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First published online January 10, 2007
doi: 10.1242/10.1242/dev.02749
1 Department of Biological Structure, University of Washington, Seattle, WA
98195-7420, USA.
2 Department of Cell Biology, Duke University Medical Center, Durham, NC 27710,
USA.
* Author for correspondence (e-mail: draible{at}u.washington.edu)
Accepted 20 November 2006
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Fgf3, Fgf8, Foxi1, Pax2a, Epibranchial placodes, Cranial ganglia, Cephalic mesoderm, Zebrafish
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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EB placode induction is likely to be a multi-step process
(Streit, 2004
). A growing body
of evidence suggests that along with the various other placodes, EB placodes
are derived from a common territory, named pre-placodal or panplacodal
ectoderm (PPE), positioned around the border of the anterior neural plate. PPE
is defined by expression of specific transcription factors, including the
homeobox factor Six1 and its binding partner Eya1
(Bessarab et al., 2004;
Pandur and Moody, 2000;
Sahly et al., 1999;
Schlosser and Ahrens, 2004;
Zou et al., 2004). Initial
formation of the PPE is regulated by Bmp, Wnt and Fgf signals
(Ahrens and Schlosser, 2005;
Brugmann et al., 2004;
Litsiou et al., 2005).
Transplantation and fate-mapping studies support the idea that the PPE
represents the earliest stage in placode induction
(Jacobson, 1963;
Kozlowski et al., 1997;
Martin and Groves, 2006;
Streit, 2002). Initially,
cells destined to form EB placode are intermingled within the PPE with
precursors of the otic placode (Streit,
2002) in what may be a PPE subdomain
(Schlosser and Ahrens, 2004).
Cells then segregate via an unknown mechanism to form EB placodes. Once
placodes are formed, Fgf3 and Bmp signal from the endodermal pouches to induce
neurogenesis (Begbie et al.,
1999; Holzschuh et al.,
2005; Nechiporuk et al.,
2005; Trokovic et al.,
2005).
Different stages of EB placode development are revealed by transcription
factor expression. Foxi1 is a forkhead-related winged helix transcription
factor expressed in EB placode precursors, and in foxi1 mutants,
placodal progenitors fail to undergo neurogenesis and subsequently die
(Lee et al., 2003). Pax2 is a
paired domain transcription factor that is upregulated in the EB placodes in
chick and Xenopus prior to neurogenesis
(Baker and Bronner-Fraser,
2000; Schlosser and Ahrens,
2004). ngn1 (also known as neurog1 - Zebrafish
Information Network) is the earliest proneural gene expressed in zebrafish EB
placodes (Andermann et al.,
2002). Similarly, neurogenin genes are expressed in EB
placodes in other species (Fode et al.,
1998; Schlosser and Ahrens,
2004). In zebrafish, Ngn1 activity is required for expression of
phox2a and phox2b
(Nechiporuk et al., 2005),
homeobox transcription factors that are in turn necessary for subsequent
differentiation of EB neurons (Dauger et
al., 2003).
Although signals that regulate EB placode formation are unknown, Fgf
signals are required for otic placode development in zebrafish, chick and
mouse (Ladher et al., 2005;
Leger and Brand, 2002;
Liu et al., 2003;
Maroon et al., 2002;
Wright and Mansour, 2003).
Loss-of-function experiments in zebrafish demonstrated that Fgf3 and Fgf8 are
redundantly required at multiple stages of otic placode induction
(Leger and Brand, 2002;
Solomon et al., 2004). Foxi1
is necessary during otic placode induction and at that point foxi1
expression is independent of Fgf signals
(Nissen et al., 2003;
Solomon et al., 2003;
Solomon et al., 2004). In
contrast, we have previously shown that Fgf signaling is necessary to maintain
expression of foxi1 in EB placodal precursors, suggesting that Fgf
signals might also be important for early specification of EB placodes
(Nechiporuk et al., 2005).
In this study, we show that global disruption of Fgf signaling blocks EB placode induction and subsequent neurogenesis. Using transplantation techniques, we demonstrate that Fgf signaling is required cell autonomously in EB placodes. Analyses of zebrafish mutants strongly suggest that inducing signals are mesoderm-derived, and that two mesodermally expressed fgf genes, fgf3 and fgf8, are required for EB placode formation. Transplantation of wild-type mesoderm into fgf3+8 morphants rescues EB ganglia, and ectopic Fgf3 or Fgf8 are sufficient to induce foxi1-positive EB precursors and phox2b-positive EB neurons. Overall, our results revealed a combined role for Fgf3 and Fgf8 during EB placode induction, and suggest a model where interactions between cranial mesoderm, ectoderm and endoderm are coordinated to assure proper development of the vertebrate head.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). The following alleles were used to produce mutant embryos:
fgf8, aceti282a
(Reifers et al., 1998);
foxi1, hsyem1 (Solomon
et al., 2003); fgf3, liat24149
(Herzog et al., 2004); and
pax2a, noitu29a (Lun
and Brand, 1998). Maternal-zygotic one-eyed pinhead
(MZoep) embryos were generated by crossing adult
oeptz57 mutant fish, which had been rescued by injecting
20 pg of oep mRNA at the one-cell stage
(Gritsman et al., 1999). The
Fgfr inhibitor, SU5402 (Calbiochem), was dissolved in dimethyl sulfoxide
(DMSO) at 50-100 mM and then further diluted to a working concentration in
embryo medium (EM) (Westerfield,
2000). Control embryos were treated with the same amount of DMSO
in EM. Embryos treated with a 60 µmol/L solution lacked trunk mesoderm,
otic vesicle, and erm and pea3 expression, without
exhibiting significant cell death. Thus, we used 60 µmol/L concentration in
all our subsequent treatments. To obtain hsp70::dn-fgfr1-positive
embryos we crossed [Tg(hsp70::dn-fgfr1)]pd1
heterozygotes (Lee et al.,
2005) to wild-type fish, heat shocked embryos at 38.5°C for 30
minutes and sorted embryos 2-12 hours later using epifluorescence.
GFP-negative embryos were used as controls.
Generation of phox2b::egfp transgenic line
We modified a phox2b-containing bacterial artificial chromosome
(BAC) clone by Escherichia coli-based homologous ET-recombination
(Zhang et al., 1998). BAC
clone C192B19 contains approximately 92 kb of sequence upstream and 32 kb
downstream of phox2b
(http://www.sanger.ac.uk/Projects/D_rerio/mapping.shtml).
Following recombination, the modified BAC clone contained an egfp
gene positioned at an endogenous start site. The accuracy of recombination was
evaluated by PCR, sequencing, and by transient expression assays.
phox2b::egfp BAC faithfully recapitulated endogenous phox2b
expression in cranial ganglia and other organs. To obtain a germline, we
injected linearized BAC DNA into zebrafish embryos, raised injected fish to
adulthood, and screened their progeny for reporter gene expression. The
germline transmission rate was 3%. The
[Tg(phox2b::egfp)]w37 strain has been outcrossed
for two generations and transmitted the transgene in a Mendelian fashion.
Morpholino 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
pressureinjected into one- or two-cell stage embryos. cas-MO,
5'-GCATCCGGTCGAGATACATGCTGTT, was injected at 2 ng/nL
(Sakaguchi et al., 2001);
fgf3-MO, 5'-CATTGTGGCATGGAGGGATGTCGGC, was injected at 0.75
ng/nL (Maroon et al., 2002);
fgf8-MOE2I2+fgf8-MOE3I3,
5'-TAGGATGCTCTTACCATGAACGTCG+5'-CACATACCTTGCCAATCAGTTTCCC, were
injected at 2+2 ng/nL each (Draper et al.,
2001).
In-situ hybridization and immunolabeling
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), eya1 (Sahly et
al., 1999), fgf3
(Kiefer et al., 1996),
fgf8 (Reifers et al.,
1998), fgfr1 (Scholpp
et al., 2004), fgfr2
(Tonou-Fujimori et al., 2002),
foxi1 (Lee et al.,
2003; Nissen et al.,
2003; Solomon et al.,
2003), ngn1 (Korzh et
al., 1998), nkx2.3
(Lee et al., 1996),
pax2a (Krauss et al.,
1991), pea3 (Raible
and Brand, 2001; Roehl and
Nusslein-Volhard, 2001), phox2a
(Guo et al., 1999),
phox2b (Shepherd et al.,
2004), anti-Hu (1:750, Sigma), anti-Pax2 (1:100, COVANCE),
anti-Prox1 (1:1000, Chemicon). For brightfield photography, embryos were
deyolked when appropriate, flat mounted in 50% glycerol plus 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). Brightness and
contrast were adjusted using Adobe Photoshop. Plastic sections of 5-6 µm
were obtained from in situ-stained embryos. For red Linbo's counterstain, dry
slides were treated with 0.5 mol/L NaOH for 5 minutes at room temperature,
then washed four times in water and dried at 73°C. Heated slides were
immersed into 2% solution of Basic Fuchsin (Allied Chemical Corporation) for 2
minutes, washed four times in water, dried and coverslipped.
Transplantation experiments
For transplants, embryos were raised in filtered EM supplemented with
penicillin (5000 U/L)/streptomycin (100 mg/L; Sigma). Donor embryos were
injected at the one-cell stage with 2% lysine-fixable fluorescein or
tetramethylrhodamine dextran (10,000 Mr; Molecular Probes)
in 0.2 mol/L KCl. Dechorionated donor and host embryos were mounted in 3.2%
methylcellulose in EM on a glass depression slide. For targeted transplants,
25-30 donor cells were inserted into the presumptive placodal domain of a
shield-staged host embryo, about 40° from the margin and 110° from the
shield. For mesodermal transplants, 25-30 cells were laid around the margin of
a 30-40% epiboly-staged embryo. Donor-derived fluorescein-labeled cells were
detected essentially as described
(Nechiporuk et al., 2005).
Fgf3 and Fgf8 misexpression
hs-fgf3myc (Maves et
al., 2002), hs-fgf8
(Roehl and Nusslein-Volhard,
2001) and hs-gfp plasmids were injected into
one-cell- or two-cell-staged embryos at 2.5 ng/µL. Embryos were
heat-shocked at 38.5°C for 30 minutes between 10 and 16 hpf, fixed at
various time points and processed to detect foxi1 or pax2a
mRNA. To detect Fgf-myc or GFP expression following RNA in-situ hybridization,
embryos were processed with mouse monoclonal anti-Myc (1:1000; Cell Signaling)
or rabbit polyclonal anti-GFP antibody (1:1000, Molecular Probes), and Alexa
568 or Alexa-488 secondary antibody (1:1000, Molecular Probes) as described
(Andermann et al., 2002).
Bead-implantation experiments were performed essentially as described
(Maves et al., 2002). Briefly,
20 µm polystyrene beads (Polysciences) were rinsed in PBS, treated with 0.5
mg/mL heparin for 20 minutes at room temperature, then incubated in 250
µg/mL mouse FGF8b (R&D Systems) with 0.5% bovine serum albumin (BSA) in
PBS for 2 hours at room temperature. Control beads were incubated in 0.5% BSA
in PBS. Embryos were mounted similar to transplantation experiments. A small
incision was made with a glass needle approximately half way between the first
somite and the anterior limit of the neural plate. One to three beads were
placed under the ectoderm either lateral to or into the neural plate tissue.
To test the efficacy of FGF8b beads, embryos with implanted beads were stained
for pea3 (Raible and Brand,
2001; Roehl and
Nusslein-Volhard, 2001). In total, 15 out of 17 embryos showed a
ring of pea3 expression around the FGF8b bead.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Schlosser and Ahrens,
2004), we analyzed its expression in zebrafish (see Fig. S1A in
the supplementary material). Expression of pax2a is found in
presumptive facial placodes beginning at 16-18 hpf (see Fig. S1A in the
supplementary material; data not shown) and extends caudally to include
presumptive glossopharyngeal and vagal placodes between 20-24 hpf. Transverse
sections of 24-hour-old embryos revealed pax2a mRNA in thickened
columnar placodal epithelium (see Fig. S1A in the supplementary material,
arrows) but only in its ventral part (see Fig. S1A in the supplementary
material, arrowheads). Between 28 and 36 hpf, pax2a expression is
maintained in individual placodes and in internalized neuroblasts, but
downregulated in differentiated Hu-positive EB neurons (see Fig. S1B in the
supplementary material; data not shown). The zebrafish pax2a null
mutant no isthmus (noitu29a)
(Lun and Brand, 1998)
displayed about 50% reduction in EB neurons (see Fig. S1C in the supplementary
material). Foxi1 activity is required for pax2a expression;
pax2a message was absent or strongly reduced in foxi1 mutant
embryos (see Fig. S1D in the supplementary material). Altogether these data
indicate that pax2a is expressed in the EB placodes prior to and
during neurogenesis and is necessary for their development.
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).
Zebrafish embryos were incubated with SU5402 or DMSO (control) beginning at 10
hpf. Alternatively, we used a zebrafish transgenic line that carries a
heat-shock-inducible form of dominant-negative Fgfr1 fused to GFP
(hsp70::dn-fgfr1) (Lee et al.,
2005). foxi1 expression was strongly reduced at 19 hpf in
both SU5402 and hsp70::dn-fgfr1 embryos
(Fig. 1). pax2a
expression in the EB placodes was completely absent at 24 hpf, and
phox2b expression was either absent or strongly reduced at 36 hpf
(Fig. 1). Thus, Fgf signaling
is required for expression of early placodal markers and subsequent
neurogenesis.
We used the SU5402 inhibitor and the hsp70::dn-fgfr1 transgenic
line to determine the temporal requirement for Fgf signaling. In zebrafish the
SU5402 block is reversible, with Fgf-mediated signaling restored within 1-2
hours after the inhibitor removal (Crump
et al., 2004; Maroon et al.,
2002; Phillips et al.,
2001). We incubated embryos in SU5402 for periods of 1.5-4.5
hours, and analyzed them at 24 hpf using Pax2 antibody
(Fig. 2A). In parallel,
hsp70::dn-fgfr1 embryos were heat shocked at the same time points as
for inhibitor application (Fig.
2B,C). In both sets of experiments, Pax2 expression in the facial
placode was either absent or strongly reduced when Fgf signaling was inhibited
between the 10- and 16.5-hour stages, with the strongest requirement found
between 10 and 11.5 hpf. Pax2 expression in the glossopharyngeal and vagal
placodes was most strongly affected later, after 11.5 hpf, suggesting that the
EB placodes are induced in an anterior to posterior fashion. Together, these
experiments demonstrate a strong Fgf requirement between 10 and 16.5 hpf for
all EB placodes (Fig. 2C), well
before the onset of neurogenesis at 24 hpf.
To determine whether Fgf signaling is active in EB placode precursors, we
analyzed expression of fgf receptors, and the Fgf transcriptional
targets erm and pea3 (see Fig. S2 in the supplementary
material). Fgfr1 has been suggested to mediate Fgf8 signals
(Scholpp et al., 2004),
whereas erm and pea3 are activated in response to both Fgf8
and Fgf3 (Raible and Brand,
2001; Roehl and
Nusslein-Volhard, 2001). Transverse sections through
13-14-hour-old embryos revealed that fgfr1, erm and pea3 are
expressed in the ectoderm in presumptive EB placode precursors (see Fig. S2 in
the supplementary material). In contrast, fgfr2, fgfr3 and fgfr4 were
not differentially expressed in the ectoderm at this stage (see Fig. S2 in the
supplementary material; data not shown). Overall, these data demonstrate that
Fgf signaling is active in EB placode precursors and is required for placode
formation.
Fgf signaling is required cell autonomously in EB placodes
Both drug treatment and the hsp70::dn-fgfr1 transgene expression
globally block Fgf signaling throughout the embryo. We therefore generated
mosaic embryos with cells from wild-type and hsp70::dn-fgfr1 lines to
determine which tissue(s) required receipt of the Fgf signal for proper EB
placode formation. We labeled hsp70::dn-fgfr1 donor embryos with
fluorescein-dextran tracer and transplanted 25-30 cells to the prospective
placodal domain of a wild-type host embryo at shield stage
(Fig. 3A). Resulting embryos
were heat shocked between 10 and 11 hpf and analyzed for Pax2 expression at 24
hpf. Whereas wild-type cells readily contributed to EB placodes in wild-type
host embryos, hsp70::dn-fgfr1 cells were largely excluded from them
(Fig. 3A and
Table 1). Moreover, we often
observed that when transgenic donor cells resided within the EB placodes, they
did not express Pax2 (Fig. 3A).
In reciprocal experiments, wild-type cells were transplanted into
hsp70::dn-fgfr1 embryos. Resulting embryos were heat shocked at
13.5-16 hpf and analyzed for Pax2 expression at 24 hpf
(Fig. 3B). The majority of
transplanted wild-type cells remained dorsally, at the level of the otic
vesicle; however, some migrated ventrolaterally and contributed to EB placodes
(Fig. 3B). When compared with
the contralateral control side, the total number of Pax2-positive cells on the
transplanted side was significantly increased (54 versus 42,
P<0.004, paired t-test;
Fig. 3B and
Table 1). If embryos were heat
shocked earlier (10-13.5 hpf), wild-type cells were completely excluded from
the presumptive EB placode region (data not shown). Overall, these data
demonstrate a cell-autonomous requirement for Fgf signaling, consistent with
our previous observations that Fgf signaling is active in the EB placodes.
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). In
contrast, embryos mutant for the sox-related transcription factor
casanova (cas) are missing endoderm
(Alexander et al., 1999;
Kikuchi et al., 2001).
foxi1 expression was severely reduced, whereas pax2a
expression was absent in MZoep mutants, a phenotype identical to that
resulting from reduction in Fgf signaling
(Fig. 4A). However,
foxi1 expression was normal and pax2a expression only
slightly altered in embryos injected with a cas morpholino (MO)
(Fig. 4A). These results
strongly suggest that mesoderm is the source of a placode-inducing signal.
These experiments and the time-course analysis described earlier suggest
that the Fgf ligands responsible for EB placode induction would be expressed
in cephalic mesoderm between 10 and 16.5 hpf. Both fgf3 and
fgf8 were expressed in two bilateral stripes adjacent to the neural
plate beginning at 10-11 hpf (Fig.
4B) (Reifers et al.,
2000). Expression began as a narrow stripe lateral to the
mid-hindbrain boundary and rhombomere 4 (r4). With time, expression extended
both rostrally and caudally adjacent to r6-7. These patterns of expression
were largely unchanged in cas-MO-injected embryos, but completely
absent in MZoep embryos (see Fig. S3 in the supplementary material),
arguing that fgf3 and fgf8 are expressed in mesoderm, not
endoderm (Fig. 4B). Transverse
sections through the 11- and 14-hour-old embryos confirmed that fgf3
and fgf8 were expressed in the underlying tissue and not ectoderm
(Fig. 4C). Interestingly, the
expansion of ectodermal foxi1 expression closely correlated with
changes in mesodermal fgf3 and fgf8 expression
(Fig. 4B). foxi1
expression was upregulated just anterior to the otic placode between 11 and 12
hpf (Fig. 4B), and then extends
posteriorly between 12 and 16 hpf (Fig.
4B). Thus, both fgf3 and fgf8 are expressed in
the cephalic mesoderm at the right stage, suggesting their involvement in EB
placode induction.
Fgf3 and Fgf8 are required for EB placode induction
To test whether Fgf3 and/or Fgf8 are required for EB placode induction, we
crossed limabsent (lia)/fgf3 mutants
(Herzog et al., 2004) with
acerebellar (ace); fgf8 mutants
(Reifers et al., 1998).
Whereas the fgf3 (liat24149) allele is null
(Herzog et al., 2004), the
fgf8 (aceit282) allele is a hypomorph that
retains about 25% of Fgf8 activity (Draper
et al., 2001). Resulting trans-heterozygous fish were intercrossed
to generate all genotypic combinations (including fgf3;fgf8 double
mutants), which were analyzed by in-situ hybridization using PPE, EB placode
and EB ganglia markers, photographed and genotyped. As expected,
pax2a expression is lost from the isthmus in fgf8 mutants
and the otic vesicle is missing in fgf3;fgf8 double mutants
(Fig. 5A)
(Leger and Brand, 2002;
Liu et al., 2003;
Maroon et al., 2002;
Reifers et al., 1998). In
addition, fgf3 mutants lacked ngn1 and phox2b
expression in the glossopharyngeal and small vagal placodes and ganglia, as
Fgf3 is needed for EB neurogenesis at later stages, as we previously reported
(Fig. 5A)
(Nechiporuk et al., 2005).
Reduction in Fgf3 and Fgf8 levels, either alone or together, did not affect formation of PPE as assessed by eya1 and six1 expression (Fig. 5A and data not shown). Ectodermal foxi1 levels were strongly reduced in fgf3;fgf8 double mutants but were not affected in either single mutant (Fig. 5A). Similarly, pax2a, ngn1 and phox2b expression in all EB placodes and ganglia was absent or strongly reduced in fgf3;fgf8 double mutants. We consistently observed some limited ngn1 and phox2a-staining, but not phox2b staining, in the large vagal placode and ganglion (Fig. 5A and data not shown). Interestingly, fgf8-/-; fgf3+/- embryos displayed an intermediate phenotype between the wild type and fgf3;fgf8 double. Because we did not observe any phenotype in fgf8-/- or fgf3+/-embryos alone, we concluded that Fgf3 and Fgf8 genetically interact.
To visualize epithelial morphology, we obtained transverse sections from wild-type and fgf3;fgf8 double mutant embryos processed for foxi1 and pax2a in situ hybridization (Fig. 5B,C). In wild type, the epithelium was well organized and displayed columnar morphology, with foxi1 expression mostly limited to the outer ectodermal layer. In contrast, epithelium was disorganized and remaining foxi1 expression extended to multiple cell layers in fgf3;fgf8 double mutants (Fig. 5B,C). Altogether, these results strongly argue that Fgf3 and Fgf8 are required for EB placode induction.
Restoration of cephalic mesoderm is sufficient to rescue EB ganglia
Both fgf3 and fgf8 are expressed in the neural tube at
the time of EB placode induction in addition to cephalic mesoderm. We
therefore performed tissue transplants to test where fgf3+8 is
required. To maximize the efficiency of these experiments, we transplanted
wild-type cells into embryos injected with fgf3+8 morpholino
oligonucleotides, rather than into fgf3;fgf8 double mutants. Because
endoderm-derived Fgf3 is required for later stages of EB placode neurogenesis
(Nechiporuk et al., 2005), we
first determined a dose of fgf3-MO that alone allowed normal
neurogenesis, but in conjunction with fgf8-MO phenocopied
fgf3;fgf8 mutants (see Fig. S4 in the supplementary material).
We injected host zebrafish embryos at the one-cell stage with
fgf3+8-MO, and wild-type donors were injected with a
fluorescein-dextran lineage tracer. At early gastrula stages (30-40% epiboly),
we transplanted 25-30 cells into the margin of fgf3+8 morphants. EB
ganglia were assessed at 54 hpf for phox2b mRNA expression
(Fig. 6A) or at 72 hpf for
phox2b::egfp transgene expression
(Fig. 6B). The
phox2b::gfp strain carries a stably integrated BAC clone with
egfp recombined into the endogenous phox2b start site.
Hindbrain transplant alone did not rescue EB ganglia
(Fig. 6A and
Table 2), but were capable of
rescuing the Fgf requirement for locus coeruleus development
(Guo et al., 1999). In
contrast, mesodermal transplants alone or together with hindbrain efficiently
rescued EB ganglia (Fig. 6A and
Table 2). We did not observe
any differences in the efficiency of the rescue between mesoderm and hindbrain
transplants versus mesoderm transplants alone
(Table 2). Although in some
cases transplants also included pharyngeal endoderm, donor endoderm did not
appear to be necessary for EB ganglia rescue in other cases
(Fig. 6B;
Table 2). When visualized at 72
hpf, it is apparent that donor cells contributed to the various facial muscles
in the rescued embryos. From these data we conclude that the cephalic
mesoderm, but not the neural tube, is sufficient to rescue EB ganglia in
fgf3+8 morphants.
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To introduce Fgf from a more localized source, we performed bead implantation experiments. The 20-µm beads were soaked in mouse recombinant Fgf8b and placed under the ectoderm just lateral to the neural plate in wild-type embryos between 11 and 13 hpf. Resulting embryos were analyzed for foxi1 expression at 19 hpf (Fig. 7B). Control embryos that received BSA-soaked beads showed no changes in foxi1 expression (Table 3 and data not shown). In contrast, we observed ectopic foxi1 expression foci in the vicinity of the Fgf8b-coated beads in 40% of the analyzed embryos (n=15; Fig. 7B and Table 3). Fig. 7C summarizes the location of ectopic foxi1 expression from all ectopic Fgf overexpression experiments. We observed ectopic foci throughout the ectoderm located ventrally to the endogenous foxi1 expression domain, but found no ectopic foci dorsal to the endogenous domain or in trunk ectoderm.
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). To
determine whether anterior expansion of foxi1 by ectopic Fgf
expression similarly affects lens specification, we assayed
hs-fgf3myc-injected embryos for a lens marker Prox1
(Tomarev et al., 1998;
Wigle et al., 1999). Almost
all embryos with rostral expansion of foxi1 expression displayed
either reduction or absence of lens Prox1 expression (15/17;
Fig. 7D). We also observed a
similar result using Fgf8b-coated beads (3/4; data not shown). This result
implies that more anterior PPE can be respecified to form EB placode
precursors, at the expense of lens precursors.
To confirm that the ectopically generated foxi1-positive
precursors could give rise to EB neurons, we assayed wild-type embryos after
bead implantation for phox2b expression at 48 hpf. Most embryos
displayed ectopic phox2b-positive foci (6/10;
Fig. 7E and
Table 3). Similarly, activation
of hs-fgf3myc or hs-fgf8 at 12-13 hpf also resulted in
formation of ectopic foci. Importantly, a number of ectopic
phox2b-positive foci were located on the ventral side of the head and
belly ectoderm, away from the endogenous phox2b-expression sites and
pharyngeal endoderm (Fig. 7F).
Analyses of phox2b::egfp embryos revealed that the ectopic cells
extended peripheral projections, confirming their neuronal identity. When
ectopic phox2b-positive foci were located adjacent to the eye, we
often observed either reduction or complete loss of lens tissue
(Fig. 7G, top panels). We
confirmed that formation of ectopic phox2b-positive neurons did not
require the presence of endodermal pouch tissue, as assayed by an endodermal
pouch marker nkx2.3 (Lee et al.,
1996). This result is consistent with previous observations
demonstrating that a subset of EB placodal cells did not require
endoderm-derived signals to undergo neurogenesis
(Holzschuh et al., 2005;
Nechiporuk et al., 2005).
Overall our data show that Fgf-induced ectopic foxi1-positive
precursors could differentiate into phox2b-positive epibranchial
neurons.
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|
DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Although mesodermal Fgf signals are necessary for both the expansion of
foxi1 and the induction of pax2a, our analysis suggests that
the critical event is the establishment of foxi1 expression. The
timing of expansion of foxi1 expression to include the presumptive EB
placode domain corresponds to the anterior and posterior spread of
fgf3 and fgf8 expression in cranial mesoderm between 10 and
14 hpf, precisely the critical period delineated by inhibitor studies and,
significantly, before the initiation of pax2a expression at 16 hpf.
Interestingly, pax2a has been similarly suggested to act downstream
of Fgf and Foxi1 signals in otic placode formation
(Hans et al., 2004).
What is the role of Pax2a in the EB placode development? Targeted
inactivation of mouse Pax2 demonstrated that it is required for ear patterning
and acoustic ganglion development (Burton
et al., 2004; Torres et al.,
1996), but its function in EB placode development was not
determined. Pax2 expression has been described in EB placodes in chick and
Xenopus (Baker and Bronner-Fraser,
2000; Schlosser and Ahrens,
2004), but not previously in zebrafish. We found that, in
zebrafish pax2a mutants, the development of some but not all EB
neurons was disrupted. It is possible that other pax genes may play
redundant roles during EB placode development, similar to the situation for
the otic placode. Whereas pax2a mutants have a very mild reduction of
the otic placode, injection of pax8 morpholino results in its almost
complete absence (Hans et al.,
2004; Mackereth et al.,
2005). Interestingly, pax8 expression has been reported
in the EB placode in Xenopus
(Schlosser and Ahrens, 2004).
Pax2b, a zebrafish ortholog of Pax2a
(Pfeffer et al., 1998), may
also play a redundant role during EB placode development.
|
;
Brugmann et al., 2004;
Litsiou et al., 2005). After
the PPE is formed, the ventral part of the competent ectoderm receives Fgf3
and Fgf8 signals from the cephalic mesoderm
(Fig. 9A). Importantly, Fgf
signals are not required for the initial induction of foxi1
expression, as foxi1 message is still present in Fgf-deficient
embryos. Residual foxi1 expression may represent a subset of cells
that will not normally contribute to EB placodes, or may indicate cells that
are stalled in the early stages of their development. Although our
misexpression experiments demonstrated that most of the PPE and ventral
embryonic ectoderm are competent to express foxi1, only the ventral
portion of the PPE normally expresses it. While the foxi1 expression
domain remains broad, pax2a expression is refined over the course of
development: pax2a is more restricted than foxi1 and is
expressed only in the individual placodes during neurogenesis. Our
observations support a multi-step model of EB placode induction where a first
step is to induce a broad field of precursors marked by foxi1
expression, which is further refined by other signals during later steps of
development. This multi-step model might explain how otic and EB precursors
interpret the same Fgf signals. Close proximity of cephalic mesoderm to the
ventral part of competent ectoderm might help to restrict foxi1+ EB
precursors. Alternatively, additional signal(s), possibly from a different
source, might distinguish EB and otic precursors. In support of this idea, a
recent study demonstrated that in addition to inductive Fgf signals, Wnt
signals, possibly from hindbrain, help refine otic placode positioning in
mouse embryos (Ohyama et al.,
2006).
|
|
). We
suggest dual roles for Fgf3 and Fgf8 in the initial development of these
tissues to ensure the subsequent developmental coordination of EB neurons and
their target of innervation. Signals from pharyngeal pouch endoderm, including
fgf3, bmp2b and bmp5, are required to initiate expression of
the early proneural genes such as ngn1 and neuroD
(nrd) in the individual EB placodes
(Fig. 9B)
(Holzschuh et al., 2005;
Nechiporuk et al., 2005).
ngn1 is also transiently expressed in migrating and condensing
neuroblasts, and is required for the subsequent formation of
phox2-positive neuronal precursors
(Andermann et al., 2002;
Nechiporuk et al., 2005).
Eventually, phox2-positive precursors condense together with
neural-crestderived glia to form cranial ganglia. Further studies will be
necessary to determine what cues guide EB neurons to their final
destination.
It has been suggested previously that pharyngeal endoderm is required for
EB placode induction in avian embryos
(Begbie et al., 1999).
Therefore, one might interpret our results showing a role for mesodermal Fgf
signaling in zebrafish placode induction as simply a reflection of the Fgf
requirement for pharyngeal pouch development
(Crump et al., 2004). However,
there are several arguments against this explanation. First, in zebrafish,
pharyngeal pouches do not begin to form until 22 hpf
(Holzschuh et al., 2005), well
after pax2a expression is established in the thickened EB ectoderm
(this study). Second, loss of pharyngeal endoderm in zebrafish
casanova mutants results not in elimination of EB placodes but rather
reduction in neurogenesis; expression of ngn1, phox2b and the Hu
antigen is lost, whereas foxi1, pax2a and morphological thickening of
the ectoderm is retained (Holzschuh et
al., 2005; Nechiporuk et al.,
2005) (this study). Although this result may reflect differences
between fish and avian species, it is important to note that only late markers
of neurogenesis (Phox2a, NF-M) were analyzed in the previous avian study
(Begbie et al., 1999), leaving
open the possibility that mechanisms might be conserved. Third, our mosaic
analysis demonstrates that Fgf signaling is required directly within the
ectoderm, suggesting that the mesodermal Fgf signal is received directly by
placodal precursors. Finally, we show that ectopic expression of Fgf results
in formation of new phox2b-positive neurons in the absence of
nkx2.3+ pharyngeal endoderm. Although we cannot rule out that
pharyngeal endoderm supplies additional EB-promoting signals, our results
strongly support a role for mesodermal Fgf directly acting upon placodal
ectoderm.
Role of Fgf signaling in specification and induction of EB placodes
FGFs play a critical role in morphogenesis of multiple organ systems by
regulating cell proliferation, differentiation, cell migration and cell
survival. Thus, Fgf signals could regulate expansion of
foxi1-positive precursors by any of the above mechanisms. Fgf signals
can act as chemoattractants during both invertebrate and vertebrate
development. In Drosophila, Fgf plays the role of a chemotactic
signal during tracheal morphogenesis
(Sutherland et al., 1996),
whereas in worms, the Fgf signal guides sex myoblasts to the gonads
(Burdine et al., 1998;
Burdine et al., 1997). Fgf4
and Fgf8 act as chemotactic signals to directly coordinate cell movements
during gastrulation in the chick embryo
(Yang et al., 2002). We
observed that foxi1 expression could be induced a few cell diameters
away from an Fgf source, arguing that Fgf might function as a long-range
signal to recruit EB placode precursors. This is consistent with recent work
in zebrafish revealing that Fgf ligand could travel as far as 16-cell
diameters away from an Fgf source (Scholpp
and Brand, 2004). Although it is attractive to speculate that Fgf
signals could promote migration, further studies are necessary to define the
exact role of Fgf3 and Fgf8 in the recruitment of EB placode precursors.
Conservation of the inductive signals in the cranial placodes
It has been argued that once the PPE domain is established, local signal(s)
promote the fate of the individual placodes
(Streit, 2004). The initial
step common to all placodes include signals from mesendoderm
(Ahrens and Schlosser, 2005;
Litsiou et al., 2005), whereas
later inductive signals for specific placodes would originate from distinct
tissues. Dorsolateral (trigeminal, lateral line and otic) placodes are in
close proximity to the neural tube, whereas ventrolateral (EB) placodes are in
close contact with the endoderm and cephalic mesoderm. Indeed, Fgf signals
from the neural tube are required during otic placode development
(Leger and Brand, 2002), and
neural tube signal(s) is sufficient to induce trigeminal placode markers in
the head ectoderm, although the nature of this signal(s) is still unknown
(Stark et al., 1997).
There are surprising similarities between otic and EB placode development.
Both otic and EB placodes require eya1 and six1
(Ozaki et al., 2004;
Xu et al., 1999;
Xu et al., 2003;
Zheng et al., 2003;
Zou et al., 2004), and their
precursors are intermingled in the PPE
(Streit, 2002). Mesodermal
Fgfs are involved in the initial induction of each placode from PPE
(Kil et al., 2005;
Wright and Mansour, 2003), and
induction is regulated by foxi1 to induce expression of
pax2a (Mackereth et al.,
2005; Nissen et al.,
2003; Solomon et al.,
2003; Solomon et al.,
2004). Finally, Fgf signals subsequently regulate neurogenesis
from both EB and otic placodes upstream of ngn1
(Alsina et al., 2004;
Nechiporuk et al., 2005).
Although it is not surprising that the six/eya/dach network is conserved in
various ectodermal placodes, as these transcription factors are expressed
during PPE formation and are important for PPE development, it is remarkable
that the same Fgf inductive signals are conserved in the EB and otic
placodes.
However, it is important to note that although Fgf3 and Fgf8 signals are
necessary during both otic and EB placode induction, we were able to separate
the timing and Fgf requirement during these two processes. First, we defined a
separate time-window requirement for Fgf signaling during EB placode
development. During the otic placode induction, Fgf signaling is not required
beyond 11 hpf, whereas it is essential until 16.5 hpf during EB placode
development. Second, ectopic activation of Fgf signaling at 13 hpf, well after
the otic placodes had been induced, promotes formation of the
foxi1-positive precursors and EB-specific phox2b-positive
neurons. Finally, analyses of MZoep mutants, which completely lack EB
placodes while their otic vesicles are only reduced, support the idea that Fgf
signaling plays distinct roles during initial stages of the otic and EB
placode development. Alternatively, because EB and otic precursors are
initially intermingled within PPE
(Schlosser and Ahrens, 2004;
Streit, 2002), it is possible
that the same signals, including Fgfs, might control initial specification of
common precursors. Interestingly, inhibition of Fgf signaling during
gastrulation in zebrafish blocks expression of the early otic placode markers
(Maroon et al., 2002;
Phillips et al., 2001). It
will be interesting to investigate whether this early Fgf block affects EB
placode development as well.
The preservation of the molecular cascades involved in each type of placode
development might be even more conserved than had previously been anticipated.
A recent report suggests that Fgfs promote olfactory placode at the expense of
lens, the ground state for cranial placodes
(Bailey et al., 2006); we found
similarly that Fgfs promote formation of foxi1+ placode precursors
while simultaneously blocking lens formation. Fgfs are also involved in
neurogenesis from olfactory epithelium
(DeHamer et al., 1994;
Kawauchi et al., 2005),
suggesting that recurrent roles for Fgfs are also a common theme in placode
formation. Surprisingly, Fgf signaling is also important during inductive
phases of the lens placode, although the identity of Fgf ligands involved
remains unknown (Faber et al.,
2001). Overall, our findings are consistent with the idea that new
placodes have arisen by co-option of similar molecular mechanisms, while the
tissue origin of the inducing signal diverged over the course of
evolution.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/134/3/611/DC1
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Ahrens, K. and Schlosser, G. (2005). Tissues and signals involved in the induction of placodal Six1 expression in Xenopus laevis. Dev. Biol. 288,40 -59.[CrossRef][Medline]
Alexander, J., Rothenberg, M., Henry, G. L. and Stainier, D. Y. (1999). casanova plays an early and essential role in endoderm formation in zebrafish. Dev. Biol. 215,343 -357.[CrossRef][Medline]
Alsina, B., Abello, G., Ulloa, E., Henrique, D., Pujades, C. and Giraldez, F. (2004). FGF signaling is required for determination of otic neuroblasts in the chick embryo. Dev. Biol. 267,119 -134.[CrossRef][Medline]
Andermann, P., Ungos, J. and Raible, D. W. (2002). Neurogenin1 defines zebrafish cranial sensory ganglia precursors. Dev. Biol. 251, 45-58.[CrossRef][Medline]
Bailey, A. P., Bhattacharyya, S., Bronner-Fraser, M. and Streit, A. (2006). Lens specification is the ground state of all sensory placodes, from which FGF promotes olfactory identity. Dev. Cell 11,505 -517.[CrossRef][Medline]
Baker, C. V. and Bronner-Fraser, M. (2000). Establishing neuronal identity in vertebrate neurogenic placodes. Development 127,3045 -3056.[Abstract]
Begbie, J., Brunet, J. F., Rubenstein, J. L. and Graham, A. (1999). Induction of the epibranchial placodes. Development 126,895 -902.[Abstract]
Bessarab, D. A., Chong, S. W. and Korzh, V. (2004). Expression of zebrafish six1 during sensory organ development and myogenesis. Dev. Dyn. 230,781 -786.[CrossRef][Medline]
Brugmann, S. A., Pandur, P. D., Kenyon, K. L., Pignoni, F. and
Moody, S. A. (2004). Six1 promotes a placodal fate within the
lateral neurogenic ectoderm by functioning as both a transcriptional activator
and repressor. Development
131,5871
-5881.
Burdine, R. D., Chen, E. B., Kwok, S. F. and Stern, M. J.</
