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First published online 21 June 2006
doi: 10.1242/dev.02455
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European Molecular Biology Laboratory (EMBL), Meyerhofstrasse 1, 69117 Heidelberg, Germany.
* Author for correspondence (e-mail: carl.neumann{at}embl-heidelberg.de)
Accepted 23 May 2006
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Limb development, Prdm1, Tbx5, Retinoic acid, Fgf, Wnt2b
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Niswander,
2002; Tickle,
2002). Limbs arise from regions of the lateral plate mesoderm
(LPM) at specific positions along the main anteroposterior body axis.
A number of studies have shown that the limb-inducing signal originates in
the axial mesoderm, and is relayed from there to the LPM (reviewed by
Capdevila and Izpisua Belmonte,
2001). An important signal shown to play a role in limb induction
in mouse, chick and zebrafish is the Vitamin A derivative retinoic acid (RA)
(Begemann et al., 2001;
Berggren et al., 1999;
Grandel et al., 2002;
Mic et al., 2004;
Niederreither et al., 1997;
Niederreither et al., 1999;
Stratford et al., 1997). RA is
synthesized mainly by the enzyme Retinaldehyde dehydrogenase 2
(Raldh2), which is expressed in the LPM and early somites
(Berggren et al., 1999;
Niederreither et al., 1997).
Inhibition of RA signaling during different time windows in zebrafish has
revealed that it is required for limb initiation during a relatively short
time span at the end of gastrulation, long before limb development commences
in the LPM (Grandel et al.,
2002). Also, mosaic experiments performed in zebrafish, where
wild-type cells were transplanted into raldh2 mutant embryos, have
shown that RA synthesis in somitic mesoderm is sufficient to trigger limb
induction in the adjacent LPM (Linville et
al., 2004). These experiments indicate that RA signaling acts very
early in the cascade of genes controlling limb induction. They also show that
the limb induction cascade is initiated in the somitic mesoderm, and suggest
that the effect of RA on limb development is probably indirect, and likely to
be mediated by secondary signals.
The T-box transcription factor Tbx5 is the earliest gene known to
be expressed in the presumptive forelimb field
(Gibson-Brown et al., 1996;
Isaac et al., 1998;
Logan et al., 1998;
Ohuchi et al., 1998;
Simon et al., 1997;
Tamura et al., 1999).
Tbx5 is crucial for forelimb induction, as loss of Tbx5
activity causes failure of forelimb initiation in mouse, chicken and zebrafish
(Agarwal et al., 2003;
Ahn et al., 2002;
Garrity et al., 2002;
Ng et al., 2002;
Rallis et al., 2003;
Takeuchi et al., 2003).
Conversely, ectopic overexpression of Tbx5 can trigger ectopic limb
outgrowth in the interlimb LPM, indicating that Tbx5 is not only
necessary, but also sufficient to initiate limb development
(Takeuchi et al., 2003). A
number of studies have shown that Tbx5 interacts both with Wnt and Fgf signals
to direct limb induction. Thus, Tbx5 is required for activation of
Fgf10 expression within the limb mesenchyme. Fgf10, in turn, signals
to the overlying ectoderm to activate Fgf8 expression in the apical
ectodermal ridge (AER) (Min et al.,
1998; Norton et al.,
2005; Ohuchi et al.,
1997; Sekine et al.,
1999). This event then leads to the establishment of a signaling
feedback loop between ectodermal Fgf8 and mesenchymal Fgf10,
which is crucial for subsequent limb outgrowth
(Min et al., 1998;
Ohuchi et al., 1997;
Sekine et al., 1999).
In addition to its role in mediating AER signaling, Fgf8 has also been
proposed to act at an earlier step in limb induction, as application of Fgf8
protein into the chicken flank is able to direct formation of an ectopic limb,
and because Fgf8 is expressed in the intermediate mesoderm (IM)
adjacent to the forelimb-forming region at the time of limb initiation
(Crossley et al., 1996;
Vogel et al., 1996). Arguing
against this hypothesis, however, is the observation that conditional removal
of Fgf8 activity from the IM has no effect on limb development in
mice (Boulet et al., 2004;
Perantoni et al., 2005). An
alternative possibility may be that Fgf8 is functionally redundant with other
members of the Fgf family expressed in the axial mesoderm. For example,
fgf17b is co-expressed with fgf8 in the somites
(Reifers et al., 2000).
Because the mosaic analysis of raldh2 mutants indicates that the
somitic mesoderm is crucial for limb induction
(Linville et al., 2004), the
somites could be a source of Fgf signaling required for limb induction. It is
therefore presently not clear whether Fgf signaling participates in relaying
the limb-inducing signal from the axial and paraxial mesoderm to the LPM.
The Wnt family of signaling molecules also plays an important role during
limb initiation. In the chick, Wnt2b is expressed in the IM and LPM,
and similar to Fgf protein application, ectopic expression of Wnt2b
or ß-catenin triggers the formation of an extra limb
(Kawakami et al., 2001;
Takeuchi et al., 2003). In
zebrafish, wnt2b is only expressed in the IM, and knock down of Wnt2b
with antisense morpholino oligonucleotides leads to failure of tbx5
expression activation in the LPM (Ng et
al., 2002). Furthermore, injection of tbx5 messenger RNA
(mRNA) can partially rescue Wnt2b knock-down embryos, whereas wnt2b
mRNA injection fails to rescue Tbx5 knock-down embryos, suggesting that Wnt2b
signaling is upstream of tbx5 during limb induction
(Ng et al., 2002). In contrast
to these results, mouse embryos mutant for Lef1 and Tcf1,
two nuclear transducers of Wnt signaling, have normal limb bud initiation and
show no effects on Tbx5 activation
(Agarwal et al., 2003;
Galceran et al., 1999). This
could either reflect a species-specific role of Wnt signaling in limb
induction, or additional Tcf genes may compensate for the loss of
these genes in the mouse (Logan,
2003).
The zebrafish has recently gained popularity as a model to study limb
development, as its paired fins are homologous to tetrapod limbs
(Grandel and Schulte-Merker,
1998). Several large-scale mutagenesis screens have led to the
isolation of zebrafish mutants affecting fin development
(van Eeden et al., 1996). The
fin primordium in zebrafish larvae is composed of a very thin layer of LPM
cells. In order to form a bud, the appropriate organ size of the fin buds is
generated not only through proliferation, but also by migration of LPM cells
towards the limb field (Ahn et al.,
2002). Fgf24, a member of the Fgf8/17/18 family of Fgf molecules,
is the earliest fgf gene known to be expressed in the zebrafish
forelimb bud, and one of its functions is to promote migration of
tbx5-positive cells towards the fin field
(Fischer et al., 2003). In the
fgf24 mutant ikarus (ika), the
tbx5-expressing LPM population does not compact and eventually
disappears at later stages of development, indicating that fgf24 is
needed on one hand for migration of tbx5-expressing cells to the limb
primordium, and on the other for the activation of fgf10, which then
relays the limb-inducing signal to the overlying ectoderm
(Fischer et al., 2003;
Norton et al., 2005).
A recent study showed that activity of the prdm1 gene is also
required for pectoral fin development, as knock down of Prdm1 leads to an
absence of pectoral fins (Wilm and
Solnica-Krezel, 2005). prdm1, also called blimp1
(B-lymphocyte induced maturation protein 1), encodes a transcriptional
repressor. Its N-terminal PR-domain possesses methyltransferase activity,
which is shared with other members of the SET domain protein family
(Kouzarides, 2002). In
addition, Prdm1 contains five Krüppel-like zinc finger domains through
which it binds to target promoters and, together with Groucho corepressors and
Histone deacetylases, causes transcriptional repression
(Makar and Wilson, 2004;
Ren et al., 1999;
Yu et al., 2000).
Prdm1 has been shown to play an essential role during the development of
several tissues. Analysis of null mutant mice has revealed a function for
Prdm1 in specification of the germ cell lineage
(Ohinata et al., 2005;
Vincent et al., 2005). In
zebrafish, Prdm1 regulates Bmp2 activity during gastrulation through the
repression of chordin (Wilm and
Solnica-Krezel, 2005), and is involved in neural crest cell
differentiation (Hernandez-Lagunas et al.,
2005; Roy and Ng,
2004). Zebrafish Prdm1 has also been shown to act downstream of
sonic hedgehog signaling during slow muscle specification
(Baxendale et al., 2004).
While it is clear that prdm1 is crucial for limb formation in
zebrafish (Wilm and Solnica-Krezel,
2005), its relationship to other genes in the limb induction
cascade has not been analysed in detail. We therefore systematically examined
the role of prdm1 in the regulatory hierarchy triggering limb
development. Second, because the regulatory relationship between RA, Wnt and
Fgf signaling in the axial mesoderm has not been fully determined, nor how
this cascade regulates Tbx5 and Fgf activity in the limb bud, we made use of
the availability of zebrafish raldh2, tbx5 and fgf24
mutants, and the Fgf-pathway inhibitor SU5402, to systematically examine the
regulatory hierarchy controlling zebrafish limb induction.
Our analysis reveals that prdm1 activation is downstream of RA, Wnt2b and Tbx5 activity in the limb primordium. Activation of prdm1 expression is also downstream of an early Fgf signaling event downstream of tbx5, directed in part by Fgf24. Following its activation in the limb bud, Prdm1 acts in a feedback loop to maintain fgf24 expression, and is required for further progression of the limb initiation cascade leading to fgf10 activation. We also find that RA signaling is necessary for wnt2b expression in the IM, whereas Fgf signaling activity is not necessary for this event. Likewise, Fgf signaling is not required for the activation of tbx5 expression in the LPM. These results indicate that Fgf signaling does not participate in the transfer of the limb-inducing signal from the axial mesoderm to the LPM, and instead plays a local role within the limb primordium downstream of tbx5. We propose a model in which RA signaling from the somitic mesoderm leads to activation of wnt2b expression in the IM, which then signals to the LPM to trigger tbx5 expression. tbx5 in turn is required for an Fgf signaling event in the limb bud leading to the activation of prdm1 expression, which then triggers activation of fgf10.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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), the tbx5 mutant heartstrings
(hst) (Garrity et al.,
2002) and the raldh2 mutant neckless
(nls) (Begemann et al.,
2001).
Morpholino injection
Antisense morpholino oligonucleotides against prdm1
(5'-TGTGTGATCTCTCCCCTGAGTGTGT-3') (Wilm et al., 2005) and
raldh2 (5'-GCAGTTCAACTTCACTGGAGGTCAT-3')
(Begemann et al., 2001) start
codon regions were purchased from Gene Tools (Corvallis, OR). Morpholinos were
diluted in distilled water and injected into one-cell stage embryos at a
concentration of 0.1 mM for MOprdm1 and 0.2 mM for MOraldh2.
Because there was some variation in the phenotypic penetrance of
prdm1 morphants, ranging from reduced fins (stumps) and unilateral
fin stumps to no fins, for all injection experiments, 10% of injected embryos
were allowed to develop until 72 hpf (hours postfertilization) to monitor the
effectiveness of prdm1 knock down. Only batches with >75% larvae
showing complete absence of fin development were further processed for in situ
hybridization.
Mosaic experiments
Bpe-GFP transgenic embryos at sphere stage were used as donors
(Higashijima et al., 1997).
Between 20 and 30 cells were transplanted into the lateral marginal zone of
sphere to dome-stage host embryos, which had previously been injected with
either MOprdm1 or MOraldh2. Rescue of pectoral fin
development and contribution of wild-type cells to anterior somites or
pectoral fin mesenchyme was monitored three days later under a fluorescent
light binocular (Leica, Cambridge, UK).
Treatment with DEAB, SU5402
Diethylaminobenzoic acid (DEAB) (Sigma) and the FGF receptor inhibitor
SU5402 (Calbiochem) were dissolved in dimethylsulfoxide (DMSO) and used at a
concentration of 10 mM and 16 µM, respectively. Incubations were carried
out in the dark at 28°C.
In situ hybridization and histochemical methods
Whole-mount in situ hybridization was performed as described (Kishimoto et
al., 1997), using the following probes: wnt2b (-22 to -1366 bp)
(Ng et al., 2002),
prdm1 (Wilm et al., 2005), tbx5
(Begemann and Ingham, 2000),
fgf24 (Fischer et al.,
2003), pea3 (Roehl
and Nüsslein-Volhard, 2001), fgf10
(Ng et al., 2002),
bmp2b (Kishimoto et al., 1997), raldh2 (Grandel et al.,
2001) and cyp26a1 (cb24 EST clone, Zebrafish International Resource
Center). BM purple (Roche) was used as a substrate. For prdm1 and
fgf24, staining reactions were performed overnight at 37°C.
Alcian Blue staining was performed according to Grandel and Schulte-Merkel
(Grandel and Schulte-Merkel,
1998).
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). As this study did not address the pectoral fin phenotype in
detail, we decided to further examine the effect of Prdm1 knock down on
pectoral fin development. Consistent with the report of Wilm and
Solnica-Krezel (Wilm and Solnica-Krezel,
2005), we find that knock down of Prdm1 causes an absence of
pectoral fins (Fig. 1A,B).
Furthermore, Alcian Blue cartilage staining reveals that all skeletal elements
of the pectoral fins are absent in prdm1 morphants at four days
postfertilization (Fig. 1C,D).
We also failed to detect any morphological signs of pectoral fin buds at
earlier stages in prdm1 morphants
(Fig. 1E,F). As these results
indicate that Prdm1 acts at an early stage in limb induction, we examined the
expression pattern of prdm1 during the time window when pectoral fin
development commences in zebrafish. Prior to fin bud formation, at the 15 to
17-somite stage, prdm1 expression is detectable in the somites and in
the posterior LPM (Fig. 2A-C).
At the 18-somite stage, which corresponds to 18 hours postfertilization (hpf),
we first detect prdm1 expression in the LPM regions close to somite
2, the region where the tbx5-positive pectoral fin mesenchyme starts
to condense (Fig. 2D-F). This
expression domain overlaps with the fgf24 expression domain. During
the next few hours, prdm1 expression increases in the fin bud
mesenchyme and, at the 23-somite stage (20.5 hpf), is clearly visible in the
fin primordia (Fig. 2G),
overlapping with fgf24 and tbx5 expression
(Fig. 2H,I).
Prdm1 acts downstream of retinoic acid signaling during pectoral fin induction
As the prdm1 morphant phenotype indicates that Prdm1 is crucial
for an early stage in pectoral fin induction, we examined the relationship
between prdm1 and raldh2, the earliest gene known to be
required for fin induction. Since prdm1, like raldh2, is
also expressed in somitic mesoderm at the level of the forelimbs, we examined
the possibility that Prdm1 is required for raldh2 expression in the
somites. However, knock down of Prdm1 activity does not affect expression of
raldh2 in the somites, nor in the LPM
(Fig. 3A-B').
Furthermore, activation of cyp26a1, a target of retinoic acid
signaling in the anterior somites
(Dobbs-McAuliffe et al.,
2004), is unperturbed in prdm1 morphants
(Fig. 3C,D). Consistent with
this observation, pectoral fin induction in prdm1 morphants is not
rescued by the administration of exogenous RA (data not shown); this is in
contrast to raldh2 mutants, which can be rescued by RA administration
(Begemann et al., 2004;
Grandel et al., 2002).
Therefore Prdm1 does not seem to act upstream of RA signaling.
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) leads
to an absence of prdm1 expression in several tissues, including the
anterior somites (Fig.
3E-F') and the pectoral fin buds
(Fig. 3G,H). Taken together,
these results indicate that prdm1 activation is downstream of RA
signaling during somite formation and limb induction.
Activation of wnt2b depends on Raldh2 activity, but not on Fgf signaling or Prdm1
Because Wnt2b is also necessary for forelimb induction, we next examined
the regulatory interactions between Wnt2b, Prdm1, RA and Fgf signaling. First,
to determine whether Prdm1 activity is needed for wnt2b expression in
the IM, we examined wnt2b expression in prdm1 morphants. No
difference in wnt2b expression could be detected in prdm1
morphants when compared with wild-type siblings
(Fig. 3I,J). We also examined
whether Fgf signaling is required for wnt2b expression, by blocking
the Fgf pathway with the Fgf receptor inhibitor SU5402
(Mohammadi et al., 1997).
Treatment of zebrafish embryos with SU5402 has been shown to block the
expression of Fgf target genes (Raible and
Brand, 2001; Roehl and
Nusslein-Volhard, 2001). As described, we find that treatment of
embryos with 16 µM SU5402 from 11.5 hpf onwards leads to an absence of
expression of the Fgf target gene pea3 at 24 hpf (data not shown). By
contrast, wnt2b is expressed normally in the same batch of
SU5402-treated embryos at 24 hpf (Fig.
3K). However, wnt2b expression in the IM is absent in
neckless (nls) mutants, which disrupts Raldh2 activity
(Fig. 3L), indicating that
wnt2b transcription is dependent on RA signaling. Loss of
wnt2b was not caused by general disruption of IM formation, as the IM
molecular marker pax2a was still expressed in nls embryos
(not shown). Taken together, these results show that wnt2b activation
in the IM depends on Raldh2 activity, but not on Fgf signaling, nor on Prdm1
activity.
Prdm1 activity is not required in the somites during pectoral fin induction
Transplantation experiments have shown that Raldh2 activity is sufficient
within the somitic mesoderm at the level of the first three somites to direct
pectoral fin induction (Linville et al.,
2004). As prdm1, like raldh2, is expressed in
the anterior somites, and expression of prdm1 in this tissue depends
on Raldh2 activity, we considered the possibility that prdm1 acts in the
somitic mesoderm to direct pectoral fin induction. However, because
prdm1 is also expressed in the nascent pectoral fin buds, an
alternative is that this latter expression domain might be necessary for fin
induction. To distinguish between these two possibilities, we performed mosaic
experiments in which we transplanted wild-type cells into prdm1
morphants. As a control, we compared this experiment to the effect of
transplanting wild-type cells into raldh2 morphants. As previously
described, pectoral fin induction can be rescued in raldh2 morphants
by wild-type cells located in anterior somites
(Fig. 4A-B''). In some
cases, we observed rescue when wild-type cells were found both in the somites
and in the fins (n=6), but in other cases we observed rescue when
cells were found only in the somites (n=3; total number of chimeric
embryos with wild-type cells in anterior somites, n=7;
Fig. 4A-B''). We did not
observe cases in which wild-type cells exclusively contributed to the rescued
limb. These results indicate that Raldh2 activity in the somites is sufficient
to direct fin induction. In the case of prdm1 morphants, we never
observed rescue in cases where wild-type cells were located only in the
somites (n=26). Even in cases were GFP expression in the anterior
somites was very strong, fin outgrowth was not restored in MOprdm1
embryos (n=19) (Fig.
4C-D''). This result indicates that Prdm1 activity, unlike
Raldh2, is not required in the somites for pectoral fin induction, but instead
suggests that it is required in the fin bud primordium.
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We also find that tbx5 expression is activated normally in the LPM
of prdm1 morphants (Fig.
5I,J). This is similar to previously reported data showing that
tbx5 is activated normally in tbx5 and fgf24
mutants (Fig. 5K)
(Ahn et al., 2002;
Fischer et al., 2003). At a
slightly later stage (24 hpf), we find that tbx5 expression in
prdm1 morphants fails to form a compact domain in the fin bud, and
the tbx5-expressing cells instead remain spread throughout the LPM
(Fig. 5L,M). The same effect is
observed in tbx5 mutants, although tbx5 downregulation is
more severe in that case (data not shown)
(Ahn et al., 2002). The
stronger downregulation of tbx5 expression in tbx5 mutants
compared with prdm1 morphants is consistent with tbx5 being
upstream of prdm1 in the fin initiation cascade and suggests that
Tbx5 activates other genes necessary for fin initiation, such as
sall4 (Harvey and Logan,
2006). Tbx5 activity is necessary for activation of fgf24
expression in the fin bud, and fgf24 mutants also fail to form a
compact tbx5-expressing domain
(Fig. 5N)
(Fischer et al., 2003). Like
fgf24, prdm1 also fails to be activated in the fin buds of the
tbx5 mutant heartstrings (hst)
(Fig. 6D,H). Taken together,
these results indicate that Tbx5 acts upstream of Prdm1, consistent with the
observation that tbx5 expression is activated in the fin primordium
earlier than prdm1 (Fig.
2F,I).
Because both Prdm1 and Fgf24 act downstream of Tbx5, we investigated whether prdm1 expression is regulated by Fgf24. We find that initiation of prdm1 transcription in the fin buds of the fgf24 mutant ikarus (ika) is both delayed and reduced. At the 23-somite stage (20.5 hpf), prdm1 expression is present in wild-type embryos, but not in ika mutants (Fig. 6A,C), but at 24 hpf, faint expression of prdm1 is present (Fig. 6E,G). However, at later stages, prdm1 expression is lost again in ika mutants (Fig. 6I,K). Loss of prdm1 expression is not due to increased cell death in the fin mesenchyme of ika mutants (Fig. 7). This indicates that activation and maintenance of prdm1 expression depends on fgf24, but that there is also fgf24-independent prdm1 expression.
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Prdm1 acts upstream of Fgf10 during fin induction
A further Fgf signaling event in the early limb bud is mediated by Fgf10
signaling from the limb mesenchyme to the overlying ectoderm. Fgf24 has been
shown to act upstream of fgf10 during limb initiation
(Fischer et al., 2003). As
Prdm1 acts downstream of fgf24, we analyzed the regulatory
relationship between prdm1 and fgf10 by making use of the
zebrafish fgf10 mutant daedalus
(Norton et al., 2005). We find
that onset of prdm1 expression and maintenance in the fin mesenchyme
of fgf10 mutants is identical to wild-type siblings
(Fig. 6A,B,E,F,I,J). However,
at 36 hpf prdm1 expression is not activated in the AER of
fgf10 mutants, although it is expressed in the wild-type AER
(Fig. 6L,M). Because
fgf10 is required for the establishment of the AER, it is likely that
the loss of prdm1 expression in daedalus mutants is due to
lack of AER formation, rather than due to a specific role of Fgf10 in
prdm1 activation. Conversely, loss of Prdm1 activity leads to a loss
of fgf10 expression (Fig.
8A,D), as has been reported for fgf24 and tbx5
mutants (Fig. 8B,C)
(Fischer et al., 2003;
Ng et al., 2002). Taken
together, these results indicate that Prdm1 acts upstream of fgf10
activation in the fin bud mesenchyme.
As Fgf24 is not required for the activation of several early genes
expressed in the fin mesenchyme, including pea3 and bmp2b
(Fig. 8E,F,I,J)
(Fischer et al., 2003), we
examined whether Prdm1 activity is necessary for these genes. We find that
both pea3 and bmp2b fail to be activated in prdm1
morphants, and observe the same absence of expression in tbx5 mutants
(Fig. 8G,H,K,L). Taken
together, these results indicate that Prdm1 is required for the activation of
fgf10, pea3 and bmp2b transcription.
The earliest Fgf signaling required for fin induction is downstream of Tbx5
To address the question whether Fgf signaling is involved in relaying the
limb-inducing signal from the axial mesoderm to the LPM, or whether it acts at
a later stage during limb induction, we used the inhibitor SU5402 to assay the
effect of Fgf pathway inhibition on the activation of early limb genes.
Treatment of embryos with 16 µM SU5402 from the one-somite stage (10.7 hpf)
onwards leads to complete downregulation of the Fgf target gene erm
throughout the embryo at 20.5 hpf and at 24 hpf
(Fig. 9A-D), indicating that
Fgf signaling is severely inhibited in these embryos. prdm1 is not
activated in the fin bud at any stage in SU5402-treated embryos
(Fig. 9E-H). By contrast, we
find that fgf24 transcription in the LPM is activated in the same
batch of SU5402-treated embryos but becomes strongly downregulated at 24 hpf
(Fig. 9I-L). At 20.5 hpf, the
tbx5 expression domain is not altered upon SU5402 treatment
(Fig. 9M,N), but at the 24 hpf
stage, we observe a failure of tbx5-expressing cells to congregate
towards the fin bud (Fig.
9O,P). As this defect is also observed in fgf24 mutants
(see Fig. 5N)
(Fischer et al., 2003), it is
likely to be due to the absence of Fgf24 activity in SU5402-treated embryos.
Also, the fact that prdm1 activation is completely blocked in
SU5402-treated embryos, but is only delayed and is partially reduced in
fgf24 mutant embryos, suggests there is an additional Fgf protein
acting downstream of Tbx5 to activate prdm1 expression, which is
semi-redundant with Fgf24. This proposal is further supported by the
observation that activation of the Fgf target pea3 is not completely
blocked in fgf24 mutants, indicating there is still Fgf signaling
present in fgf24 mutants (Fig.
8F).
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DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Chapman et al., 1996;
Gibson-Brown et al., 1996;
Simon et al., 1997), followed
by fgf24, and then prdm1 a few hours later. Interestingly,
tbx5 is expressed more broadly in the LPM than are the other two
genes. Both prdm1 and fgf24 are expressed in a small patch
of cells corresponding to the nascent fin primordium, whereas tbx5 is
also expressed in surrounding cells that later migrate to the fin bud
(Ahn et al., 2002;
Fischer et al., 2003).
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) (this study).
During plasma cell differentiation, Prdm1 has been shown to act as a
repressor, directly repressing the transcription of cmyc (previously
known as c-myc), PAX5 and CIIITA
(Lin et al., 2002;
Lin et al., 1997;
Piskurich et al., 2000). This
suggests that the activation of limb genes downstream of prdm1 would
have to be indirect, via repression of another repressor. We have observed
that prdm1 morphants have elevated levels of prdm1
transcripts (data not shown), suggesting that during zebrafish development,
Prdm1 can act as a repressor of its own transcription. However, it has also
been proposed that Prdm1 could also act as a transcriptional activator
(Baxendale et al., 2004), and
we therefore cannot exclude that it might directly activate target gene
expression during fin initiation. To discriminate between these options,
further work needs to be carried out.
A cascade of inductive events originating in the anterior somites leads to initiation of forelimb development in the LPM
We have systematically analyzed the hierarchical relationship between the
genes and signaling pathways required for zebrafish pectoral fin induction.
This group of genes includes raldh2, wnt2b, tbx5, prdm1, fgf24 and
fgf10, and our results support a model in which these genes form a
linear hierarchy controlling the transfer of the limb-inducing signal from the
anterior somites to the LPM (Fig.
10). The earliest gene known to function in pectoral fin induction
is raldh2 (Begemann et al.,
2001; Grandel et al.,
2002). In the absence of Raldh2 activity, all other limb genes
fail to be expressed, including wnt2b (this study) and tbx5
(Begemann et al., 2001). This
is consistent with the observation that Raldh2 activity is necessary for limb
induction at early segmentation stages
(Grandel et al., 2002), which
is well before the earliest fin bud marker, tbx5, is expressed in the
LPM. Furthermore, as Raldh2 activity is required in the first three somites
(Linville et al., 2004) (this
study), this indicates that the signaling cascade leading to pectoral fin
induction originates in the somitic mesoderm during early segmentation
stages.
|
|
).
|
). Further work will be necessary to identify the
complete set of zebrafish fgf genes acting during limb
initiation.
Maintenance of fgf24 expression becomes dependent on Prdm1
activity soon after its initial activation, indicating that Prdm1 operates in
a feedback loop to regulate fgf24 maintenance. The failure of
tbx5-expressing LPM cells to congregate towards the fin bud in the
absence of Prdm1 activity is most likely due to the failure of fgf24
maintenance, as Fgf24 is required for this migratory event
(Fischer et al., 2003).
Finally, Prdm1 activity is required for the downstream activation of fgf10 expression, which then relays the limb initiation signal from the mesenchyme to the ectoderm, to direct AER development and limb outgrowth.
The earliest requirement for Fgf signaling during forelimb induction is downstream of tbx5 activation
An important issue remaining unresolved so far is whether Fgf signaling is
required for the transfer of the limb-inducing signal from the axial mesoderm
to the LPM. We addressed this question by using the Fgf pathway inhibitor
SU5402 to define the earliest step at which Fgf signaling is required for
forelimb induction. Our results reveal that Fgf signaling is necessary neither
for expression of wnt2b in the IM, nor for the activation of
tbx5 expression in the LPM, suggesting that the transfer of the
limb-inducing signal from the axial mesoderm to the LPM is independent of Fgf
signaling. This is consistent with the observation that conditional removal of
Fgf8 activity from the IM in the mouse has no effect on limb development
(Boulet et al., 2004;
Perantoni et al., 2005).
Similarly, the zebrafish fgf8 mutant acerebellar does not
show impaired pectoral fin development
(Reifers et al., 1998). Our
results indicate that the earliest requirement for Fgf signaling during limb
induction is for the activation of prdm1 but not for onset of
tbx5 expression. Taken together, these data suggest that Fgf
signaling is not required for the transfer of positional information from the
somites or IM to the LPM during limb induction, and instead plays a local role
within the limb primordium. They also show that the Fgf signaling cascade is
established downstream of Tbx5 activity.
Conservation of the limb induction cascade among vertebrate species
The expression pattern of prdm1 during limb development is
conserved between zebrafish and tetrapods. In chick and mouse, Prdm1
is also initially expressed within the limb mesenchyme, and later becomes
activated in the overlying AER (Ha and
Riddle, 2003; Vincent et al.,
2005). In contrast to prdm1 knock down in zebrafish,
Prdm1 null mutant mice do not display any defects in limb bud
initiation (Vincent et al.,
2005). This difference may be due to redundancy of Prdm1
function with a related gene in the mouse, or it may reflect a
species-specific role of prdm1 in zebrafish limb induction. It will
be interesting to analyze the effect of Prdm1 loss-of-function and
gain-of-function in the chick, to determine whether Prdm1 activity plays a
role during limb induction in this species.
In contrast to Prdm1 mutants, mouse mutants for Raldh2
(Mic et al., 2004;
Niederreither et al., 1999),
Tbx5 (Agarwal et al.,
2003; Rallis et al.,
2003) or Fgf10 (Min
et al., 1998; Sekine et al.,
1999) all display failure of limb induction similar to the
corresponding zebrafish mutants (Begemann
et al., 2001; Garrity et al.,
2002; Grandel et al.,
2002; Norton et al.,
2005), thus indicating that the limb induction cascade is broadly
conserved between tetrapods and teleost fish. However, there are clearly also
differences. For example, mouse Wnt2b does not play a role in limb
induction (Ng et al., 2002),
and no gene corresponding to zebrafish fgf24 is present in tetrapod
genomes (Draper et al., 2003).
In both cases, other members of their respective gene families may fulfill
their role in the mouse. Alternatively, specific steps in the limb induction
cascade may have changed during evolution. To answer this question, it will be
important to understand the regulation of early limb genes in several
vertebrate species at the level of their promoter activity, as changes in
signals regulating limb induction should be reflected in altered regulation of
the promoters of their target genes.
|
ACKNOWLEDGMENTS |
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