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First published online 14 June 2006
doi: 10.1242/dev.02438
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Lehrstuhl für Zoologie und Evolutionsbiologie, Department of Biology, University of Konstanz, Konstanz, Germany.
* Author for correspondence (e-mail: gerrit.begemann{at}uni-konstanz.de)
Accepted 10 May 2006
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Retinoic acid, Raldh2, Limb, Pectoral fin, Somites, Spadetail, No tail, Neckless, Zebrafish
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Logan, 2003;
Tickle, 2003).
Fibroblast growth factors (FGFs) have been shown to play an important role
in limb induction. When beads are soaked in a range of fibroblast growth
factors and are placed in the flank of chick embryos, they induce the
formation of ectopic limb buds (Cohn et
al., 1995; Ohuchi et al.,
1995). Competence of the LPM to respond to these signals is
initially distributed along most of the AP axis of the LPM in chick and mouse
embryos (Tanaka et al.,
2000).
The search for the axial sources of FGF expression in the chick embryo lead
to the observation that the intermediate mesoderm (IM), situated between the
somites and the LPM, is required for cell proliferation in the adjoining limb
bud mesenchyme (Geduspan and Solursh,
1992; Smith et al.,
1996); furthermore, FGF8 is produced in the intermediate mesoderm
at the time of limb induction and, when applied to the chick flank, induces
the development of additional limbs
(Crossley et al., 1996;
Vogel et al., 1996). On the
other hand, when the induction of mesonephros, a component of IM, is blocked
rostral to the future limb field, limb buds nevertheless form and develop
normally (Fernandez-Teran et al.,
1997). In addition, elimination of FGF8 from the IM does not
affect limb initiation or outgrowth
(Boulet et al., 2004). In
addition, genes of the Wnt family are differentially expressed in the chick IM
and LPM and are capable of inducing limb outgrowth
(Kawakami et al., 2001).
Specifically, forelimb initiation requires Wnt2b expression in the IM
and/or LPM of the chick, while its source in zebrafish appears to be the
somites flanking the pectoral fin field
(Ng et al., 2002). Thus, the
identity of axial signals required for limb development as well as their
source(s) remain to be determined.
Retinoic acid (RA) is required for a variety of processes during vertebrate
embryonic development. Its effect is transmitted by retinoic acid receptors
(RARs) at the level of regulating the expression of target genes (reviewed by
Morriss-Kay and Ward, 1999).
Three enzymes, Aldh1a1-3 (previously designated Raldh1-3), catalyze the final
oxidative step by which vitamin A (retinol) is converted to RA, but only
Aldh1a2 has been shown to be responsible for RA synthesis during early stages
of embryogenesis. During gastrulation, aldh1a2 is expressed in
embryonic mesoderm and is later found in mesodermal derivatives, such as
somites, IM and LPM (Niederreither et al.,
1997; Berggren et al.,
1999; Swindell et al.,
1999; Begemann et al.,
2001; Chen et al.,
2001; Grandel et al.,
2002).
Mouse and zebrafish mutants in aldh1a2 have been used to show that
the somitic mesoderm acts a source of RA that patterns adjacent axial tissues,
such as the hindbrain and pancreas; importantly, aldh1a2 mutants also
lack forelimb buds (Begemann et al.,
2001; Grandel et al.,
2002; Niederreither et al.,
2002; Linville et al.,
2004; Molotkov et al.,
2005; Stafford et al.,
2006). The absence of forelimb buds in aldh1a2 mutants is
associated with the loss of tbx5 expression, the earliest known
marker of the developing forelimb field
(Gibson-Brown et al., 1996;
Tamura et al., 1999;
Begemann and Ingham, 2000). In
the mouse, Tbx5 first serves to establish forelimb bud outgrowth by
initiating the expression of regulatory loops of Wnt and Fgf proteins
(reviewed by Capdevila and Izpisua
Belmonte, 2001; Logan,
2003); while in the zebrafish, tbx5 is required for the
migration of mesenchymal LPM cells to the pectoral fin bud. As a result,
mutants in both vertebrates do not form forelimb buds
(Ahn et al., 2002;
Garrity et al., 2002;
Agarwal et al., 2003;
Rallis et al., 2003). The
failure to induce tbx5 expression in the absence of early RA
signaling therefore indicates an essential early role for RA during the
establishment of the limb/fin field.
In zebrafish, aldh1a2 is expressed in the posterior mesenchyme of
the developing pectoral fin and in the LPM
(Grandel et al., 2002;
Emoto et al., 2005). RA has
been detected in the limb bud mesenchyme
(Thaller and Eichele, 1987;
Niederreither et al., 2002);
however, RA production in the cells of the polarizing region of the limb bud
has not been demonstrated. Expression analyses in zebrafish and mouse embryos
mutant for Aldh1a2 have shown that RA is required to induce sonic
hedgehog (Shh), the protein responsible for the polarizing activity (reviewed
by Tickle, 2003), during
forelimb outgrowth (Begemann et al.,
2001; Grandel et al.,
2002; Niederreither et al.,
2002), and suggest that RA signaling may be involved in the
establishment of AP polarity in the forelimb. Indeed, maternal RA
supplementation of Aldh1a2 mutant mice rescues forelimb development,
but does not restore normal AP patterning of the limb
(Niederreither et al., 2002).
Rescued limbs are characterized by a lack or abnormal distal distribution of
Shh. More recent studies, however, have proposed a different role for RA in
limb patterning. Using mice mutant for Aldh1a2,
Mic et al. (Mic et al., 2004)
have shown that RA signaling is required first to initiate forelimb
development and later to expand the apical ectodermal ridge (AER) along the
distal ectoderm. A study by Yashiro et al.
(Yashiro et al., 2004)
similarly suggested that a gradient of RA signaling is required to determine
proximodistal identity in the developing mouse forelimb.
In this study, we determine the crucial time period for limb induction and the source of RA. By analyzing pectoral fin development in the absence of RA, we uncover that prepatterning by RA occurs several hours prior to the budding of the pectoral fin to establish a functional zone of polarizing activity. Our findings point to an essential role for somite-derived RA in fin field induction, apical fold formation and in establishing posterior cell fates within the condensing fin mesenchyme.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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),
ntltc41 (Odenthal et
al., 1996) and sptb104
(Kimmel et al., 1989) were
reared and staged at 28.5°C according to
Kimmel et al. (Kimmel et al.,
1995).
Pharmacological treatments
Embryos were incubated in the dark at 28.5°C in 10-8 M
all-trans retinoic acid (Sigma), diluted in embryo medium, from a
10-2 M stock solution in DMSO. The pan-retinoic acid receptor
antagonist BMS493 (a kind gift of Bristol Myers Sqibb) was diluted to
10-5 M and 5x10-6 M from a 10-2 M stock
solution in ethanol. DEAB (4-diethylaminobenzaldehyde) (Fluka) was applied at
a concentration of 10-5 M from a 10-2 M stock in DMSO,
without shielding from daylight. As controls, wild-type embryos were treated
with equivalent concentrations of DMSO. Genotyping to detect homozygosity of
the nls allele was performed as previously described
(Begemann et al., 2001).
In situ hybridization
Whole-mount in situ hybridization was performed as previously described for
aldh1a2 (Begemann et al.,
2001), using the following additional probes: dlx2a
(Akimenko et al., 1994),
hand2 (Yelon et al.,
2000), hoxc6a (Molven
et al., 1990), hoxd11a, hoxd12a
(Sordino et al., 1995),
msxc (Ekker et al.,
1992), myod (Weinberg
et al., 1996), shh
(Krauss et al., 1993),
tbx5 (Begemann and Ingham,
2000), tpm1 (Zebrafish Information Network) and
pax2a (Krauss et al.,
1991).
Mosaic analysis
Donor embryos were injected at the one-cell stage with 2.5% lysine fixable
tetramethyl-rhodamin-dextran and 3.0% lysine fixable biotin-dextran
(Mr 100,000)(Molecular Probes) dissolved in 0.2 M KCl. At
late blastula stages, groups of 10-30 donor cells were transplanted into
unlabelled host embryos of the same stage, derived from pair-matings of
nls heterozygotes, and placed along the margins of the blastoderm,
which gives rise to the mesendoderm
(Kimmel et al., 1990).
Transplants were carried out blindly, and host genotypes determined at 24 hpf.
Transplanted cells were examined at 48 hpf for fluorescence in all
nls hosts prior to or after fixation and in situ hybridization.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Kudoh et al., 2002;
Stafford and Prince, 2002;
Keegan et al., 2005). In order
to determine in more detail the developmental stages during which RA signaling
is required to induce the pectoral fin field, we employed two complementary
strategies: rescuing neckless (nls) through exogenous RA;
and inhibiting fin field formation by blocking RA signaling in wild-type
zebrafish.
Initiation of forelimb bud formation is characterized by the expression of
tbx5 in the anterior lateral plate mesoderm and precedes the
emergence of visible forelimb buds
(Ruvinsky et al., 2000;
Ahn et al., 2002;
Garrity et al., 2002;
Ng et al., 2002). Pectoral fin
development is characterized by the appearance of an apical fold that
expresses dlx2a (Akimenko et al.,
1994). We treated wild-type embryos with 10-6 M BMS493,
which inhibits the activation of all three RA-receptor (RAR) subforms in
amniotes by reinforcing co-repressor binding to RAR
(Wendling et al., 2000;
Dupé and Lumsden, 2001)
from 10 hpf onwards, and confirmed that fin buds do develop and express both
tbx5 and dlx2a (Grandel
et al., 2002) (Fig.
1B,F). However, when treated with 5x10-6 M BMS493
or with 10-5 M diethylaminobenzaldehyde (DEAB), a competitive
reversible inhibitor of retinaldehyde dehydrogenases
(Begemann et al., 2004) from 10
hpf onwards, no fin buds emerged and none of the genes were expressed
(Fig. 1C,D,G,H). We next
refined these tests of RA requirement by incubations in 10-5 M DEAB
or in a range of 10-5 to 5x10-6 M BMS493. We
observed that blocking RAR activation after 13 hpf, or inhibiting endogenous
RA synthesis after 12 hpf, has no effect on the onset of tbx5
expression in the pectoral fin, while treatments commencing earlier disrupt
fin field induction (Table 1A).
These results suggest that RA signaling is required for fin development up to
13 hpf.
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;
Begemann and Ingham, 2000)
(Fig. 1Q). nls mutants
were identified either through expansion of krox20 (egr2b - Zebrafish
Information Network) expression in the hindbrain when treated after 10 hpf
(not shown), or by genotyping when treated prior to 10 hpf. In agreement with
a requirement for RA after 10 hpf, treatments initiated as late as 12 hpf are
sufficient to rescue tbx5 expression to approximately wild-type
levels, while treatment at 13 hpf or later is not sufficient
(Table 1B). To confirm the hypothesis that RA signaling after gastrulation is required for fin induction, RA synthesis was inhibited with DEAB in wild-type embryos starting from 5 hpf onwards, and at 11 hpf was supplanted with exogenous RA; fin bud induction in these fish proceeded indistinguishably from wild-type development (Fig. 1I,J). Together, these experiments suggest that pectoral fin field development requires RA signaling after the end of gastrulation and prior to the 12 hpf (six-somite stage). The sufficiency of this short period of treatment indicates that RA is required for induction, rather than maintenance of the pectoral fin field.
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) and is
expressed in the fin mesenchyme at 24 hpf
(Ng et al., 2002). In
RA-rescued fins of nls mutants, fgf10 is expressed strongly
(Fig. 1K,L), suggesting that
its upstream component fgf24 must also be active. tbx15 is
strongly expressed throughout the pectoral fin mesenchyme starting from 30 hpf
(Begemann et al., 2002) and is
detected in RA-rescued nls fins
(Fig. 1M,N). We conclude that
genes downstream of tbx5 in the fin mesenchyme are activated
following RA-treatment in nls. By contrast, expression of
wnt2b, which is required for tbx5 expression and at 22 hpf
is found in the ventral part of somites flanking the pectoral fin bud
(Ng et al., 2002), is not
abolished in wild type fish treated with DEAB or a pan-antagonist of all
RA-receptors from 11-22 hpf (Fig.
1O,P and not shown), which includes the time of fin induction and
early patterning. Thus, RA is dispensable for somitic wnt2b
expression. Like aldh1a2, wnt2b acts upstream of tbx5 during
fin induction (Ng et al.,
2002), so that both genes might operate in parallel pathways,
Alternatively, earlier wnt2b expression outside the somites may be
required to regulate tbx5.
Somitic mesoderm is required for pectoral fin induction
During segmentation stages, aldh1a2 is expressed in the somitic
and intermediate mesoderm (Begemann et al.,
2001; Grandel et al.,
2002). As both these embryonic structures are in close proximity
to the future pectoral fin field, we wanted to distinguish whether the source
of RA that triggers pectoral fin induction is the intermediate or somitic
aldh1a2 expression domain. To this end, we genetically ablated the
somitic mesoderm in embryos double mutant for spadetail
(spt) and no tail (ntl)
(Amacher et al., 2002;
Goering et al., 2003), taking
advantage of the fact that double-mutants for the allelic combination of
sptb104 and ntltc41 lack somitic
mesoderm, but retain a reduced intermediate mesoderm (IM), as marked by the
expression of pax2a, a marker of the pronephric duct and the
glomerulus (Majumdar et al.,
2000) (Fig. 2A-D).
Similarly, somitic aldh1a2 expression is reduced in spt and
is absent in sptb104/ntltc41
(Fig. 2E-H'). Because a
faint aldh1a2 staining remains in double mutants, we wanted to
identify unambiguously double-mutant embryos by simultaneously monitoring both
myod and aldh1a2 expression. At 13 hpf, when RA signaling is
required for pectoral fin field induction, aldh1a2 is expressed in
the wild-type IM, and can be clearly detected at reduced levels in the IM of
sptb104/ntltc41
(Fig. 2J,J'). These
results establish that in
sptb104/ntltc41 the IM is likely to
produce RA. sptb104/ntltc41 double
mutants do not develop any morphological signs of pectoral fin development and
tbx5 expression in the pectoral fin field is indistinguishable from
wild type in ntl (Fig.
3B). In agreement with the reduction of aldh1a2
expression in trunk mesoderm, tbx5 expression is slightly reduced in
spt (Fig. 3C). By
contrast, double-mutant embryos lack tbx5 expression
(Fig. 3D), suggesting that fin
field induction requires RA signaling from somites rather than from IM.
RA is sufficient to rescue fins in the absence of somites
As the lack of tbx5 expression in ntl/spt could either be
due to the lack of somite-derived signals distinct from RA or because the LPM
may not be competent to form fins, we asked if exogenous application of RA
after gastrulation would rescue pectoral fin field induction. Interestingly,
application of 10-8 M RA, beginning at 10 hpf, efficiently rescues
tbx5 expression in
sptb104/ntltc41, suggesting that the
LPM is competent to respond to a somite-derived signal to form fins
(Fig. 3I,K). As RA application
after gastrulation does not reconstitute somite development in double mutants
(Fig. 3J-M), RA is identified
as the signal emanating from somites that induces the pectoral fin field.
Moreover, these findings further suggest that somite-derived signals other
than RA are not required to initiate tbx5 expression.
RA from anterior somites induces pectoral fin field formation
In order to determine more directly the source of RA, we next investigated
whether somitic cells of wild-type origin were able to induce tbx5
expression in the lateral plate mesoderm of nls mutants. We
transplanted lineage-labeled wild-type cells at blastula stages into the
lateral marginal zone of nls mutant hosts, a region fated to give
rise to trunk somites (Melby et al.,
1996). nls hosts were identified by their hindbrain
phenotype at 22-24 hpf and scored for the number and distribution of
transplanted cells (Table 2).
In such an experiment, we not only identified the nls hosts with
rescued fins, but the presence of wild-type cells in somites without rescue is
equally informative. None of the nls hosts had donor cells in
derivatives of the intermediate mesoderm. In one host with rescued fins
(n=2), the majority of muscle fibers in somites 5-7 were donor
derived, with only a few wild-type cells in somite 3 and 4
(Fig. 4A-C). In another
successful rescue, the majority of muscle fibers in somites 3-6 were wild-type
derived (Fig. 4D-F). In hosts
with only a few wild-type cells occupying somites 4-7 (n=6), or where
donor cells gave rise to part or all of the muscle fibers in somites posterior
to somite 8 (n=6), no rescue was observed
(Table 2;
Fig. 4H). Interestingly, one
transplanted embryo showed very strong contribution of wild-type cells to the
dorsal halves of somites 1-8, yet did not lead to pectoral fin development
(Fig. 4G).
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Sources and requirement of RA in the developing pectoral fins
To resolve the role of RA in pectoral fin bud development and during the
outgrowth phase, we first determined the expression pattern of
aldh1a2 in more detail and in later stages of fin development than
previously shown. aldh1a2 is first re-expressed at 28 hpf in the
posterior-most part of the pectoral fin bud and in the adjacent LPM
(Fig. 5A,B). Between 32 and 48
hpf, aldh1a2 is expressed in the entire pectoral fin mesenchyme
(Fig. 5C,D) and becomes
restricted proximally at later stages (Fig.
5E,H). Thus, during initiation of fin outgrowth, RA is produced
from a posterior source of aldh1a2 expression, is synthesized
uniformly in the entire fin at later stages, and becomes gradually restricted
to the proximal anterior fin mesenchyme by day 5.
We next investigated the roles of RA signaling during later stages of pectoral fin development. Wild-type embryos treated with either BMS493 (not shown) or DEAB from 16 hpf onwards develop pectoral fin buds, although they are smaller than in wild type (Fig. 6). This suggests that the initiation of bud outgrowth is independent of RA, although normal cell proliferation within the pectoral fin bud may require full RA signaling.
To determine to what extent fin patterning is affected in the absence of
RA, we examined gene expression in the fin bud mesenchyme and apical fold. The
bHLH transcription factor hand2 is required for the expansion of the
pectoral fin-forming region of the LPM and is expressed in the pectoral
fin-bud mesenchyme, with the exception of its anteriormost aspect
(Yelon et al., 2000). In the
absence of RA, hand2 is not expressed
(Fig. 6A,B). Because mutants in
hand2 fail to express sonic hedgehog (shh), we next
analyzed shh expression. In wild type, shh is expressed in
the posterior fin bud mesenchyme at 28 hpf, co-localizing with the zone of
polarizing activity (ZPA). In the absence of RA, shh is strongly
downregulated (Fig. 6C,D). Both
shh and hand2 remain downregulated at least up to 48 hpf
(not shown). Thus, downregulation of shh expression may be due to the
immediate lack of RA, or may be a consequence of the loss of hand2.
hoxd11a and hoxd12a are markers of the posterior mesenchyme
of wild-type fin buds at 40 hpf (Sordino
et al., 1995) (Fig.
6E,G), the expression of which does not depend on shh
(Neumann et al., 1999). When
RA synthesis is suppressed, expression of hoxd11a and
hoxd12a is downregulated (Fig.
6E-H). hoxc6a is marker of the anterior half of the fin
mesenchyme at 28 hpf and is restricted more anteriorly as the fin bud grows
(Molven et al., 1990)
(Fig. 6I). When RA signaling is
abolished, hoxc6a is expanded posteriorly to cover the anterior half
of the mesenchyme at 38 hpf (Fig.
6J). These findings indicate that early AP patterning of the
developing fin bud is compromised in the absence of RA, leading to an
expansion of the anterior marker hoxc6a and the downregulation of
markers of the posterior fin bud mesenchyme.
To test whether loss of RA signaling affects the development of the apical
fold, a structure required for fin outgrowth, we examined dlx2a
expression. As in wild type, dlx2a is expressed along the AP axis of
the apical fold during fin budding in the absence of RA synthesis
(Akimenko et al., 1994)
(Fig. 6K,L). In a subset of
embryos treated with DEAB from 14 hpf, rather than 16 hpf, onwards, slight
variations in the AP-extent of dlx2a expression was observed, such as
downregulation in the anterior and posterior apical fold (not shown). This
suggests that full RA signaling is required to establish a complete apical
fold.
|
;
Mic et al., 2004;
Yashiro et al., 2004). We
therefore determined if pectoral fin patterning is mediated by RA synthesized
within the fin bud mesenchyme, by blocking signaling after initiation, but
prior to aldh1a2 expression in the fin bud. Wild type were incubated
in 10 µM DEAB from 14, 18 and 22 hpf onwards, and assayed for shh
expression at 32 hpf. Inhibiting RA signaling from 14 hpf results in reduced
fin buds devoid of detectable shh expression and inhibition from 18
hpf onwards lead to a reduction of most shh expression
(Fig. 6O,P). By contrast,
blocking RA signaling after 22 hpf had little effect on shh
expression (Fig. 6M,N). This
suggests that AP patterning within the pectoral fin field occurs throughout
early to mid-somitogenesis and prior to the formation of the limb bud. As
aldh1a2 is expressed in the fin bud after its AP pattern has been
established, somite-derived RA is required to pattern the developing field
along with the condensation of the fin mesenchyme.
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DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Linville et al.,
2004). Our findings also explain the results of a previous study,
in which treatment of zebrafish embryos with citral, an unspecific inhibitor
of retinaldehyde dehydrogenases, from 80% epiboly to the two-somite stage
leads to morphologically normal fish that lack the pectoral fins
(Vandersea et al., 1998).
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), and RA is required while these somites are formed.
Similarly, in the mouse embryo the forelimbs develop adjacent to somites 8-12,
and limited maternal RA treatment that includes the development of the first
4-8 somites in Aldh1a2-mutant mice rescues initiation of the forelimb
field (Mic et al., 2004).
Considering the variation inherent to rescue studies in the mouse in general,
and that most RA may not be cleared immediately, limb field induction in mice
can be viewed as taking place concurrently with the emergence of flanking
somites. This observation holds particularly true for the chick embryo, where
forelimb induction has occurred by the 20-25 somite stage, and the wing bud
develops adjacent to somites 15-20 (reviewed by
Martin, 1998). We therefore
propose a general mechanism by which somite formation and forelimb field
induction are coupled through a window of RA responsiveness.
In zebrafish, the pelvic fins develop more than 2 weeks after completion of
somitogenesis, in a position ventrolateral to the ninth and tenth myotome
(Grandel and Schulte-Merker,
1998). If RA is equally required to induce pelvic fin development,
pelvic fin induction may either occur shortly after the formation of the
corresponding somites, or may be concomitant with the formation of the
mesenchymal layer present in the prospective pelvic area that is present
during the third week postfertilization
(Grandel and Schulte-Merker,
1998). In the latter case, a heterochronic shift in signaling from
RA alone or in conjunction with another signal may have enabled the delay in
pelvic fin development in zebrafish. Alternatively, RA-mediated induction may
not be in place for the hindlimbs. Appropriate RA rescue and inhibition assays
will have to be performed to test the extent of conservation in the molecular
mechanisms governing fore- and hindlimb induction.
RA is a permissive factor for fin development
Unlocalized exogenous RA rescues tbx5-expressing fin buds in
nls or RA-depleted embryos to an extent indistinguishable from wild
type (Fig. 1). RA thus cannot
be an instructive signal, but has to act as a permissive factor whose ectopic
presence does not interfere with fin formation.
As RA is sufficient to rescue fin induction in the absence of somitic
mesoderm, other somite-derived signals are not required to allow fin field
induction. This finding appears to be in disagreement with a previously
proposed role of wnt2b, which acts genetically upstream of tbx5.
wnt2b is expressed in the ventral part of somites flanking the pectoral
fin bud at 22 hpf (Ng et al.,
2002), several hours after fin induction. Morpholino-induced
inactivation of wnt2b leads to a failure of expression of
tbx5, as well as of fgf10 and fgf24, in the fin bud
mesenchyme, which can be rescued by injection of tbx5 mRNA
(Ng et al., 2002;
Fischer et al., 2003). Our
findings therefore imply that wnt2b may be expressed outside the
somites during pectoral fin field induction, in LPM or IM. Unfortunately,
wnt2b is only weakly expressed, hampering detection of expression
outside the somites or during the early segmentation period (not shown).
RA acts over a short range during pectoral fin induction
The somitic mesoderm and LPM are in close proximity during pectoral fin
field induction, separated by the IM. Diffusion of RA from forming somites
must therefore travel through IM or the ectoderm to the LPM. The occurrence of
one transplant with considerable contribution of wild-type cells to the dorsal
myotome that fails to rescue fin induction suggests that RA may diffuse over a
short distance only (Fig. 8).
In agreement with the temporal requirement of RA, aldh1a2-expressing
cells posterior to the eighth somite either emerge too late or are located too
far from the fin field for induction. Alternatively, the LPM may have lost the
competence to respond to RA once the embryo has passed the eight-somite
stage.
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; Begemann and Ingham,
2000; Ruvinsky et al.,
2000). The temporal requirement for RA signaling coincides with
the onset of tbx5 expression in the LPM, therefore RA may directly
induce tbx5 expression in the presumptive fin field. However, we have
been unable to identify sequences of putative binding sites for RA receptors
(RA-responsive elements) in the putative promoter region of human
Tbx5 (the genomic sequence of the zebrafish tbx5 region is
currently incomplete) that could mediate direct regulation of tbx5.
Agarwal et al. (Agarwal et al.,
2003) have proposed that in the mouse an axial signal may exist
that serves to support Tbx5 expression, or that might confer
competence to the LPM to induce forelimb bud outgrowth by Tbx5. Our
experiments in zebrafish suggest that RA is synthesized at the right time and
place to qualify as such a signal.
Limb initiation through axial signals
Axial signals that regulate limb development are likely to be derived from
paraxial or intermediate mesoderm (IM). The finding that tbx5
expression, and thus the presumptive fin field, is absent in ntl/spt
embryos, identifies somitic aldh1a2 expression, rather than
expression in the IM, as the earliest genetically confirmed source of an axial
signal that triggers fin field initiation.
A role of IM in limb induction is still debated. Experiments using foil
barriers and ablation of IM tissue in the chick embryo have suggested that the
IM provides important signals for limb development
(Stephens and McNulty, 1981;
Strecker and Stephens, 1983;
Geduspan and Solursh, 1992).
However, this view has been challenged by the observation that wing buds form
in experiments blocking chick mesonephric differentiation, and more
specifically IM-expressed FGF8 is dispensable for mouse limb induction
(Fernandez-Teran et al., 1997;
Boulet et al., 2004); finally,
the loss of IM cells does not affect normal development of limbs in
Pax2-/-/Pax8-/- mouse embryos
(Bouchard et al., 2002).
Although we did not observe transplanted wild-type cells in the IM of
nls mutants, the rescue of fin development by somites alone suggests
that IM-derived RA signaling is not required for pectoral fin initiation in
wild type.
Continuous RA signaling prior to fin outgrowth is required for AP-pre-patterning and apical fold formation
Taking advantage of the relative ease with which RA signaling can be
manipulated using exogenous agents in the zebrafish, we have uncovered that
following RA-mediated induction of the pectoral fin field at 12-13 hpf, RA
remains to be required continuously throughout the pre-budding stage to
establish posterior cell fates in the fin field. Importantly, this RA
dependency occurs several hours prior to the development of a visible
mesenchymal condensation and of apical fold formation. This is exemplified by
the absence of shh expression in fin buds devoid of RA prior to 22
hpf (Fig. 6M-P). As
aldh1a2 is not expressed within the fin field until a visible bud
emerges at 26-28 hpf, the strong expression of aldh1a2 in adjacent
somites is most likely to provide RA to the developing pectoral fins.
In addition, hoxc6a, a marker of the anterior pectoral mesenchyme,
is expanded posteriorly in RA-depleted fins. We interpret this as a
consequence of loss of shh, which is required to repress the
posterior expression of hoxc6a
(Neumann et al., 1999). By
contrast, posterior expression of hoxd11a and hoxd12a is
independent of shh (Neumann et
al., 1999), yet both genes are never expressed in fin buds lacking
RA. Similarly, hand2 is not expressed in the absence of RA, and
forelimbs in both zebrafish and mice that are mutant for
hand2/Hand2 resemble limbs that develop without RA, in that
they never express hoxd11a and hoxd12a in the fin mesenchyme
(Yelon et al., 2000) or
Shh in the limb bud
(Charité et al., 2000).
hand2 expression in the LPM has been implicated in regulating AP
prepatterning prior to the formation of the ZPA and is required for early
expansion of the presumptive fin field
(Yelon et al., 2000).
Similarly, in Aldh1a2-/- mutant mice, Hand2 is
not expressed in forelimbs, but expression can be rescued by RA
(Niederreither et al., 2002;
Mic et al., 2004). Our results
thus suggest that continued RA signaling from somites acts via hand2
expression to establish a prepattern in the fin field mesenchyme. This process
occurs during early somitogenesis and advocates a requirement of
somite-derived RA for the maintenance of hand2 expression in the LPM
(Fig. 8).
It has been suggested that Aldh1a2 expression in proximal limb
mesoderm of the mouse establishes a proximodistal gradient needed for AER
formation in the forelimb buds (Mic et
al., 2004). We find that zebrafish embryos blocked immediately
after the initiation stage (starting at 14 hpf) infrequently formed incomplete
apical folds, while fin buds that developed during a RA block starting at 16
hpf did not exhibit apical fold defects (not shown and
Fig. 6K,L). At this early
stage, somite-derived RA-signaling is unlikely to form a gradient in the fin
mesenchyme, and has been detected throughout the pre-budding mouse forelimb
(Mic et al., 2004). This
suggests that RA signaling is required between 14-16 hpf, when dlx2a
is first expressed in the fin field
(Akimenko et al., 1994), for
full AP extension of dlx2a expression in the mature apical fold.
Likewise, AER-formation in mammalian forelimbs is more likely to be a
consequence of early deficits in full RA signaling, rather than the failure to
establish a gradient of RA activity over the outgrowing limb bud. This notion
is further supported by the expression of zebrafish aldh1a2
throughout the entire fin bud mesenchyme during the outgrowth phase.
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Agarwal, P., Wylie, J. N., Galceran, J., Arkhitko, O., Li, C.,
Deng, C., Grosschedl, R. and Bruneau, B. G. (2003). Tbx5 is
essential for forelimb bud initiation following patterning of the limb field
in the mouse embryo. Development
130,623
-633.
Ahn, D. G., Kourakis, M. J., Rohde, L. A., Silver, L. M. and Ho, R. K. (2002). T-box gene tbx5 is essential for formation of the pectoral limb bud. Nature 417,754 -758.[CrossRef][Medline]
Akimenko, M. A., Ekker, M., Wegner, J., Lin, W. and Westerfield, M. (1994). Combinatorial expression of three zebrafish genes related to distal-less: part of a homeobox gene code for the head. J. Neurosci. 14,3475 -3486.[Abstract]
Amacher, S. L., Draper, B. W., Summers, B. R. and Kimmel, C. B. (2002). The zebrafish T-box genes no tail and spadetail are required for development of trunk and tail mesoderm and medial floor plate. Development 129,3311 -3323.
Begemann, G. and Ingham, P. W. (2000). Developmental regulation of Tbx5 in zebrafish embryogenesis. Mech. Dev. 90,299 -304.[CrossRef][Medline]
Begemann, G., Schilling, T. F., Rauch, G. J., Geisler, R. and Ingham, P. W. (2001). The zebrafish neckless mutation reveals a requirement for raldh2 in mesodermal signals that pattern the hindbrain. Development 128,3081 -3094.
Begemann, G., Gibert, Y., Meyer, A. and Ingham, P. W. (2002). Cloning of zebrafish T-box genes tbx15 and tbx18 and their expression during embryonic development. Mech. Dev. 114,137 -141.[CrossRef][Medline]
Begemann, G., Marx, M., Mebus, K., Meyer, A. and Bastmeyer, M. (2004). Beyond the neckless phenotype: influence of reduced retinoic acid signaling on motor neuron development in the zebrafish hindbrain. Dev. Biol. 271,119 -129.[CrossRef][Medline]
Berggren, K., McCaffery, P., Dräger, U. and Forehand, C. J. (1999). Differential distribution of retinoic acid synthesis in the chicken embryo as determined by immunolocalization of the retinoic acid synthetic enzyme, RALDH-2. Dev. Biol. 210,288 -304.[CrossRef][Medline]
Bouchard, M., Souabni, A., Mandler, M., Neubuser, A. and
Busslinger, M. (2002). Nephric lineage specification by Pax2
and Pax8. Genes Dev. 16,2958
-2970.
Boulet, A. M., Moon, A. M., Arenkiel, B. R. and Capecchi, M. R. (2004). The roles of Fgf4 anf Fgf8 in limb bud initiation and outgrowth. Dev. Biol. 273,361 -372.[CrossRef][Medline]
Capdevila, J. and Izpisua Belmonte, J. C. (2001). Patterning mechanisms controlling vertebrate limb development. Annu. Rev. Cell Dev. Biol. 17, 87-132.[CrossRef][Medline]
Charité, J., McFadden, D. G. and Olson, E. N. (2000). The bHLH transcription factor dHAND controls Sonic hedgehog expression and establishment of the zone of polarizing activity during limb development. Development 127,2461 -2470.[Abstract]
Chen, Y., Pollet, N., Niehrs, C. and Pieler, T. (2001). Increased XRALDH2 activity has a posteriorizing effect on the central nervous system of Xenopus embryos. Mech. Dev. 101,91 -103.[CrossRef][Medline]
Cohn, M. J., Izpisua-Belmonte, J. C., Abud, H., Heath, J. K. and Tickle, C. (1995). Fibroblast growth factors induce additional limb development from the flank of chick embryos. Cell 80,739 -746.[CrossRef][Medline]
Crossley, P. H., Minowada, G., MacArthur, C. A. and Martin, G. R. (1996). Roles for FGF8 in the induction, initiation, and maintenance of chick limb development. Cell 84,127 -136.[CrossRef][Medline]
Dupé, V. and Lumsden, A. (2001). Hindbrain patterning involves graded responses to retinoic acid signalling. Development 128,2199 -2208.
Ekker, M., Akimenko, M. A., Bremiller, R. and Westerfield, M. (1992). Regional expression of three homeobox transcripts in the inner ear of zebrafish embryos. Neuron 9, 27-35.
Emoto, Y., Wada, H., Okamoto, H., Kudo, A. and Imai, Y. (2005). Retinoic acid-metabolizing enzyme Cyp26a1 is essential for determining territories of hindbrain and spinal cord in zebrafish. Dev. Biol. 278,415 -427.[CrossRef][Medline]
Fernandez-Teran, M., Piedra, M. E., Simandl, B. K., Fallon, J. F. and Ros, M. A. (1997). Limb initiation and development is normal in the absence of the mesonephros. Dev. Biol. 189,246 -255.[CrossRef][Medline]
Fischer, S., Draper, B. W. and Neumann, C. J.
(2003). The zebrafish fgf24 mutant identifies an additional level
of Fgf signaling involved in vertebrate forelimb initiation.
Development 130,3515
-3524.
Garrity, D. M., Childs, S. and Fishman, M. C.
(2002). The heartstrings mutation in zebrafish causes heart/fin
Tbx5 deficiency syndrome. Development
129,4635
-4645.
Geduspan, J. S. and Solursh, M. (1992). A growth-promoting influence from the mesonephros during limb outgrowth. Dev. Biol. 151,242 -250.[CrossRef][Medline]
Gibson-Brown, J. J., Agulnik, S. I., Chapman, D. L., Alexiou, M., Garvey, N., Silver, L. M. and Papaioannou, V. E. (1996). Evidence of a role for T-box genes in the evolution of limb morphogenesis and the specification of forelimb/hindlimb identity. Mech. Dev. 56,93 -101.[CrossRef][Medline]
Goering, L. M., Hoshijima, K., Hug, B., Bisgrove, B., Kispert,
A. and Grunwald, D. J. (2003). An interacting network of
T-box genes directs gene expression and fate in the zebrafish mesoderm.
Proc. Natl. Acad. Sci. USA
100,9410
-9415.
Grandel, H. and Schulte-Merker, S. (1998). The development of the paired fins in the zebrafish (Danio rerio). Mech. Dev. 79,99 -120.[CrossRef][Medline]
Grandel, H., Lun, K., Rauch, G. J., Rhinn, M., Piotrowski, T., Houart, C., Sordino, P., Kuchler, A. M., Schulte-Merker, S., Geisler, R. et al. (2002). Retinoic acid signalling in the zebrafish embryo is necessary during pre-segmentation stages to pattern the anterior-posterior axis of the CNS and to induce a pectoral fin bud. Development 129,2851 -2865.
Kawakami, Y., Capdevila, J., Buscher, D., Itoh, T., Rodriguez Esteban, C. and Izpisúa-Belmonte, J. C. (2001). WNT signals control FGF-dependent limb initiation and AER induction in the chick embryo. Cell 104,891 -900.[CrossRef][Medline]
Keegan, B. R., Feldman, J. L., Begemann, G., Ingham, P. W. and
Yelon, D. (2005). Retinoic acid signaling restricts the
cardiac progenitor pool. Science
307,247
-249.
Kimmel, C. B., Kane, D. A., Walker, C., Warga, R. M. and Rothman, M. B. (1989). A mutation that changes cell movement and cell fate in the zebrafish embryo. Nature 337,358 -362.[CrossRef][Medline]
Kimmel, C. B., Warga, R. M. and Schilling, T. F.
(1990). Origin and organization of the zebrafish fate map.
Development 108,581
-594.
Kimmel, C. B., Ballard, W. W., Kimmel, S. R., Ullmann, B. and Schilling, T. F. (1995). Stages of embryonic development of the zebrafish. Dev. Dyn. 203,253 -310.[Medline]
Krauss, S., Johansen, T., Korzh, V. and Fjose, A. (1991). Expression of the zebrafish paired box gene pax[zf-b] during early neurogenesis. Development 113,1193 -1206.[Abstract]
Krauss, S., Concordet, J. P. and Ingham, P. W. (1993). A functionally conserved homolog of the Drosophila segment polarity gene hh is expressed in tissues with polarizing activity in zebrafish embryos. Cell 75,1431 -1444.[CrossRef][Medline]
Kudoh, T., Wilson, S. W. and Dawid, I. B. (2002). Distinct roles for Fgf, Wnt and retinoic acid in posteriorizing the neural ectoderm. Development 129,4335 -4346.
Linville, A., Gumusaneli, E., Chandraratna, R. A. and Schilling, T. F. (2004). Independent roles for retinoic acid in segmentation and neuronal differentiation in the zebrafish hindbrain. Dev. Biol. 270,186 -199.[CrossRef][Medline]
Logan, M. (2003). Finger or toe: the molecular
basis of limb identity. Development130,6401
-6410.
Majumdar, A., Lun, K., Brand, M. and Drummond, I. A. (2000). Zebrafish no isthmus reveals a role for pax2.1 in tubule differentiation and patterning events in the pronephric primordia. Development 127,2089 -2098.[Abstract]
Martin, G. R. (1998). The roles of FGFs in the
early development of vertebrate limbs. Genes Dev.
12,1571
-1586.
Melby, A. E., Warga, R. M. and Kimmel, C. B. (1996). Specification of cell fates at the dorsal margin of the zebrafish gastrula. Development 122,2225 -2237.[Abstract]
Mic, F. A. and Duester, G. (2003). Patterning of forelimb bud myogenic precursor cells requires retinoic acid signaling initiated by Raldh2. Dev. Biol. 264,191 -201.[CrossRef][Medline]
Mic, F. A., Sirbu, I. O. and Duester, G.
(2004). Retinoic acid synthesis controlled by Raldh2 is required
early for limb bud initiation and then later as a proximodistal signal during
apical ectodermal ridge formation. J. Biol. Chem.
279,26698
-26706.
Molotkov, A., Molotkova, N. and Duester, G. (2005). Retinoic acid generated by Raldh2 in mesoderm is required for mouse dorsal endodermal pancreas development. Dev. Dyn. 232,950 -957.[CrossRef][Medline]
Molven, A., Wright, C. V., Bremiller, R., De Robertis, E. M. and Kimmel, C. B. (1990). Expression of a homeobox gene product in normal and mutant zebrafish embryos: evolution of the tetrapod body plan. Development 109,279 -288.[Abstract]
Morriss-Kay, G. M. and Ward, S. J. (1999). Retinoids and mammalian development. Int. Rev. Cytol. 188,73 -131.[Medline]
Neumann, C. J., Grandel, H., Gaffield, W., Schulte-Merker, S. and Nusslein-Volhard, C. (1999). Transient establishment of anteroposterior polarity in the zebrafish pectoral fin bud in the absence of sonic hedgehog activity. Development 126,4817 -4826.[Abstract]
Ng, J. K., Kawakami, Y., Buscher, D., Raya, A., Itoh, T., Koth, C. M., Rodriguez Esteban, C., Rodriguez-Leon, J., Garrity, D. M., Fishman, M. C. et al. (2002). The limb identity gene Tbx5 promotes limb initiation by interacting with Wnt2b and Fgf10. Development 129,5161 -5170.
Niederreither, K., McCaffery, P., Dräger, U. C., Chambon, P. and Dolle, P. (1997). Restricted expression and retinoic acid-induced downregulation of the retinaldehyde dehydrogenase type 2 (RALDH-2) gene during mouse development. Mech. Dev. 62, 67-78.[CrossRef][Medline]
Niederreither, K., Vermot, J., Schuhbaur, B., Chambon, P. and
Dolle, P. (2002). Embryonic retinoic acid synthesis is
required for forelimb growth and anteroposterior patterning in the mouse.
Development 129,3563
-3574.
Odenthal, J., Haffter, P., Vogelsang, E., Brand, M., van Eeden, F. J., Furutani- Seiki, M., Granato, M., Hammerschmidt, M., Heisenberg, C. P., Jiang, Y. J. et al. (1996). Mutations affecting the formation of the notochord in the zebrafish, Danio rerio. Development 123,103 -115.[Abstract]
Ohuchi, H., Nakagawa, T., Yamauchi, M., Ohata, T., Yoshioka, H., Kuwana, T., Mima, T., Mikawa, T., Nohno, T. and Noji, S. (1995). An additional limb can be induced from the flank of the chick embryo by FGF4. Biochem. Biophys. Res. Commun. 209,809 -816.[CrossRef][Medline]
Rallis, C., Bruneau, B. G., Del Buono, J., Seidman, C. E.,
Seidman, J. G., Nissim, S., Tabin, C. J. and Logan, M. P.
(2003). Tbx5 is required for forelimb bud formation and continued
outgrowth. Development
130,2741
-2751.
Ruvinsky, I., Oates, A. C., Silver, L. M. and Ho, R. K. (2000). The evolution of paired appendages in vertebrates: T-box genes in the zebrafish. Dev. Genes Evol. 210, 82-91.[CrossRef][Medline]
Smith, D. M., Torres, R. D. and Stephens, T. D. (1996). Mesonephros has a role in limb development and is related to thalidomide embryopathy. Teratology 54,126 -134.[Medline]
Sordino, P., van der Hoeven, F. and Duboule, D. (1995). Hox gene expression in teleost fins and the origin of vertebrate digits. Nature 375,678 -681.[CrossRef][Medline]
Stafford, D. and Prince, V. E. (2002). Retinoic acid signaling is required for a critical early step in zebrafish pancreatic development. Curr. Biol. 12,1215 -1220.[CrossRef][Medline]
Stafford, D., White, R. J., Kinkel, M. D., Linville, A.,
Schilling, T. F. and Prince, V. E. (2006). Retinoids signal
directly to zebrafish endoderm to specify insulin-expressing
ß-cells. Development
133,949
-956.
Stephens, T. D. and McNulty, T. R. (1981). Evidence for a metameric pattern in the development of the chick humerus. J. Embryol. Exp. Morphol. 61,191 -205.[Medline]
Strecker, T. R. and Stephens, T. D. (1983). Peripheral nerves do not play a trophic role in limb skeletal morphogene
-tropomyosin-1 (tpm1)
expression (L,M) in RA-treated double mutants. Four rescue experiments, each
with 40 embryos, were performed (total numbers of phenotypes: 32 spt,
35 ntl, 11 ntl/spt, 82 siblings without mutant
phenotype).
