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doi: 10.1242/10.1242/dev.00455
DEVELOPMENT AND DISEASE |
1 Center for Cancer Research and Department of Biology, Massachusetts Institute
of Technology, Cambridge, MA 02139, USA
2 Genome Technology Branch, National Human Genome Research Institute, National
Institutes of Health, Bethesda, MD 20892, USA
* Author for correspondence (e-mail: burgess{at}nhgri.nih.gov)
Accepted 20 February 2003
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRT-PCRRESULTSDISCUSSIONREFERENCES |
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Key words: Foxi1, Otic placode, Pharyngeal pouch, fgf8, fgf3, pax8
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRT-PCRRESULTSDISCUSSIONREFERENCES |
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). The
placode cavitates to form a lumen and hair cells begin to differentiate,
projecting bristles into the liquid filled interior
(Haddon and Lewis, 1996). Over
the next few days, columns of tissue grow out from the walls of the otic
vesicle, fusing in the center of the space to generate semicircular canals.
Many genes have been shown either to be expressed in the developing zebrafish
ear, or to be involved in the proper formation of the organ
(Whitfield et al., 2002).
pax2a and dlx3b are expressed in the developing placodes and
roles in ear development for each of these genes have been demonstrated or
inferred (Krauss et al., 1991;
Lun and Brand, 1998;
Solomon and Fritz, 2002). Fgf3
and Fgf8 have been implicated directly in the induction of the otic placode
(Leger and Brand, 2002;
Maroon et al., 2002;
Phillips et al., 2001).
Inhibition of either one of these signaling molecules causes marked reductions
in the size of the placode, while inhibiting both causes an elimination of the
ear primordia. pax8 is the earliest known molecular marker for cells
that will become the otic placode, but its actual role in induction is not
known (Pfeffer et al.,
1998).
As in all vertebrates, the zebrafish ear serves the dual functions of
linear acceleration detection and hearing
(Bever and Fekete, 2002).
Although functionally similar to the human ear, many structures are not
present including the cochlea and middle ear. Remarkably, the middle ear
structures first arose during the water-to-land transition and their origins
are believed to be structures derived from the gills (spiracular pouch) and
the jaw (hyomandibular bone) of fish
(Webster et al., 1992).
The pharyngeal cartilages are derived from three streams of ventrolaterally
migrating cranial neural crest (NC) cells. The pharyngeal skeleton shows both
an anterior-to-posterior (AP) segmented polarity, with each segment deriving
from an embryonic pharyngeal arch, as well as a dorsal-to-ventral (DV)
polarity within individual AP segments
(Trainor and Krumlauf, 2001).
The first mandibular arch gives rise to a ventral Meckel's cartilage, which
articulates with a dorsal palatoquadrate. The second hyoid arch is serially
homologous to the first arch with a ventral ceratohyal that articulates via
the interhyal with the hyosymplectic. The hyosymplectic is composed of a
ventral symplectic rod region and a dorsal plate-like hyomandibular region.
The arches and pouches form dynamically with the ectoderm forming an AP series
of bilateral surface `in-pockets' complementary to bilateral endodermal
out-pockets that together form the pharyngeal pouches separating adjacent
arches along the AP axis. The migratory NC cells fill the spaces between the
forming pouches adopting a cylindrical morphology encasing central cores of
paraxial mesoderm (Kimmel et al.,
2001).
Pharyngeal pouches likely signal to adjacent NC cells
(Le Douarin and Ziller, 1993;
Veitch et al., 1999) and
essential roles for endothelin1 (edn1) in forming ventral
cartilages has been shown in zebrafish and mice
(Clouthier et al., 1998;
Kurihara et al., 1994;
Miller et al., 2000). Evidence
for a dorsal cartilage-patterning signal is provided by the zebrafish
fgf8 mutant, acerebellar (ace), which has a reduced
hyomandibular region. However, early neural tube defects and absence of
ace expression in the pouches makes interpretation difficult
(Roehl and Nusslein-Volhard,
2001). Studies on zebrafish casanova (cas)
mutants and Fgf3 morpholino antisense oligonucleotide knockdowns suggest that
loss of Fgf3 pouch expression results in early apoptotic elimination of
posterior NC cells (Alexander et al.,
1999; David et al.,
2002). How an Fgf signal might function in DV patterning within an
arch is still unclear.
In a large, insertional mutagenesis screen performed at MIT
(Amsterdam et al., 1999;
Golling et al., 2002), we
isolated four insertional alleles of the zebrafish ortholog of the forkhead
related transcription factor Foxi1 (FREAC6, FKHL10, HFH-3, Fkh10)
(Avraham et al., 1995;
Chen et al., 2002;
Clevidence et al., 1993;
Hulander et al., 1998;
Larsson et al., 1995) that
show specific defects in both ear and jaw development. The otic placodes are
severely reduced in size, often split into two smaller placodes, and the
semicircular canals fail to form properly, often resulting in a single large
cavity instead of the normal three distinct chambers. The dorsal first arch
derivative, the palatoquadrate, is mildly reduced in mutant animals, whereas
the ventral Meckel's cartilage is indistinguishable from wild type. The dorsal
second arch derivative, the hyomandibular region, is severely reduced in
mutant animals while the ventral symplectic rod region and ceratohyal appear
only mildly affected. Of the posterior arches, the third and fourth arches
show substantial variable reductions while the fifth to seventh branchial arch
cartilages appear relatively unaffected. Interestingly, targeted mutations of
Foxi1 in mice show defects in ear development and homozygous mutant mice are
born with both hearing and vestibular defects, as well as a lower survival
rate after birth (Hulander et al.,
1998), suggesting some conservation of the developmental pathway
of the ears between zebrafish and mice.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRT-PCRRESULTSDISCUSSIONREFERENCES |
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). The
retroviral insertion junction fragment cloning and animal genotyping
procedures were as previously described
(Golling et al., 2002;
Sun and Hopkins, 2001). The
primers used for genotyping carrier fish and embryos were:
). The
primers used for 5'RACE amplification were
5'-GATGGTGCAGGTTCTGCTGGTACATGCTG-3' followed by
5'-TCTGACCTGCTGAGTGTGCTCGTCTCTGAGG-3'. The primers used for
3'RACE amplification were 5'-GAGAAAAAGAAGAGCCGACGGAAAC-3'
followed by 5'-GTCTGTGAAATCCGAGGACGCG-3'.
Zebrafish BAC library screening
High-density filters to the CHORI-211 BAC library were hybridized with a
radioactive probe made from the EcoRI-digested plasmid containing a
partial foo cDNA. The positive BAC clones were identified by PCR
amplifications and used for fingerprinting and automated sequencing.
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RT-PCR |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRT-PCRRESULTSDISCUSSIONREFERENCES |
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)
and RNA was prepared using Trizol reagent (ROCHE) with subsequent first-strand
synthesis as previously described (Sun and
Hopkins, 2001). The RT-PCR primers used for amplifying the
sequence containing all foo proviral insertion sites were 1321-start
and 1321-RT2 5'-CCTAAGATGACAAGAATGGGTGTTGG-3'. For RT-PCR
amplification of the internal non-flanking sequence the primers used were
3239-gap1 5'-CGCTCAGTCAGATTTATCAGTACGTGG-3' and 3239-gap2. The
primers used for amplifying the b-actin control sequence were
5'-CATCAGCATGGCTTCTGCTCTGTATGG-3' and
5'-GACTTGTCAGTGTACAGAGACACCCTG-3'.
Plasmids
The plasmid pCR2.1-foxi1 3'RACE was isolated by performing
3'RACE with the previously described primers. It was digested with
NotI and antisense probe for in situ hybridization synthesized using
T3 RNA polymerase as previously described
(Sun and Hopkins, 2001).
Previously described plasmids for probes were hand2
(Angelo et al., 2000),
dlx2 and dlx3b (Akimenko
et al., 1994), gsc
(Schulte-Merker et al., 1994),
krox20 (egr2 – Zebrafish Information Network)
(Oxtoby and Jowett, 1993),
nkx2.3 (Lee et al.,
1996) and pax2a
(Krauss et al., 1991). Plasmid
for making fgf3 probe was constructed by PCR amplification from 0-24
hpf cDNA using primers fgf3-f 5'-CTTGTTGTTACTGAGCTTCTTGGATCCGAG-3'
and fgf3-r 5'-CCTCCAGATTTCAGTGTCAAACAATGCC-3', followed by
subcloning into pCRII-BLUNT to yield pCR-fgf3. To make antisense probe,
pCR-fgf3 was digested with NotI and transcribed using SP6 RNA
polymerase. Plasmid for making pax8 probe was constructed by PCR
amplification from 0-24 hpf cDNA using primers pax8-f
5'-GCTTCCGGAGGTGATCCGGCAAAGG-3' and pax8-r
5'-CTGGAGTTGGTGAATCTCCAGGCCTCG-3' followed by subcloning into
pCRII-BLUNT to yield pCR-pax8. To make antisense probe, pCR-pax8 was digested
with BamHI and transcribed using T7 RNA polymerase.
Embryo injections
Embryos were injected at the one- to four-cell stages using pulled glass
needles and a picospritzer II (Parker Instrumentation) with the Fgf3-MO
(Phillips et al., 2001).
Calibration of injection needles indicated an average injection volume of 1 nl
per 50 msecond pulse. Morpholino sequence was
5'-ACTCATGTTGACTACTCCTCCCACT-3'; four-base mismatch control was
5'-ACTCATCTTCACTACTGCTCCCAGT-3'.
In situ hybridization and immunohistochemistry
Whole-mount in situ hybridization was performed as previously described
(Thisse et al., 1994).
Two-color reactions were performed as previously described
(Hauptmann and Gerster, 1995;
Sun and Hopkins, 2001).
Whole-mount immunohistochemistry was performed as previously described
(Hanneman et al., 1988).
For TUNEL assays, fixed dehydrated embryos were rehydrated and acetone
permeabilized for 10 minutes at –20°C, then rehydrated in PBST,
washed in PBS + 0.25 mg/ml BSA and preincubated in TdT buffer for 2 hours at
room temperature. TdT/fluorescein-dUTP reactions (Roche product 1-767-291)
were incubated overnight at room temperature. Embryos were then washed twice
for 45 minutes in PBST+1 mM EDTA at 65°C, 4 times for 45 minutes in PBST
at room temperature, and processed with anti-fluorescein antibodies for
alkaline phosphatase activity as previously described
(Thisse et al., 1994).
Cartilage staining
Alcian Blue staining was performed as previously described
(Golling et al., 2002).
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRT-PCRRESULTSDISCUSSIONREFERENCES |
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), the
mutant phenotype was linked to a single proviral integration for each of four
independent isolates of foo (not shown). The genomic DNA adjacent to
the proviral integration events was cloned, sequenced, and used to determine
linkage of the insertions to the mutant phenotype
(Fig. 1E) (see
Golling et al., 2002) All
alleles were crossed and failed to complement. Linkage data placed the
mutation responsible for the foo/foo mutant phenotype within 0.9 cM
of a proviral insertion. The sequences adjacent to all four proviruses show
identity to a single zebrafish EST (Accession Number BI891904). We isolated
several bacterial artificial chromosomes (BACs) and sequence from this genomic
region showed significant homology at the DNA and protein level to the mouse
forkhead related transcription factor Foxi1 (Fkh10) which has presumed
orthologs in several species, including human (Fkhl10, FREAC6), rat (HFH-3)
and frogs (Foxi1c). Among all species, including zebrafish, a genomic
structure of two exons with a single intron (in the same location) is
conserved (Fig. 1F). In one
allele, hi3747, the mutagenic provirus lies in sequences encoding a
poly-histidine stretch near the 5' end of the gene and should result in
a truncation before the conserved DNA-binding domain
(Fig. 1F). We performed RT-PCR
on mutant and wild-type embryos for each allele with two sets of primers: one
set spanning the entire gene and located outside all four viral integration
sites; and a second internal set that did not flank any of the viral
integration sites (Fig. 1F).
The longer amplicon was not successfully amplified from any of the alleles,
although a significantly reduced amount of RNA was detected with the smaller
amplicon in hi1321, hi3239 and hi3747. Almost no RNA was detected in the
hi1359A allele. In situ hybridization of foo antisense RNA to mutant
embryos was negative compared with wild-type siblings
(Fig. 2D,E), suggesting a
destabilized mRNA. The severity of the ear phenotype was variable within each
mutant line, making comparisons of allele strength difficult. However, the
allele with the most consistent phenotype was hi3747, the mutant with a
proviral integration closest to the 5' end of the gene and before the
DNA binding domain. In hi3747, the otic vesicles were substantially reduced
and often split (as in Fig.
1D).
|
;
Nasevicius and Ekker, 2000).
As further confirmation that mutations in the foo gene are
responsible for the observed phenotypes, we designed two morpholino antisense
oligonucleotides to foo mRNA and injected them into embryos. In 50%
of the embryos, the phenotype generated was indistinguishable from the mutant
alleles (39/78) in ear or jaw defects, while the control morpholino showed no
effect (data not shown).
Expression of Foxi1 in the early embryo
By RT-PCR, foo transcripts were not detected in unfertilized
oocytes but were readily detected by the sphere stage (data not shown).
Location of foo expression by in situ hybridization was first
detected at the dome stage (Fig.
2A,B). By the shield stage, expression was in the presumptive
ventral ectoderm near the animal pole that is fate mapped to be placodal
precursors (Fig. 2C)
(Kozlowski et al., 1997;
Woo et al., 1995). By 90%
epiboly, foo expression was split into bilateral regions
(Fig. 2D). As convergence
continued, foo expression appeared to be restricted to a region
destined to become the otic placode (Fig.
2F). Expression of foo precedes expression of
pax8, making it the earliest known marker of otic placode
formation.
Impact of foo mutations on otic placode patterning
Because of the early expression of foo in the developing otic
placode, we examined the expression in our mutant of other genes known to be
expressed early in the otic placode. Mutant embryos showed reduced expression
of dlx3b and pax2a that, in addition, was often split into
several small patches of expression (Fig.
3D,F,H, arrows). Expression of dlx3b and pax2a
in other areas of the developing embryo were unaffected
(Fig. 3D,F,H, arrowheads). The
strongest effect we detected was on the expression of pax8. At the
one-somite stage, no pax8 expression could be detected in the
presumptive otic placode of one quarter of the embryos
(Fig. 3B). However, we saw
normal expression of pax8 in the mesoderm of presumptive kidney
(Fig. 3B, arrowhead). This
suggests that foo is upstream of pax8 in the otic vesicle
and is required for proper expression of pax8 in this region.
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). The ventral expression of hand2 appeared
largely unaffected in the first and second arches consistent with the near
normal formation of ventral cartilage elements in the anterior arches, but,
mirroring the dlx2/3 defects, expression of hand2 was
markedly reduced in the third and fourth arches of foo/foo embryos
(Fig. 7C,D). Taken together,
altered expression in foo/foo embryos of these three markers indicate
a severe disruption in the proper patterning of the third and fourth
arches.
|
). gsc expression in wild-type embryos shows
distinct dorsal NC cell expression in the first and second arches flanking
either side of pp1 as well as a distinct second arch ventral expression domain
(Miller et al., 2000). Outside
the arches, gsc is also expressed within the developing otic vesicle.
Strikingly, the dorsal first and second arch gsc expression domains
were not detected in foo/foo embryos
(Fig. 7E,F, arrows). Expression
in the otic vesicles was likewise not detected in foo/foo embryos.
Conversely, the olfactory and ventral second arch gsc expression
domains appeared unaffected in mutants. Consistently, the homeobox
transcription factor hoxa2, which is uniformly expressed in the
second arch NC cells, also showed a dorsally restricted second arch NC cell
defect (data not shown). Thus, defects in markers for the dorsal, but not
ventral, NC cells of the first and second arches precedes the hyomandibular
defect.
foo is required for survival of certain NC cell
populations
To determine whether the loss of expression of NC cell markers and absence
of corresponding cartilages was the result of NC cell apoptosis, we performed
terminal deoxynucleotidyl transferase fluorescein-dUTP nick end labeling
(TUNEL) assays to visualize apoptotic cells in developing embryos. When
compared with wild-type embryos, foo/foo embryos display a
substantial, transient increase in apoptosis in the pharyngeal arch region
(Fig. 8A-G). The transient wave
of cell death peaks around 26 hpf at 
20-fold over the intensity observed
in wild-type embryos, and is then reduced by 28 hpf
(Fig. 8H). Notably, no
increased apoptosis was observed at 22 hpf or at earlier time points during
otic development (Fig. 8H; data
not shown). Based on their sub-ectodermal location in transverse sections, at
least some of these TUNEL-positive cells appear to be post-migratory NC cells
(Fig. 8E-G, sections located as
approximately indicated in 8D). However, we cannot rule out that some of these
TUNEL-positive cells might be endodermal pouch cells.
|
).
Therefore, we examined the dynamic expression of fgf3 in the
pharyngeal pouches. By 15 somites to at least 34 hpf of development,
fgf3 was expressed in the endoderm two pouches at a time with
downregulation in the most anterior pouch coincident with up regulation in the
next most posterior pouch on a roughly four hour cycle
(Fig. 8I,K,M,O, arrowheads;
data not shown). At 16- and 20-somite stages, expression of fgf3 was
not significantly different between wild-type and mutant animals indicating
that initiation of fgf3 expression was not compromised, and that
fgf3-positive cells are at least initially present in mutant animals
(Fig. 8I,J). However, by the
24-somite stage, foo/foo embryos showed a premature loss of
fgf3 expression from pp1, the anterior-most pharyngeal pouch
(Fig. 8L, red arrowhead).
Examination of later developmental time points also shows premature loss of
fgf3 expression from the anterior-most pouch
(Fig. 8N,P, blue then first
black arrowhead). Thus, although initiation of fgf3 pouch expression
is always observed, the maintenance of fgf3 pouch expression appears
compromised. Expression of fgf3 in other regions of the embryo, such
as the isthmic region, was unaffected, which indicates the specificity of this
defect.
As morpholinos that inhibit fgf3 are known to cause posterior
pharyngeal arch defects similar to those observed in foo/foo embryos,
we analyzed fgf3 morpholino injected animals for hyomandibular
defects (David et al., 2002).
Consistent with personal communications (L. Maves and C. B. Kimmel,
unpublished) we observed not only the posterior gill arch defects but also a
reduction of the hyosymplectic (Fig.
8Q,R, arrows). Out of 39 fgf3 morpholino-injected animals
selected at 30 hpf for a reduced otic vesicle phenotype and raised to 4 days
of age, 10 showed all cartilages severely reduced (not shown), 16 displayed
the strong phenotype (Fig. 8R) and 13 showed the mild phenotype (Fig.
8Q). We also analyzed fgf3 morpholino injected animals
for apoptosis by TUNEL assay and observed substantially increased levels of
apoptosis in morphant animals compared with controls. However, extensive cell
death in fgf3 morpholino injected animals made conclusive
interpretations of the TUNEL assays regarding specific NC cell apoptosis
effects difficult (data not shown).
To determine the extent of the pharyngeal pouch patterning defect, we examined the staining pattern of zn-8, a monoclonal antibody that recognizes the pouch ectoderm, and we examined the expression pattern of the transcription factor nkx2.3, a later marker for posterior pouch endoderm. At 34 hpf, the zn-8 antibody effectively labeled pp1-pp4 in the wild-type animal but foo/foo embryos displayed significant reductions in pp2-pp4 (Fig. 9A,B). Although slightly reduced, pp1 was labeled by zn-8 in foo/foo embryos indicating that complete patterning of pp1 may depend upon additional factors, reflecting the higher complexity of the cartilage structures developing adjacent to this pouch. Consistent with this interpretation, ephrin B2a expression in pp1 was unaffected in foo/foo embryos (data not shown). The disrupted expression of nkx2.3 from pp2-pp5 provides further indication that posterior pouch patterning requires foo (Fig. 9C,D).
|
). To
determine whether the foo-pax8 pathway is present in the pharyngeal
pouches we examined pax8 expression in foo/foo embryos.
Although we were unable to detect expression of pax8 in pp1 or pp2 at
the 15-somite stage (data not shown), there was clear expression of
pax8 in the posterior part of pp2 at the 21-somite stage and that
expression domain was absent in foo/foo embryos
(Fig. 9E,F, blue arrowhead).
Likewise, at 30 hpf, pax8 expression was observed in more posterior
pouches of normal sibling embryos but absent in foo/foo embryos (data
not shown). This apparent AP progression of pax8 pouch expression was
reminiscent of both the foo and fgf3 expression profiles.
Interestingly, at 34 hpf, pax8 expression in the thyroid precursor
cells was detected in foo/foo embryos indicating that not all
pharyngeal endoderm expression of pax8 is dependent on foo
activity (data not shown). |
DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRT-PCRRESULTSDISCUSSIONREFERENCES |
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; Woo et al.,
1995). By the tail bud stage, foo expression was
localized in two distinct bilateral regions that mark the future otic
placodes. Prior to this study, pax8 was the earliest known marker of
the otic placodes. Remarkably, not only does foo expression in
presumptive placodal tissue precede pax8 expression, but
foo/foo embryos also show a complete loss of pax8 expression
in the otic placode as well as reduction and disorganization of later otic
placode markers. This places pax8 downstream of foo, which
makes it the earliest known marker of otic placode development and gives
foo a demonstrated role in the induction of a specific otic vesicle
marker.
At tail bud stage, dlx3b is normally expressed in all placodal
tissue forming a discrete lateral band of expression that encircles the
anterior neural plate with higher expression over the future otic placode
(Akimenko et al., 1994). By the
10-somite stage, dlx3b is normally strongly expressed in the otic
placode, although its expression is no longer detected laterally between the
otic placode and the anteriormost domain. Although 10 somites stage
foo/foo embryos show the reduced otic placode expression of
dlx3b, the mutant embryos apparently also retain from the tail bud
stage some of the lateral band of dlx3b expression surrounding the
anterior neural plate (see Fig.
3), suggesting that foo may function in restricting or
maintaining the coherence of the expression domains of placodal markers. This
failure to tightly restrict the expression domains of otic placode markers
such as dlx3b and pax2a might explain why foo/foo
embryos often display split and/or duplicated otic vesicles at pharyngula and
later stages. Thus, this Foxi1-dependent pathway has an essential function for
the developing otic placode but not a significant role in initial
induction.
Three recent papers show that induction of the otic placode requires both
fgf3 and fgf8 (Leger and
Brand, 2002; Maroon et al.,
2002; Phillips et al.,
2001). The combined data from the three papers suggest that
pax8 expression is induced by fgf3 signaling, but the amount
of signal required to induce pax8 is much lower than other otic
placode markers.
Removing fgf3/8 signals completely eliminates otic induction, but
foo/foo embryos still show an induction of several markers. Although
other models are possible, the simplest model places foo activity
between fgf3/8 and pax8 in the induction. Because of the
observed phenotype, we propose that fgf3/8 signaling to the otic
placode induces parallel pathways. The Foxi1-independent pathway begins very
early and would be responsible for the initial induction and expansion of the
otic placode. As foo/foo embryos completely lacked pax8
expression in the otic placode, yet were still able to induce placodal
structures that ultimately formed an ear, our data suggests that in zebrafish,
pax8 and foo function are not required for otic placode
induction. There is early pax8 expression in mouse otic placodes
(Pfeffer et al., 1998), and a
mouse line with a targeted deletion of pax8 has been made, but there
no data have yet been reported regarding ear defects in these mice
(Mansouri et al., 1998).
The second, Foxi1-dependent pathway has at least the one downstream effect:
that of inducing pax8 expression. Based on data obtained by treating
embryos with the Fgf receptor inhibitor SU5402
(Leger and Brand, 2002),
initiation of this pax8 induction would take place sometime after 70%
epiboly, as SU5402 added at 70% epiboly can block pax8 expression.
The role of the earlier initiating Fgf signal would be to provide an
activating signal for foo-expressing cells to induce pax8
and perhaps other unidentified downstream genes. It is also important to note
that foo expression is likely to be independent of fgf3/8
signaling even though pax8 expression is not.
We suggest that foo/foo mutant embryos demonstrate an uncoupling of otic placode induction from its subsequent morphogenesis (Fig. 10). This organizational or `integrity-maintenance' activity would hold the placode together with the predicted effect of this model being similar to what we actually see in the foo mutants, that is, smaller dissociated placodal precursors. Because the regions of pax2a and dlx3b appear to be somewhat reduced, we also propose that foo-expressing cells generate positive feedback to the Fgf signals (Fig. 10). This is an idea we will return to more strongly in the discussion of the role of foo in the developing jaw.
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;
Smith et al., 1997), the
observed third stream migration defect is perhaps secondary to the previously
described otic placode defects. Without the maintained integrity of tight
cell-cell interactions, the otic placode may no longer be capable of
deflecting the ventral movement of third stream NC cells. The extent of the
third stream migration defect appeared limited to invasion of otic placode
territory because no krox20-expressing third stream cells were
detected in post-migratory second stream territory
(Fig. 4D, dorsal view, the area
lateral to r2-r4). It is important to emphasize that a significant fraction of
the third stream NC cells do not undergo apoptosis because, although reduced,
posterior cartilages are readily detected in foo/foo embryos.
Alternatively, these migration defects could be intrinsic to NC cells lacking
Foo activity, a question that will require cell transplantations and cell
autonomy studies for a clear answer. Because first and second stream NC cell migration appeared normal in foo/foo embryos, the hyomandibular defects probably arose from defects at later stages of jaw development. By the 15-somite stage, foo expression was readily observed in the endoderm of the pharyngeal pouches. At around 20 somites stage, foo/foo embryos showed a loss of pax8 expression from the pouches followed shortly at the 24 somites stage by a premature loss of fgf3 expression. The combined action of strong Fgf signaling and foo activity would prefigure the pouch pax8 expression domains, as fgf3 is expressed in only the posterior half of pp2 as is pax8. Consistent with this observation, pax8 expression in the otic placode is strongly dependent on both Fgf signaling and foo activity. Thus, in both the ear and the jaw, we propose that foo expression is responsible for the modification of downstream cellular responses to Fgf signaling leading to the induction of pax8 expression.
Interestingly, loss of pax8 expression in foo/foo embryos precedes the loss of fgf3 maintenance in pp2. It is possible that foo and pax8 function to maintain pouch endoderm integrity of markers such as nkx2.3 in a manner analogous to otic placode integrity (Fig. 10). A future direction will be to determine whether feedback maintenance of Fgf signaling depends on pax8 function. fgf3 is expressed in the pharyngeal pouches in a roughly AP progressive wave. From 15 somites until around 34 hpf, fgf3 expression is maintained in two pouches at a time with attenuation of the anterior-most pouch coinciding with the up regulation of the next posterior-most pouch. In foo/foo embryos, initial induction of fgf3 expression in any particular pharyngeal pouch appeared to be only modestly affected. However, the onset of signal attenuation appeared prematurely, which is consistent with a role for foo in the maintenance of fgf3 pouch expression. Thus, foo expression is not required for initial induction of fgf3 in pouches but instead is part of a transient positive feedback mechanism that maintains fgf3 signaling. It is possible that this maintenance is either at the transcriptional level causing cells to continue to express fgf3, or at the survival level, providing positive feedback signals that prevent fgf3 cells from apoptosis. Further experiments will be required to determine which of these is true.
At 26 hpf, a 20-fold increase in NC cell apoptosis was observed in the arches. This transient wave of cell death is probably a direct result of the lost fgf3 maintenance because both cas mutants, which lack all endoderm (including fgf3 pouch expression), and SU5402-treated embryos, in which Fgf receptor signaling is blocked, display extensive NC cell apoptosis. The apoptosis in foo mutant embryos is not as severe as these other cases because some residual fgf3 signaling is still present. Consistently, fgf3-MO animals appear to display jaw defects that are very similar to and slightly more severe than those observed in foo/foo embryos (Figs 5, 8). Therefore, the failure to maintain fgf3 expression in foo/foo embryos is what causes the widespread but transient NC cell apoptosis, causing reductions in the dorsal structures of both the mandibular and hyoid arches, and variable reductions in the more posterior branchial arches.
The apoptotic death of NC cells probably explains the later absence of the
dorsal first and second arch gsc expression domains as well as the
loss of posterior arch defects in dlx2, dlx3b and hand2
expression. Notably, dlx2 expression is likewise lost in posterior
arches of cas/cas and fgf3-MO animals
(David et al., 2002). The
unperturbed patterning of the ventral NC cells is indicated by the normal
expression of dlx2, dlx3b, hand2, gsc and hoxa2 gene
expression in ventral NC cells as well as the equivalence in size of Meckel's
cartilage between foo/foo and wild-type embryos. Interestingly,
ventral NC cells of both the mandibular and hyoid arches do not require an
fgf3-mediated signal for survival and/or proliferation, yet are
absent in SU5402-treated embryos, implicating an essential role for a
different as yet unidentified Fgf ligand. Similarly, it is worth noting that
in spite of the fgf3 maintenance defects, which extend into the more
posterior pouches such as pp3 to pp5, that the most posterior fifth to seventh
arches appear unaffected in foo/foo embryos. A possible explanation
for the normal patterning of these posterior arches is an undetermined Fgf
signal that can compensate for the loss of fgf3. Consistent with this
explanation, fgf3-MO animals often show normal cartilages from the
fifth to seventh arches that are missing in SU5402-treated embryos
(David et al., 2002).
The complexity of the pharyngeal endoderm patterning pathways is further highlighted by the dual observations of lost pp2 pax8 expression around the 20 somites stage (Fig. 9E,F) and retained thyroid follicular precursor cell pax8 expression in 34 hpf foo/foo embryos (data not shown). As both tissues are pharyngeal endoderm derived, alternate pathways must exist for inducing pax8 expression not only in tissues such as the eyes, MHB and pronephric ducts but also in more medial regions of the pharyngeal endoderm. It is possible or likely that foo expression in the pharyngeal pouches has other important functions that will require further experimentation to establish.
There are several noteworthy implications for the observed failure to
maintain fgf3 expression on possible mechanisms of establishing DV or
AP polarity within an arch. First, it suggests that the relative size of a
particular cartilage element can be directly influenced by the duration of a
particular Fgf signal; the shorter the Fgf pulse, the fewer cells
survive/proliferate and the smaller the structure. It is therefore possible
that either a different Fgf signal might mediate survival of a different
population of cartilage precursors or that a short but intense pulse of an Fgf
signal could facilitate survival near the point source but with little effect
at a distance. Second, it suggests that regional functionality of Fgf
signaling could direct cartilage shape. The patterned induction of a variety
of Fgf antagonist molecules, such as sef or sprouty family
members, could direct cartilage shape by eliminating specific Fgf signals from
specific NC cells. In support of this, sprouty4 has been shown to be
expressed in the pouches (Furthauer et
al., 2001). The combined action of multiple patterned Fgf agonists
and multiple antagonists could therefore generate fairly complex DV, AP and ML
matrixes of survival, proliferation and death cues. Furthermore, we suggest
small changes in this complex interplay of signals could be one of the
mechanisms for the evolutionary adaptation of jaw structures.
Evolutionary implications
Our results show that in zebrafish, foo is a very early regulator
of both ear and jaw development and is upstream of pax8 induction in
both structures. pax2, pax5 and pax8 are believed to have
arisen from a single ancestral pax2/5/8 gene
(Pfeffer et al., 1998). Two
recent publications have identified a single pax2/5/8 gene in
ascidians and amphioxus that is expressed in the region of the primordial
pharynx, a region analogous to the pharyngeal arches of vertebrates
(Kozmik et al., 1999;
Wada et al., 1998). Given the
observed commonality of gene expression between the otic vesicle and the
arches, we argue for a synthesis between two interpretations of the
pax2/5/8 expression data in these primitive organisms
(Kozmik et al., 1999;
Wada et al., 1998). The
ascidian cupulae could be considered gill slits that developed a placode-like
function that differentiates into the ciliated primary sensory cells involved
in feeding. The functional and spatial separation of these sensory regions
away from the underlying arches as chordates evolved might explain why certain
molecular markers that characterized the original gill slit ectoderm are
maintained in vertebrate placode-derived structures. Because at least one
vertebrate, the zebrafish, co-expresses foo and pax8 in the
otic placode ectoderm and also in the pouch endoderm, one might imagine that
the primordial gill slit would express foo and pax8 in both
ectoderm and endoderm layers. The ability to perform gene knockdown studies in
ascidians will allow for direct testing of conservation of gene regulatory
networks (Heasman, 2002;
Satou et al., 2001).
Even if the jaw and ear do not have a common origin, sharing gene
regulatory networks helps explain how, across the water-to-land transition,
the hyomandibular might transform into the stapes
(Webster et al., 1992). If the
hyoid NC cells lost purpose in the jaw but were still responsive to many of
the same signaling molecules, such as Fgfs, then it is possible that the
presumptive hyoid NC cells began responding to signals relating to the otic
placode instead of the forming jaw. Without the similarities in gene
expression, the mechanism for this jaw to middle ear NC cell shift would have
to have happened in a more random and uncontrolled way. To address this issue
directly is challenging; however, a simple, testable prediction of this
jaw-ear crossover theory is that Foxi1–/– mice
should display middle ear bone defects, perhaps specific to the stapes.
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ACKNOWLEDGMENTS |
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