1 EMBL, Meyerhofstrasse 1, D-69117 Heidelberg, Germany
2 Howard Hughes Medical Institute, Fred Hutchinson Cancer Research Center, PO
Box 19024, 1100 Fairview Avenue North, Seattle, WA 98109 USA
* Author for correspondence (e-mail: carl.neumann{at}embl-heidelberg.de)
Accepted 11 April 2003
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SUMMARY |
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Key words: Zebrafish, Limb development, fgf24, fgf10, tbx5, wnt2b, ikarus, Pectoral fin, Apical ectodermal ridge
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INTRODUCTION |
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Another gene involved in limb induction is Fgf10. Fgf10 is
expressed in the limb mesenchyme beginning at very early stages, and when
ectopically expressed, can induce additional limbs in the chicken
(Ohuchi et al., 1997).
Furthermore, Fgf10 mutant mice fail to form limbs
(Min et al., 1998
;
Sekine et al., 1999
). These
mutant embryos do not show activation of markers expressed in the limb bud
ectoderm, suggesting that Fgf10 relays limb induction from the
mesenchyme to the ectoderm. Fgf10 belongs in a subclass of the Fgf family with
highest affinity for the Fgf receptor 2 isoform b, Fgfr2b
(Ornitz et al., 1996
), which
is expressed in epithelial cells
(Orr-Urteger et al., 1993
)
(reviewed by Xu et al., 1999
).
Fgfr2b mutant mice share many phenotypes with Fgf10 mutants
(DeMoerlooze et al., 2000
;
Ohuchi et al., 2000
), further
supporting a model in which mesenchymally expressed Fgf10 activates Fgfr2b in
the overlying ectoderm (reviewed by Xu et
al., 1999
). Fgf2, Fgf4 and Fgf8, however, have highest affinity
for Fgfr2c (Ornitz et al.,
1996
), which is mesenchymally expressed
(Orr-Urteger et al., 1993
).
This scenario suggests that the limb-inducing activity of Fgf8 and similar
Fgfs is mediated by Fgf10, which relays the inductive event to the ectoderm.
Consistent with this proposal, Fgf10 is able to induce ectodermal limb markers
in the chicken flank even in the absence of mesenchyme, while Fgf2 and Fgf4
are not (Yonei-Tamura et al.,
1999
). The induction of Fgf4 and Fgf8 in the
ectoderm by Fgf10 is not direct, and appears to be mediated in the chicken by
WNT3A, which is activated in the ectoderm in response to Fgf10
(Kengaku et al., 1998
;
Kawakami et al., 2001
).
Once the limb bud has formed, Fgf4 and Fgf8 are expressed
in the apical ectodermal ridge (AER), a signaling center that directs
outgrowth of the limb bud, and these Fgfs have been shown to mediate the
activity of the AER in the chicken and the mouse
(Laufer et al., 1994;
Niswander et al., 1994
;
Sun et al., 2002
).
Fgf10 continues to be expressed in the mesenchymal cells underneath
the AER, and forms a feedback loop of mutual dependence with the ectodermally
expressed Fgfs (reviewed by Xu et
al., 1999
). Fgf10 thus also directs ectodermal expression
of Fgf4 and Fgf8 during the outgrowth phase of the limb.
Tbx5 encodes a T-box transcription factor that is expressed in the
forelimb mesenchyme at very early stages, and has been shown to participate in
the specification of limb identity in the chicken
(Rodriguez-Esteban et al.,
1999; Takeuchi et al.,
1999
). Recent results have indicated that Tbx5 is also
involved in limb bud initiation. Targeted knockdown of tbx5, or
mutagenesis of the tbx5 locus, leads to zebrafish embryos that lack
pectoral fin buds (Ahn et al.,
2002
; Garrity et al.,
2002
; Ng et al.,
2002
). These mutants fail to activate expression of ectodermal fin
bud markers, which correlates with the absence of fgf10 in the
mesenchyme. Furthermore, targeted knockdown of wnt2b causes loss of
tbx5 expression in the zebrafish pectoral fin primordium, suggesting
that Tbx5 acts downstream of Wnt2b to induce fgf10 during limb
initiation (Ng et al.,
2002
).
The zebrafish has recently become established as a model system to study
the development of vertebrate paired appendages, and a number of zebrafish
mutants affecting the development of paired fins have been isolated in a
large-scale genetic screen (van Eeden et
al., 1996). We report the molecular and phenotypic analysis of one
of these mutants, named ikarus (ika; znfn1a1
Zebrafish Information Network). We show that ika encodes
fgf24, which is a new member of the Fgf8/17/18 subfamily of Fgf
ligands, and has highest sequence similarity to Fgf18. In the absence of
fgf24 activity, we observe activation of early mesenchymal fin bud
markers, such as tbx5, but the absence of all genes expressed in the
fin bud ectoderm. We show that fgf24 acts downstream of tbx5
to activate fgf10 expression. These results identify an additional
layer controlled by Fgf signaling in the genetic hierarchy initiating limb
development.
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MATERIALS AND METHODS |
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Meiotic mapping and sequencing of ika alleles
We mapped the ika mutation to linkage group 14 by using a standard
panel of SSLP markers (Knapik et al.,
1998). For fine mapping single ika mutant embryos (938 in
total) were tested against individual SSLP markers in the crucial interval.
The cloning and physical mapping of fgf24 are described elsewhere
(B.W.D., D. W. Stock and C. B. Kimmel, unpublished). For sequencing of the
fgf24 gene, total RNA was extracted from wild-type or ika
embryos at 36 hpf. RT-PCR was performed using the Superscript kit (Invitrogen)
with the following primers: fgf24up (5'-TCCGGGGTTTTGTTTGTGAG-3')
and fgf24down (5'-TCTTTTCGGTAGCCATTGTTTATT-3'). PCR products from
four independent PCR reactions on two different RNA samples were sequenced on
both strands and analyzed using the MacVector software.
Morpholino injections
Morpholinos were purchased from GeneTools LLC. The following morpholinos
were used: anti-tbx5 oligonucleotide for the coding sequence, as
described by Ahn et al. (Ahn et al.,
2002). Anti-wnt2b oligonucleotide, as described by Ng et
al. (Ng et al., 2002
). For
fgf24, we used an oligonucleotide targeted against the translation
start site with the following sequence: 5'
GACGGCAGAACAGACATCTTGGTCA-3'. As a control we used the standard control
oligonucleotide available from GeneTools. All oligonucleotides were
solubilized in 1xDanieau's solution and injected into one-cell stage
zebrafish embryos at concentrations ranging from 5-10 ng/embryo.
Transplantation
Donor embryos were injected with 2.5% rhodamine-dextran, and cells
transplanted into hosts at 30-70% epiboly. To target wild-type cells to the
fin mesenchyme, transplantation was carried out as described previously
(Ahn et al., 2002). To target
the ectoderm, we transplanted cells to a region opposite the shield in early
gastrula embryos.
Histochemical methods
Whole-mount in situ hybridization was performed as previously described
(Kishimoto et al., 1997),
using the following probes: tbx5
(Begemann and Ingham, 2000
);
msxc (Akimenko et al.,
1995
); dlx2 (Akimenko
et al., 1994
); pea3 and erm1
(Roehl and Nusslein-Volhard,
2001
); fgf8 (Reifers
et al., 1998
); fgf10
(Ng et al., 2002
);
shh (Krauss et al.,
1993
); bmp2
(Kishimoto et al., 1997
); and
hand2 (Yelon et al.,
2000
). Alcian Blue staining was performed described previously
(Grandel and Schulte-Merker,
1998
). Histological sections were obtained by staining
cryosections with Methylene Blue (Humphrey
and Pittman, 1974
).
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RESULTS |
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The stop codon in ikahx118 leaves a truncated protein
lacking more than half of its C terminus
(Fig. 2D), thus removing most
of the core region, which has been shown to interact with the receptor in the
case of other Fgf proteins (reviewed by
Ornitz and Itoh, 2001).
ikahx118 is thus likely to be a null allele, which is
supported by the full phenotypic penetrance observed with this allele. The
cysteine converted in ikatm127c to phenylalanine is one of
six amino acids that are conserved in the core region of all Fgf proteins
(reviewed by Ornitz and Itoh,
2001
), suggesting that it is crucial for normal function.
Nevertheless, this allele appears to retain some activity, as it shows
variable phenotypic penetrance and expressivity
(van Eeden et al., 1996
).
To further test the possibility that ika encodes fgf24,
we designed an antisense morpholino oligonucleotide to block translation of
fgf24 (Nasevicius and Ekker,
2000). Injection of morpholinos targeted against fgf24
generated larvae that specifically lack pectoral fins (n=72), but
otherwise appear normal, thus phenocopying the ika mutation
(Fig. 2D) (B.W.D., D. W. Stock
and C. B. Kimmel, unpublished). We injected morpholinos within the range of
5-10 ng per embryo, and within this range, all injected embryos displayed
complete absence of pectoral fins. The injection of a control morpholino had
no effect (data not shown), and yielded individuals identical to the wild-type
larva shown in Fig. 1A.
From these data, we conclude that ika encodes fgf24 and hereafter refer to the mutant by its molecular name. The GenBank Accession number for fgf24 is AY204859.
fgf24 is expressed in the mesenchyme of early
pectoral fin buds
To better define the role played by fgf24 during pectoral fin
development, we localized the fgf24 transcript using in situ
hybridization. We first detect fgf24 in the region of the pectoral
fin primordia at 18 hpf (Fig.
3A). Apart from tbx5, which is first detected in this
region at 17 hpf (Begemann and Ingham,
2000; Ruvinsky et al.,
2000
), fgf24 is the earliest marker for the pectoral fin
primordia. At this stage, the transcript is detectable only in the mesenchyme
(Fig. 3A). Expression is still
present in the fin bud mesenchyme at 24 hpf
(Fig. 3B), but becomes
downregulated in these cells between 28 and 30 hpf
(Fig. 3C). At the same time,
fgf24 is activated in the overlying AER
(Fig. 3C).
|
tbx5 is the earliest markers for pectoral fin development, and is
expressed in the mesenchymal compartment of the fin buds
(Fig. 4A) (Begemann and Ingham, 2000;
Ruvinsky et al., 2000
). We
find that it is also expressed in these cells at 24 hpf in fgf24
mutants, although the expression appears weaker than in wild-type embryos
(Fig. 4B).
|
dlx2 is an early marker for the AER in zebrafish. We find that
dlx2 is activated in the ectoderm of wild-type pectoral fin buds
already at 20-22 hpf (Fig. 4E;
data not shown), which precedes formation of the AER
(Grandel and Schulte-Merker,
1998). At early stages, dlx2 appears to be expressed
throughout the entire fin bud ectoderm, and later becomes restricted to the
AER (data not shown). We find that dlx2 expression is not detectable
in the pectoral fin buds of fgf24 mutants at any time
(Fig. 4F; data not shown).
shh is first expressed in pectoral fins at 24 hpf. We detect no shh expression in the pectoral fins of fgf24 mutants at 24 hpf (Fig. 4G,H), or at later stages (see Fig. 8A,B).
|
erm1 and pea3 encode transcription factors that have been
shown to be dynamically expressed in many tissues during zebrafish development
(Roehl and Nuesslein-Volhard,
2001). We find that both genes are expressed throughout the
pectoral fin bud mesenchyme, and that pea3 is additionally expressed
in the ectoderm (Fig. 5A,C). Both erm1 and pea3 are expressed in the mesenchyme of
fgf24 mutants, but pea3 fails to be activated in the
ectoderm (Fig. 5B,D). Like
msxc, erm1 and pea3 expression starts to fade around 30 hpf
in fgf24 mutants, and is no longer detectable at later stages (data
not shown).
|
|
fgf24 acts downstream of wnt2b
and tbx5 and upstream of fgf10
during limb bud initiation
The fgf24-/- pectoral fin phenotype bears strong
resemblance to that of zebrafish embryos lacking wnt2b or
tbx5 activity (Ahn et al.,
2002; Garrity et al.,
2002
; Ng et al.,
2002
). To investigate the relationship of fgf24 with
these two genes, we examined the expression of fgf24 in the absence
of wnt2b or tbx5 activity, by using a morpholino knockdown
approach (Nasevicius and Ekker, 2002). We fail to detect fgf24
expression at 20 hpf or 24 hpf in the pectoral fin bud primordia in the
absence of either wnt2b or tbx5 activity
(Fig. 6A-C; data not shown).
Injection of control morpholinos yielded embryos showing wild-type
fgf24 expression identical to that shown in
Fig. 6A. Together with the
observation that tbx5 expression is initiated in fgf24
mutants (Fig. 4B), these
results indicate that fgf24 acts downstream of tbx5 in the
genetic cascade initiating pectoral fin bud development. As tbx5
fails to be activated in the absence of wnt2b activity
(Ng et al., 2002
), and as
fgf24 activation depends on tbx5
(Fig. 6B), it is not surprising
that fgf24 also depends on wnt2b activity.
|
Taken together, these results suggest that fgf24 acts downstream of tbx5 to activate fgf10 expression during limb bud initiation (Fig. 6F).
fgf24 activity is required in the mesenchyme to
activate fgf10
As fgf24 is expressed both in the mesenchyme and in the AER, we
wished to determine in which cells fgf24 activity is required for
fgf10 activation. We therefore transplanted wild-type cells into
fgf24 mutant embryos. We observed rescue of fin bud outgrowth when
wild-type cells were located in the fin bud mesenchyme
(Table 1, n=3). These
fin buds also showed an AER at 36 hpf, and fgf10 expression. In one
case, we did not observe rescue when wild-type cells were located in the
lateral plate mesoderm at the level of the pectoral fin bud (data not shown).
This may be due to the fact that not enough wild-type cells were present in
this case. We never observed rescue of the fgf24 phenotype when
wild-type cells were located in the ectoderm at the pectoral fin level
(Table 1, n=6).
|
fgf24 activity is required for the migration
of tbx5- expressing mesenchymal cells to the fin
primordium
As tbx5 is required for the movement of mesenchymal cells in the
lateral plate mesoderm to the pectoral limb bud
(Ahn et al., 2002), and as
fgf24 is activated downstream of tbx5, we asked whether
fgf24 might play a role in mediating this effect of tbx5. To
this end, we compared the distribution of tbx5-expressing cells in
the lateral plate mesoderm in wild-type and fgf24 mutant embryos
between 18 hpf and 32 hpf. At 18 hpf, tbx5 expression is
indistinguishable in wild-type and fgf24 mutants
(Fig. 7A,B). However, at 27
hpf, the tbx5-expressing cells have congregated towards the pectoral
fin bud in wild-type embryos, but remain dispersed in fgf24 mutants
(Fig. 7C,D). This phenotype is
even more striking at 32 hpf (Fig.
7E,F).
This observation indicates that fgf24 activity is required for the correct movement of tbx5-expressing cells in the lateral plate mesoderm to the pectoral fin bud.
Anterior/posterior polarity in fgf24 mutant pectoral fin
buds
The activation of shh expression in the posterior mesenchyme of
chick limb buds depends on Fgfs secreted from the AER
(Laufer et al., 1994;
Niswander et al., 1994
;
Sun et al., 2002
). Because
fgf24 mutants do not show any sign of AER formation, we examined
whether shh is activated in the absence of fgf24 activity.
We find that shh expression is not detectable in fgf24
mutants at any stage, consistent with the absence of AER in these mutants
(Fig. 4G,H;
Fig. 8A,B). msxc is
initially expressed throughout the mesenchyme of the pectoral fin buds, but
expression becomes restricted to the anterior by 28-30 hpf in wild-type
embryos (Fig. 4A;
Fig. 8C). In fgf24
mutant pectoral fin buds, this repression in the posterior mesenchyme fails to
occur (Fig. 8D). This is
probably due to the absence of Shh, because the same phenotype is observed in
shh mutant pectoral fin buds
(Neumann et al., 1999
).
hoxd13 and hoxa13 also fail to be activated in
fgf24 mutant pectoral fin buds (data not shown). Again, this
phenotype is similar to what is observed in the absence of shh
activity (Neumann et al.,
1999
).
bmp2 is expressed both in the AER and in the posterior mesenchyme
(Fig. 8E). We find that
bmp2 is activated in the posterior mesenchyme in fgf24
mutants, but not in the ectoderm (Fig.
8F). This correlates with the observation that activation of
bmp2 in the posterior mesenchyme is independent of shh
activity (Neumann et al.,
1999). The shh-independent anterior/posterior (AP)
polarity of pectoral fin buds has been shown to be directed by the
hand2 gene, which encodes a bHLH transcription factor
(Yelon et al., 2000
).
Consistent with this observation, we find normal posterior activation of
hand2 in fgf24 mutants
(Fig. 8G,H), although
hand2 expression gradually fades after 30 hpf (data not shown).
These data suggest that the early aspects of AP polarity are established in
the fgf24 mutant pectoral fin primordium. Those aspects of AP
polarity that are lost in fgf24 mutants are ones which have been
shown to depend on shh activity
(Neumann et al., 1999), which
correlates well with the failure to activate shh expression in
fgf24 mutant fin buds, while the shh-independent AP polarity
is unaffected by the loss of fgf24.
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DISCUSSION |
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In this study, we have shown that the zebrafish fgf24 gene, which
is disrupted by the ikarus mutation, acts downstream of tbx5
to activate fgf10 in the LPM, thus identifying another Fgf acting
early in the cascade of limb induction. Consistent with this model,
fgf24 is expressed very early during pectoral fin development, and is
activated in the LPM at a similar stage as tbx5 (this study)
(Begemann and Ingham, 2000;
Ruvinsky et al., 2000
).
Together with the observation that Tbx5 is activated within 1 hour of
implanting an Fgf-soaked bead into the flank of a chick embryo
(Isaac et al., 2000
), these
results suggest that Tbx5 and Fgf24 act early in the limb-inducing cascade.
Fgf10, however, is not activated until 17 hours after Fgf application
(Ohuchi et al., 1997
),
consistent with the proposal that its induction requires an additional
signaling event. A more direct effect of mouse TBX5 on Fgf10
transcription has been recently proposed, based on the finding that there is
at least one potential TBX5-binding site upstream of the mouse Fgf10
promoter, and that the Fgf10 promoter can be upregulated by
co-expressing Tbx5 in cultured cells
(Agarwal et al., 2003
).
However, this direct model does not explain why the activation of
Fgf10 by Fgf bead application takes so much longer than the
activation of Tbx5, and it remains to be seen what the role of the
TBX5-binding sites are in vivo. One possibility is that the Fgf10
promoter integrates several different signals, and that direct binding by TBX5
is necessary, but not sufficient for activation in vivo. Our data indicate
that one of the additional requirements for Fgf10 activation in the
zebrafish is the exposure of these cells to Fgf24.
The data presented here and elsewhere suggest that zebrafish Wnt2b, Tbx5,
Fgf24 and Fgf10 act sequentially in a linear pathway in which Wnt2b induces
Tbx5 expression, which then induces Fgf24, which in turn induces Fgf10
(Fig. 6F). In addition to its
role in this linear cascade, zebrafish Tbx5 has been shown to be required for
the correct migration of lateral plate mesenchymal cells to the pectoral fin
primordium (Ahn et al., 2002).
Here we have shown that Fgf24 is also required for this process, as
tbx5-expressing cells fail to congregate to the pectoral fin
primordium in the absence of fgf24 activity. The dispersal of
tbx5-expressing cells is not as dramatic in the absence of
fgf24 activity as it is in the absence of tbx5 activity,
suggesting that Fgf24 does not mediate all effects of Tbx5 on this migratory
event. This is consistent with the observation that tbx5 activity is
cell-autonomously required for the correct movement of cells to the fin bud
(Ahn et al., 2002
).
The pectoral fin phenotype of fgf24 mutants is similar to that of
zebrafish raldh2 mutants (Begemann
et al., 2001; Grandel et al.,
2002
). As raldh2 has been shown to act upstream of
tbx5 activation in the fin bud
(Begemann et al., 2001
), it is
also likely to act upstream of fgf24 activation.
Fgf8-related Fgfs control several aspects of vertebrate limb
induction
The Fgf24 protein is a new member of the Fgf8/17/18 subfamily of Fgf
ligands (B.W.D., D. W. Stock and C. B. Kimmel, unpublished). Furthermore,
Fgf24 also shows functional overlap with Fgf8, as both genes are expressed at
early stages in the posterior mesoderm of the embryo, and
fgf8/fgf24 double mutants display developmental defects in
this region that are not observed in either fgf8 or fgf24
single mutants, thus indicating that these two Fgfs have very similar
activities (B.W.D., D. W. Stock and C. B. Kimmel, unpublished). Hence, the
analysis of the fgf24 mutant phenotype provides the first
loss-of-function data demonstrating a role for an Fgf8-like gene in limb
initiation, and complements the gain-of-function experiments which show that
ectopic application of Fgf2, Fgf4 and Fgf8 can trigger the development of
additional limbs (Cohn et al.,
1995; Ohuchi et al.,
1995
; Crossley et al.,
1996
; Vogel et al.,
1996
; Yonei-Tamura et al.,
1999
).
Members of the Fgf8 subclass of Fgfs appear to be expressed in all three
tissues involved in limb initiation, and seem to have important functions at
three distinct steps in early limb development. First, Fgf8, which is
expressed in the chicken IM, initiates WNT2B expression in the IM.
Secondly, Fgf24, which is expressed in the zebrafish LPM, activates
fgf10 expression in the LPM, and thirdly, Fgf4 and Fgf8, which are
expressed in the chicken and mouse AER, direct outgrowth of the limb bud and
maintain Fgf10 expression in the mesenchyme. Interestingly, our data
suggest that Fgf24 signals in an autocrine manner to activate fgf10
in the LPM, as both genes appear to be co-expressed in the same region,
although the activation of fgf24 precedes that of fgf10.
However, on the basis of these data, we cannot distinguish whether Fgf24
produced by an individual cell signals to the same cell, or whether there is
signaling between cells in the same tissue. Our data also suggest that
mesenchymally expressed Fgf24 activates mesenchymal shh expression at
24 hpf. This situation is clearly different from the chick and the mouse,
where Fgf8 secreted from the AER has been shown to activate
shh in the mesenchyme (Laufer et
al., 1994; Nieswander et al., 1994;
Sun et al., 2002
). Consistent
with this observation, the zebrafish fgf8 gene is activated in the
AER at a much later stage than in tetrapods (12 hours after the activation of
shh in the mesenchyme), and zebrafish fgf8 mutants have no
effect on fin development (Reifers et al.,
1998
), although this may be a hypomorphic mutation.
It is also noteworthy in this context that fgf24 is activated in the AER after limb bud initiation has taken place, and at the same time is downregulated in the mesenchyme (Fig. 3C). This observation raises the possibility that Fgf24, together with the other Fgfs expressed in the AER, continues to direct fgf10 expression in the mesenchyme during limb outgrowth, this time by a paracrine mechanism. This scenario is not supported by our transplantation results, however, because the fgf24 mutant phenotype is rescued only by wild-type cells located in the fin mesenchyme, but not in the ectoderm, and rescue leads to restoration of fgf10 expression even at 36 hpf, when fgf24 is expressed in the AER, and not the mesenchyme. The possibility remains that at this stage, Fgf24 functions redundantly with other Fgfs expressed in the AER, such as Fgf4 and Fgf8.
Genetic differences between forelimb and hindlimb development
It is interesting to note that some genes are specifically required for the
development of either forelimbs or hindlimbs, while other genes function in
both types of limbs. Fgf24, for example, is only required for the development
of pectoral fins, but not pelvic fins, and shares this characteristic with
chicken Tbx5 and Wnt2b (Rodriguez-Esteban
et al., 1999; Takeuchi et al.,
1999
; Kawakami et al.,
2001
). Fgf10, however, appears to play an equivalent role
in both fore- and hindlimbs in mice and chicken
(Min et al., 1998
;
Sekine et al., 1999
). It is
likely that chicken Tbx4 and Wnt8c, or related genes, play a role in hindlimb
development that is similar to the role of Tbx5 and Wnt2b, respectively, in
forelimb development (Rodriguez-Esteban et
al., 1999
; Takeuchi et al.,
1999
; Kawakami et al.,
2001
). Hence, our data suggest that a zebrafish Fgf closely
related to Fgf24 fulfills a similar function during pelvic fin
development.
Evolutionary conservation of genes involved in vertebrate limb
induction
There appears to be strong evolutionary conservation of the developmental
mechanism of limb bud initiation. For example, inactivation of the
Tbx5 gene leads to similar reductions of the forelimbs in chicken,
zebrafish, human and mouse (Newbury-Ecob
et al., 1996; Ahn et al.,
2002
; Garrity et al.,
2002
; Ng et al.,
2002
; Agarwal et al.,
2003
). In addition, the role of Wnt2b in limb initiation
appears highly conserved in chicken and zebrafish
(Kawakami et al., 2001
;
Ng et al., 2002
). By contrast,
however, there appears to be no role for Wnt2b in initiating mouse
limb development (Ng et al.,
2002
), nor is there a role for Wnt3a in inducing the
mouse AER (Barrow et al.,
2003
). Nevertheless, the phenotype of the
Lef1/Tcf1 double mutant mouse is consistent with WNT
signaling being necessary for AER formation in the mouse
(Galceran et al., 1999
). These
results suggest that different Wnt proteins, comparable in activity to the
chicken Wnt2b and Wnt3a proteins, fulfill their respective roles in the mouse.
Consistent with this proposal, it has recently been shown that the mouse
Wnt3 gene fulfills the function of chicken WNT3A during limb
development (Barrow et al.,
2003
).
This idea is similar to the one that different genes of similar activity fulfill the same function during fore- and hindlimb development, and suggests that the existence of gene families with similar activities has allowed the functional replacement of specific genes during evolution. By the same reasoning, our data suggest that an Fgf comparable in activity with Fgf24 occupies a similar position in the genetic cascade that initiates limb development in other vertebrate species. However, as both the mouse and human genomes have been sequenced, it is clear that they contain no ortholog of the zebrafish fgf24 gene. Although it is possible that zebrafish Fgf24 fulfills the role attributed to tetrapod Fgf8 in limb initiation, this is unlikely, because Fgf8 is never expressed in limb mesenchyme in any vertebrate examined to date. An alternative possibility is that another tetrapod Fgf family member with similar activity to Fgf8 is transiently expressed in the limb bud mesenchyme, and that this expression has so far gone undetected. Future experiments will hopefully resolve this issue.
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ACKNOWLEDGMENTS |
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