. Changes in competence to respond to
inducing or survival signals can occur by simply changing receptor expression
(Birren et al., 1993), or at
the other extreme by changing chromatin structure
(Song and Ghosh, 2004). The
next challenge in the study of cranial placode induction will be to understand
the molecular basis of these two different competent cell states.
|
ACKNOWLEDGMENTS |
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REFERENCES |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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