Department of Biological Structure, University of Washington, Seattle, WA 98195-7420, USA
* Author for correspondence (e-mail: draible{at}u.washington.edu)
Accepted 22 April 2005
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SUMMARY |
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Key words: Fgf3, Foxi1, Epibranchial placodes, Cranial ganglia, Endoderm, Pharyngeal pouches, Placode induction, Neurogenesis
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Introduction |
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Several studies have identified transcription factors that regulate
different steps of EB placode development. In mouse, specification and
delamination of neuroblasts from EB placodes is controlled by expression of
the atonal-related basic helix-loop-helix (bHLH) transcription factor
neurogenin 2 (Fode et al.,
1998), while neurogenesis from other cranial placodes is
controlled by neurogenin 1 (Neurog1) (Ma
et al., 1998
; Ma et al.,
2000
). These roles may be reversed in amphibian and avian species
(Schlosser and Northcutt,
2000
; Begbie et al.,
2002
). By contrast, a single neurogenin, Ngn1 (Neurog1
Zebrafish Information Network) is required for the development of all ganglia
in zebrafish (Andermann et al.,
2002
). The homeobox transcription factors phox2a and
phox2b are necessary for the subsequent differentiation of EB neurons
(Guo et al., 1999
;
Morin et al., 1997
;
Pattyn et al., 1997
;
Pattyn et al., 1999
).
Expression of ngn1 is regulated in EB placodes by two other sets of
transcription factors, Eya1 and Six1 in mouse (Zou, 2004),
and the forkhead-related winged helix transcription factor foxi1 in
zebrafish (Lee et al., 2003
).
In foxi1 mutants, placodal progenitors fail to express both
ngn1 and phox2a. However, foxi1 is also expressed
in the pharyngeal pouches during the time of EB placode neurogenesis, and
mutant embryos show defective pouch morphology and marker expression
(Nissen et al., 2003
;
Solomon et al., 2003
). Thus,
it remains to be determined whether the phenotype observed in foxi1
mutants is caused by defective Foxi1 in ectoderm or by abnormal signaling from
the pharyngeal pouches.
Much less is known about the inductive signals that regulate EB placode
development. Previous studies have suggested that pharyngeal endoderm acts as
a source of signals for EB placodes. Landacre
(Landacre, 1931) first noted a
close spatial and temporal association between EB ganglia and pharyngeal
endoderm in axolotl. In chick, tissue recombination experiments demonstrated
that pharyngeal endoderm promotes neuron production from cranial ectoderm
(Begbie et al., 1999
). BMP7,
which is expressed in chick pharyngeal pouch endoderm, also induces
neurogenesis in cranial ectoderm, and the BMP-inhibitor follistatin blocks the
neurogenic effect of pharyngeal endoderm
(Begbie et al., 1999
). However,
BMP7 is not sufficient for inducing neurons from trunk ectoderm, while trunk
ectoderm is competent to form EB neurons when grafted to the head region
(Vogel and Davies, 1993
). This
suggests that other signal(s), in addition to BMP7, are required for EB
placode induction and/or neurogenesis.
We postulated that possible candidates for signals that regulate EB
neurogenesis include members of the Fgf signaling family. Fgfs play important
roles in embryogenesis (e.g. development of limb bud, heart, tooth, lung, otic
vesicle) as well as in the adult organism (e.g. tissue repair, angiogenesis).
To date 23 members have been isolated from human and mouse
(Javerzat et al., 2002). Our
recent literature and database searches have revealed at least 25 candidate
Fgf members in zebrafish
(http://www.sanger.ac.uk/Projects/D_rerio).
The effects of vertebrate Fgfs are mediated by four transmembrane receptors
(Fgfr1 through Fgfr4), which are members of the receptor tyrosine kinase (RTK)
superfamily (Ornitz, 2000
;
Scholpp et al., 2004
;
Sleptsova-Friedrich et al.,
2001
; Thisse et al.,
1995
; Tonou-Fujimori et al.,
2002
). Upon ligand binding, Fgfrs trigger, by way of Ras, a
sequential activation of the mitogen-activated kinase (MAPK)/extracellular
activated kinase (ERK) signaling cascade
(Cobb and Goldsmith, 1995
),
which in turn leads to the activation of nuclear transcription. In zebrafish,
the ETS transcription factor genes erm and pea3 are
transcriptional targets of this pathway
(Raible and Brand, 2001
;
Roehl and Nusslein-Volhard,
2001
).
Although not implicated in EB placode development, Fgfs are well
established as regulators of development of the otic placode
(Riley and Phillips, 2003;
Wright and Mansour, 2003
). In
particular, Fgfs have been implicated in otic placode neurogenesis in chick
(Alsina et al., 2004
).
Inhibition of Fgf signaling blocks ngn1 expression and formation of
the acoustic ganglion, while ectopic Fgf10 promotes expression of the
proneural genes neuroD and neuroM. A recent study indicated
that zebrafish Foxi1 modulates cellular responses to Fgf signaling
(Nissen et al., 2003
),
suggesting that a similar situation may occur in EB placode development.
Here, we use a genetic approach to test whether endoderm is required for EB ganglia development. We show that endoderm-deficient zebrafish embryos lack the majority of the EB ganglia sensory neurons. Mosaic analyses demonstrated that endoderm restoration could rescue EB ganglia and specifically identified endodermal pouches as a source of the inductive signal. We further implicate Fgf3 signaling as a major endodermal determinant required for the neurogenesis of the EB placodes. Tissue transplantation experiments confirmed that Fgf3 activity is specifically required in the endodermal pouches. Finally, ectopic expression experiments demonstrated that Fgf3 is sufficient for inducing sensory neurons in both wild-type and endoderm-deficient morphants. Altogether, our results demonstrate a requirement for the pharyngeal pouch endoderm and endoderm-derived Fgf3 in the neurogenesis and survival of the majority of EB sensory neurons.
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Materials and methods |
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Embryo injections
Antisense morpholino oligonucleotides (MO) were obtained from GeneTools
(Corvalis, OR), diluted to a working concentration in Danieau buffer (58
mmol/l NaCl, 0.7 mmol/l KCl, 0.4 mmol/l MgSO4, 0.6 mmol/l
Ca(NO3)2, and 5 mmol/l HEPES, pH 7.6), and 2-3 nl were
pressure-injected into one-cell stage embryos. cas-MO,
5'-GCATCCGGTCGAGATACATGCTGTT, was injected at 2 ng/nl
(Sakaguchi et al., 2001);
fgf3-MO1, 5'-CATTGTGGCATGGAGGGATGTCGGC, was injected at 0.75
ng/nl (Maroon et al., 2002
);
fgf3-MO2, 5'-CAGTAACAACAAGAGCAGAATTATA, was injected at 5 ng/nl
(Raible and Brand, 2001
);
fgf4-MOE1I1, 5'-AACTTACTGTAGCGGTTTTCGTTGT, was injected at 3
ng/nl (Jackman et al., 2004
);
fgf8-MOE2I2+fgf8-MOE3I3,
5'-TAGGATGCTCTTACCATGAACGTCG+5'-CACATACCTTGCCAATCAGTTTCCC, were
injected at 0.5 ng/nl each (Draper et al.,
2001
); fgf24-MOE3I3, 5'-AGGAGACTCCCGTACCGTACTTGCC,
was injected at 2.5 ng/nl (Draper et al.,
2003
); foxd3-MO, 5'-TGCTGCTGGAGCAACCCAAGGTAAG, was
injected at 2 ng/nl (Lister et al., unpublished); ngn1-MO,
5'-ACGATCTCCATTGTTGATAACCTGG, was injected at 5 ng/nl
(Andermann et al., 2002
).
In-situ hybridization, immunolabeling, TUNEL assay and Richardson's stain
In-situ hybridization and immunolabeling experiments were performed
according to the published protocols
(Andermann et al., 2002). We
used the following riboprobes and antibodies: erm
(Raible and Brand, 2001
;
Roehl and Nusslein-Volhard,
2001
), fgf3 (Kiefer
et al., 1996
), fgfr1
(Scholpp et al., 2004
),
foxi1 (Lee et al.,
2003
; Nissen et al.,
2003
; Solomon et al.,
2003
), ngn1 (Korzh et
al., 1998
), nrd (neurod Zebrafish
Information Network) (Korzh et al.,
1998
), pea3 (Raible
and Brand, 2001
; Roehl and
Nusslein-Volhard, 2001
), phox2a
(Guo et al., 1999
),
phox2b (Shepherd et al.,
2004
), anti-Hu (1:750, Molecular Probes), anti-Zn-5 (1:200, The
Zebrafish International Resource Center, Eugene, OR). For TUNEL, embryos were
fixed for 1 hour at room temperature in 4% paraformaldehyde in PBS (pH = 7.3).
Embryos were then washed 3x with TBST (PBS with 2% Triton X-100 and 5%
Tween-20, Sigma), dehydrated and rehydrated through a methanol:TBST series and
transferred back into TBST. Embryos were treated with 10 µg/ml Proteinase K
for 5 minutes, postfixed for 20 minutes, and then washed several times in
TBST. Following equilibration in TdT dilution buffer, embryos were first
incubated in the fluorescein-TUNEL labeling mix (Roche) for 1 hour on ice and
then for 1 hour at 37°C. Embryos were then washed with TBST and processed
to detect fluorescein using NBT/BCIP chromogenic substrate (Roche). For
brightfield photography, embryos were deyolked when appropriate, flat mounted
in 50% glycerol/50% PBS and photographed on a Nikon SMZ 1500 stereoscope or
Zeiss Axioplan microscope using Spot CCD camera (Diagnostic Instruments).
Fluorescent images were obtained using an LSM-5 Pascal confocal microscope
(Zeiss). Images were processed in Adobe Photoshop. Some embryos were
dehydrated, embedded in Araldite resin (Polysciences) and sectioned at 4-8
µm. For Richardson's stain, 26-hour-old embryos were fixed in 4% PFA in PBS
for 2 hours at room temperature, dehydrated and embedded. Dry 4 µm sections
were heated to 73°C on a hot plate and dipped into an aqueous solution of
0.0005% Azure II, 0.001% Methylene Blue, and 0.001% Sodium Borate for 80
seconds, then washed, air dried and mounted in Araldite resin.
Grafting experiments
For transplants, embryos were raised in filter-sterilized EM supplemented
with penicillin (5000 U/l)/streptomycin (100 mg/l; Sigma). mRNAs were
synthesized using mMessage Machine Kit (Ambion) following the manufacturer's
protocol. Donor embryos were injected at the one-cell stage with 2%
lysine-fixable fluorescein dextran (10,000 Mr; Molecular Probes) or 80 pg of
gfp mRNA and 1.2 pg of tar* mRNA (Peyreiras et al., 1998) in
0.2 mol/l KCl. Dechorionated donor and host embryos were mounted in 3%
methylcellulose in Ringer's solution
(Westerfield, 2000) on a glass
depression slide. Ten to 20 donor cells were inserted into a host embryo. For
in-situ hybridization, donor-derived fluorescein-labeled cells were detected
essentially as described (Prince et al.,
1998
), except INT Red chromogenic substrate (Iodo Nitrotetrazolium
Violet, Sigma) in PVA (Polyvinyl Alcohol) was used instead of Fast Red.
Fgf3 misexpression
HS-fgf3 (Maves et al.,
2002) and HS-gfp
(Halloran et al., 2000
)
plasmids were co-injected into one-cell-stage embryos at 2.5 ng/µl. At this
concentration, plasmid uptake by early blastomeres is very mosaic, leading to
small clones of fgf-expressing cells upon heat shock. Embryos were
heat shocked at 37°C for 1 hour at 22 hpf, fixed at 36 hpf and assayed for
phox2a and gfp expression. Control embryos injected with
HS-gfp alone showed normal phox2a expression. To detect GFP
expression following RNA in-situ hybridization, embryos were processed with
anti-GFP antibody (1:200, Molecular Probes) and Alexa 568 anti-mouse secondary
antibodies (1:500, Molecular Probes) as described
(Andermann et al., 2002
).
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Results |
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To determine whether endoderm is necessary for the development of EB
neurons, we injected zebrafish embryos with a specific MO directed against
casanova (cas) mRNA
(Sakaguchi et al., 2001). Cas
is a Sox-related factor that is required for endoderm formation
(Alexander et al., 1999
;
Kikuchi et al., 2001
). One
hundred percent of injected embryos exhibited cardiabifida and loss of the
pancreas (monitored by nrd expression; data not shown), confirming
loss of endoderm. In addition, the pharyngeal pouches were completely absent
in all cas-MO embryos examined
(Fig. 1A), as detected by
expression of Zn-5, a DM-GRASP cell-surface antigen
(Fashena and Westerfield,
1999
). When compared to uninjected controls, cas
morphants completely lacked ngn1, nrd, phox2a and phox2b
expression in the glossopharyngeal and small vagal placodes and ganglia, while
expression in the facial and large vagal placodes and ganglia were
significantly reduced (Fig. 1A,
Table 1). To confirm that the
above phenotype did not result from developmental delay, we stained
80-hour-old wild-type and cas morphants with a pan-neuronal anti-Hu
antibody. While the facial ganglion is difficult to ascertain at this stage
because it is often fused with trigeminal and anterior lateral line ganglia,
glossopharyngeal and small vagal ganglia were clearly absent and the large
vagal ganglion was significantly reduced in cas-MO embryos
(Fig. 1A). Analyses of Hu
expression at 36 hpf revealed that trigeminal and lateral line ganglia were
not affected (data not shown), indicating a specific defect in the EB ganglia
in cas morphants. These experiments demonstrate that endoderm is
required for neurogenesis in the EB placodes.
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To determine if the EB placode cells undergo cell death in cas-MO
embryos, we processed these embryos for TdT-mediated dUTP nick-end labeling
(TUNEL) assay (Fig. 1D). While
little cell death was observed in controls, an increase in cell death was
observed in cas morphants at 26 hpf. Previous study indicated that
endoderm is required for survival of the arch cartilage precursors
(David et al., 2002). Indeed,
we found a number of TUNEL-positive neural crest cells in the cas
morphants. However, we also detected TUNEL-positive cells within ectoderm in
the location of the EB placodes (Fig.
1D, inset). In addition to TUNEL, transverse sections through the
head region of cas morphants revealed the presence of fragmented
nuclei in the EB placodes (see Fig.
5I), indicating cell death. Thus, we concluded that endoderm is
also required for survival of at least some EB placode cells.
Expression of Fgf signaling members in the EB placodes
The above experiments suggested that a diffused signal from the endoderm
promotes EB placode neurogenesis. We set out to investigate whether the Fgf
signaling pathway is activated in EB placodes. We incubated zebrafish embryos
with the pharmacological Fgfr inhibitor SU5402
(Mohammadi et al., 1997)
beginning at 19 hpf, well before any onset of ngn1 expression.
Inhibitor-treated embryos did not express ngn1, phox2a and
phox2b in the glossopharyngeal and small vagal placode and ganglia,
while facial and large vagal placode and ganglia were significantly reduced
(Fig. 2A). Zn-5 expression
revealed that SU5402-treated embryos had abnormal pouch morphology
(Fig. 2A) and segmentation.
However, pharyngeal pouch segmentation is not absolutely necessary for the
development of EB ganglia (Piotrowski and
Nusslein-Volhard, 2000
). We used the inhibitor to determine the
timing requirement of the Fgf signal. We treated embryos every 2 hours from 18
hpf until 30 hpf and then assayed them for nrd expression at 36 hpf
(data not shown). The EB ganglia were not affected in the embryos treated
after 26 hpf. By contrast, these ganglia were significantly reduced or absent
in the embryos treated before 24 hpf (data not shown). This indicated that EB
placode neurogenesis is regulated by an Fgf signal before or around 24
hpf.
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|
Fgf3 is required for neurogenesis in the EB placodes
To determine if Fgf3 is required for EB placode neurogenesis, we injected
morpholinos directed against fgf3 mRNA. As controls, we assayed
uninjected embryos as well as embryos injected with a combination of
morpholinos directed against fgf8
(Fig. 3 and data not shown). We
used two non-overlapping fgf3 morpholinos, termed here
fgf3-MO1 and fgf3-MO2, which have been extensively
characterized by others (David et al.,
2002; Leger and Brand,
2002
; Maroon et al.,
2002
; Phillips et al.,
2001
; Raible and Brand,
2001
; Walshe and Mason,
2003a
; Walshe and Mason,
2003b
). In our hands, fgf3-MO1 displayed a more penetrant
phenotype than fgf3-MO2, including significant reduction in
pax2.1 expression in the isthmus and otic placode at 18 somites as
well as significant reduction of the otic vesicle, as previously described
(data not shown). Therefore we used fgf3-MO1 in the majority of our
assays. Strikingly, fgf3 morphants lacked ngn1, phox2a and
phox2b expression in glossopharyngeal and small vagal placodes and
ganglia, and had reduced expression in the facial and large vagal placodes and
ganglia (Fig. 3A,
Table 1), similar to the
phenotype observed in cas morphants. When assayed at 80 hpf with Hu
antibody, sensory neurons of the glossopharyngeal and small vagal ganglia were
absent, while the large vagal ganglion was reduced
(Fig. 3A). Importantly,
fgf3-MO1 did not interfere with the pharyngeal pouch development, as
indicated by normal Zn-5 expression (Fig.
3B). This is consistent with recent studies demonstrating that
fgf3 morpholino injections did not interfere with the pharyngeal
pouch development (Crump, 2004; David et
al., 2002
). However, embryos that carry a null allele in the
fgf3 gene have defects in the pharyngeal pouch segmentation
(Herzog et al., 2004
). This
indicates that fgf3 morphants used in this study are hypomorphic with
respect to the pharyngeal endoderm segmentation.
|
We investigated whether other Fgfs are involved in EB placode neurogenesis.
fgf8 is expressed in the endodermal pouches of chick, mouse and
zebrafish (Crossley and Martin,
1995; Hidalgo-Sanchez et al.,
2000
; Mahmood et al.,
1995a
; Mahmood et al.,
1995b
; Reim and Brand,
2002
; Veitch et al.,
1999
; Wall and Hogan,
1995
). To determine whether Fgf8 is required for zebrafish EB
placode development, we analyzed the mutant acerebellar
(ace), which harbors a mutation in the fgf8 gene
(Reifers et al., 1998
).
Because the existing aceti282a allele is hypomorphic, we
also analyzed fgf8-MO-induced phenotypes. Expression analyses of
ngn1, phox2a and phox2b demonstrated that all EB placodes
and ganglia were present in ace mutants and fgf8 morphants
(see Fig. S1 in the supplementary material; data not shown). Moreover, all EB
ganglia appeared grossly normal when assayed at 80 hpf by Hu antibody (see
Fig. S1 in the supplementary material). Occasionally, we noticed a shift or a
fusion in the phox2a or phox2b expression domains marking
glossopharyngeal and small vagal ganglia, which develop in close proximity to
the otic vesicle (see Fig. S1 in the supplementary material). As Fgf8 is
required for induction of the otic vesicle as well as segmentation of the
branchial arches (Crump, 2004; Leger and
Brand, 2002
; Liu et al.,
2003
; Maroon et al.,
2002
; Phillips et al.,
2001
), we suspect these differences in EB neuron distribution may
be indirect. Similar analyses revealed that neither fgf4 nor
fgf24, which are also expressed in the pharyngeal endoderm at 24 hpf
(David et al., 2002
;
Draper et al., 2003
), were
required for EB placode neurogenesis (data not shown).
Chondrogenic neural crest is not required for early neurogenesis in the EB placodes
A previous study demonstrated a requirement for Fgf3 in the development of
the posterior arch cartilages (David et
al., 2002). Consequently, abnormal EB placode neurogenesis found
after blocking Fgf3 function could be indirect. Thus, we sought to disrupt the
migration of the posterior chondrogenic precursors by means other than
reduction in Fgf3 activity. The winged helix transcription factor Foxd3 is
required for the development of many NC derivatives, including posterior arch
cartilages (J. A. Lister and D.W.R., unpublished). Zebrafish embryos injected
with a specific morpholino directed against foxd3 displayed defects
in the posteriormost dlx2-positive NC stream (see Fig. S2 in the
supplementary material), a phenotype nearly identical to that observed in
fgf3 morphants (David et al.,
2002
; Walshe and Mason,
2003a
). By contrast, analyses of ngn1 expression did not
reveal any defects in early EB placode neurogenesis in foxd3
morphants (see Fig. S2 in the supplementary material). However, at 80 hpf, Hu
antibody staining revealed that various cranial ganglia, including the
trigeminal, lateral line and EB ganglia, were reduced in size, and many
Hu-positive neurons failed to coalesce into distinct ganglia (see Fig. S2 in
the supplementary material). This observation is consistent with the
previously proposed idea that foxd3-positive glia are required for
normal organization of the cranial ganglia
(Begbie and Graham, 2001
). We
concluded that the chondrogenic neural crest is not required for early
neurogenesis in EB placodes.
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|
Fgf3 is sufficient for inducing phox2a-positive neurons in wild-type embryos and cas morphants
To determine if Fgf3 is sufficient for inducing EB sensory neurons, we used
a heat-shock (HS) promoter to drive fgf3 expression in wild-type
embryos and cas morphants
(Halloran et al., 2000;
Maves et al., 2002
). Embryos
were co-injected with HS-fgf3 and HS-gfp plasmids at the
one-cell stage, heat shocked for 1 hour beginning at 22 hpf, and then assayed
for phox2a and gfp expression at 36 hpf
(Fig. 7,
Table 3). As controls,
wild-type embryos and cas morphants were injected with the same
amount of HS-gfp plasmid alone and subjected to the same treatment.
HS-fgf3 plasmid was injected at 2.5 ng/µl, because at higher
concentrations (beyond 5 ng/µl), a majority of embryos were dorsalized,
probably due to a leaky expression from the HS promoter. Any dorsalized
embryos were excluded from the analyses. Strikingly, a significant number of
wild-type embryos (21%) displayed ectopic phox2a-positive foci
(Fig. 7,
Table 3). By contrast, we found
only one (2%) phox2a-positive site that we classified as ectopic in
HS-gfp-injected controls. Ectopic neurons were induced in the
vicinity of all EB ganglia, including facial, glossopharyngeal and vagal
ganglia (Table 3). Similarly,
27% of the cas morphants displayed either ectopic
phox2a-positive foci or rescue of the facial or large vagal ganglia
(compared with only 2% in controls; Fig.
7, Table 3). However, by contrast to the wild-type embryos, we did not observe any
phox2a-positive neurons in the third or fourth arch, which would
indicate rescue of the glossopharyngeal and small vagal ganglia
(Table 3). In both wild-type
and cas-MO injected embryos, the majority of ectopic
phox2a-positive neurons (71%) were adjacent to the
gfp-positive cells. These experiments indicate that fgf3
expression is sufficient to induce phox2a-positive EB neurons.
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Discussion |
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The onset of zebrafish EB neurogenesis is highlighted by expression of
ngn1, which is expressed in all EB placodes and transiently in
delaminating neuroblasts (Fig.
8B,C). ngn1 expression is required for the subsequent
expression of three other transcription factors, nrd, phox2a and
phox2b. The timing and position of nrd expression suggests
that it is upstream of phox2a and phox2b
(Andermann et al., 2002) (data
not shown), although this idea has not been tested directly. In zebrafish,
both Fgf3-dependent and Fgf3-independent neurogenic precursors give rise to
phox2a- and phox2b-expressing cells, although it is unclear
whether all EB neurons express these markers. It will be important to
investigate whether the Fgf3-dependent and Fgf3-independent subpopulations
represent particular functional subtypes within the facial and large vagal
ganglia. Development of the molecular markers that recognize different
subtypes of the EB sensory neurons in zebrafish will help to address this
question.
Interestingly, while fgf3 expression in wild-type embryos was sufficient to induce phox2a-positive neurons in all arches, in cas morphants it was not sufficient to rescue any glossopharyngeal or small vagal neurons, located in the third and fourth arches, respectively. This result may indicate that another endoderm-derived factor, in addition to Fgf3, is required for neurogenesis of the glossopharyngeal and small vagal placodes. Another explanation is that foxi1 expression, which is required to respond to Fgf signals, was lost in the third and fourth arches in cas morphants (Fig. 5B).
In addition to promoting neurogenesis, endoderm and endoderm-derived Fgf3 may play a role in promoting survival of the EB placodal cells. Interestingly, placodal cells in foxi1 mutants undergo cell death between 28 and 36 hpf, right after they fail to undergo neurogenesis. We observed a similar effect in cas and fgf3 morphants. On one hand, these observations may indicate that the above factors are responsible for suppressing the apoptotic pathway and/or promoting survival of the EB neurons. On the other hand, programmed cell death may be induced in the EB placodes when they fail to undergo their normal developmental program. We favor the latter explanation, because fgf3 expression is sufficient for inducing phox2a expression, supporting the argument that Fgf3 is directly involved in EB placode neurogenesis.
Previous studies in avian embryos have implicated pharyngeal endoderm in
the induction of EB placodes (Begbie et
al., 1999). However, this study used markers that were expressed
in differentiating neurons, rather than placodal precursors. Thus, it was not
clear whether endoderm was required for EB placode induction or subsequent
neurogenesis. Interestingly, in these studies cranial ectodermal explants
generated some phox2a-positive neurons even in the absence of
pharyngeal endoderm, while trunk ectoderm never produced neurons with or
without inducing tissue. By contrast, trunk ectoderm is capable of EB neuron
production after transplant in vivo (Vogel
and Davies, 1993
). These results are consistent with our model for
two inductive signals: an endoderm-independent signal involved in placode
formation, and an endoderm-dependent signal for neurogenesis.
It has been suggested that endodermally derived BMP7 plays a role in the
generation of EB neurons in avian embryos
(Begbie et al., 1999;
Begbie and Graham, 2001
). In
zebrafish, bmp7 is expressed in endoderm at 15 somites (16.5 hpf) and
thus may play a role in the EB placode development even before Fgf3-dependent
neurogenesis (Dick et al.,
2000
). Alternatively, Bmp7 signaling may represent a parallel
pathway required during EB placode neurogenesis. Interestingly, expression of
phox2a in the locus coeruleus requires both Bmp and Fgf signals
(Guo et al., 1999
). It will be
important to determine the exact timing and pattern of bmp7
expression and its role during EB placode induction in zebrafish.
Role of Foxi1 in EB placode development
A number of previous studies demonstrated a Foxi1 requirement during otic
placode, jaw and EB placode development
(Lee et al., 2003;
Nissen et al., 2003
;
Solomon et al., 2003
). During
ear development, foxi1 is expressed in the otic placode precursors
and is required for these cells to respond to Fgf-inducing signals, suggesting
that Foxi1 might play the role of competence factor
(Solomon et al., 2004
). Our
experiments demonstrated that in the absence of Foxi1 activity EB placodes
were still induced, while neurogenesis was impaired. Moreover, a downstream
effector of Fgf signaling, pea3, was not activated in the EB
placodes, strengthening the notion that upregulation of Foxi1 is required in
EB precursors to respond to Fgf signals, a role similar to the one postulated
for otic placode induction. Importantly, Lee et al. demonstrated that
foxi1 mutants undergo massive cell death in the cranial placodes
between 28 and 36 hpf, shortly after neurogenesis is induced
(Lee et al., 2003
). This is
consistent with the idea that Foxi1 may render ectodermal cells competent to
undergo neurogenesis. Finally, phox2a and phox2b expression
analysis in foxi1 mutants revealed a phenotype distinct from the one
observed in cas and fgf3 morphants. While foxi1
mutants displayed a more severe loss of phox2a expression,
phox2b expression was only mildly affected, often persisting even in
smaller glossopharyngeal and vagal ganglia. These observations indicate that
phox2a and phox2b might be expressed in overlapping, but not
identical, populations of the EB neurons, and that these neuronal populations
might be differentially regulated by Fgf signals.
|
In addition to roles in early otic placode induction, Fgfs are also
important for otic placode neurogenesis in chick
(Alsina et al., 2004).
Inhibition of Fgf signaling blocks formation of the acoustic ganglion, while
ectopic Fgf10 promotes ear expression of the proneural genes such as Nrd and
NeuroM. Similarly, we showed that Fgf signaling is essential to induce
neurogenesis in the EB placode and yet an unidentified Fgf ligand may be
required for maintaining ectodermal foxi1 expression.
Common signaling pathways may specify neurogenic placodes
It is becoming increasingly apparent that placode development in
vertebrates is a multi-step process that involves a multitude of signaling
pathways, many of which are similar in different placodes
(Bhattacharyya and Bronner-Fraser,
2004; Streit,
2004
). For example, in zebrafish and mouse, foxi genes
are expressed in otic and epibranchial placodes
(Hans et al., 2004
;
Hulander et al., 1998
;
Lee et al., 2003
;
Nissen et al., 2003
;
Ohyama and Groves, 2004
;
Solomon et al., 2003
). Loss of
zebrafish foxi1 results in severe disruption of otic and EB placode
development (Lee et al., 2003
;
Nissen et al., 2003
;
Solomon et al., 2003
). Fate
maps in zebrafish and chick demonstrate that otic and EB placode precursors
might be intermingled during the early stages of development
(Bhattacharyya et al., 2004
;
Kozlowski et al., 1997
;
Streit, 2002
), and careful
analysis of gene expression in Xenopus supports this idea
(Schlosser and Ahrens, 2004
).
It is tempting to speculate that despite clear differences in timing of
induction, otic and EB placodes might share a common field of precursors, and
in both cases foxi activity may confer ectoderm competence to respond
to Fgf signals. It will be interesting to investigate whether overexpression
of foxi1 together with fgf3 will induce ectopic sensory
neurons in atypical locations in the embryo, including the trunk. Among other
early markers, various members of Pax/Eya/Six/Dach network are required for
the development of both EB and otic placodes and probably act downstream of
Foxi1 in both systems (Riley and Phillips,
2003
; Streit,
2004
; Zou, 2004). Additional experiments in Xenopus,
chick and mouse embryos support the idea that pax, eya, six and
dach genes act early during epibranchial placode specification
(Abu-Elmagd et al., 2001
;
Baker and Bronner-Fraser, 2000
;
Ishii et al., 2001
;
Schlosser and Ahrens, 2004
;
Zou, 2004), but their role in zebrafish EB placode development remains to be
determined.
Overall, an emerging picture supports the idea that multiple signals from different tissues are required to specify various sensory placodes. The availability of markers that define various steps of otic placode induction, including earliest steps in conjunction with loss-of-function experiments has been particularly useful in defining functions of various inducers. Thus, identification of additional early markers involved in EB placode development in zebrafish should help to pinpoint signals that induce EB placodes.
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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/132/16/3717/DC1
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