1 Gene Expression Laboratory, The Salk Institute for Biological Studies, La
Jolla, CA 92037-1099, USA
2 Unidade de Desenvolvimento, Instituto Gulbenkian de Ciencia, Rua Da quinta
Grande 6 Aptdo 14. 2780-901 Oeiras, Portugal
3 The Institute of Molecular and Cellular Biosciences, The University of Tokyo,
1-1-1 Yayoi, Bunkyo-ku, Tokyo, 113-0032, Japan
* Author for correspondence (e-mail: belmonte{at}salk.edu)
Accepted 28 June 2004
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SUMMARY |
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Key words: Apical ectodermal ridge, Sp transcription factor, Fgf8, Fgf10, Wnt, Vertebrate limb
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Introduction |
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Limb outgrowth requires the formation and maintenance of three different
signaling centers: the apical ectodermal ridge (AER) controls proximodistal
growth; the zone of polarizing activity, which is located in the posterior
mesenchyme is responsible for anteroposterior pattern formation; and the
non-ridge ectoderm directs formation of the dorsoventral axis. Their
coordinated action constructs the three-dimensional morphology of the limb
(reviewed by Capdevila and Izpisua
Belmonte, 2001; Niswander,
2003
; Tickle,
2002a
). Among these signaling centers in the limb bud, the AER,
which is a thickened epithelial structure positioned at the distal edge of the
limb bud at the dorsoventral boundary, is pivotal for maintaining limb
outgrowth. Surgical removal of the AER results in cell death in the mesenchyme
and abrogates limb outgrowth (Dudley et
al., 2002
; Rowe et al.,
1982
; Sun et al.,
2002
). The importance and necessity of the AER in limb outgrowth
is a conserved feature of vertebrate development as illustrated in mice, chick
and zebrafish (Grandel and Schulte-Merker,
1998
; Tickle,
2002b
). Despite recent extensive studies, the molecular and
genetic mechanisms that control initiation and maintenance of the AER in these
model organisms is far from being understood.
The morphogenesis of the AER can be divided into two processes.
(1) The induction of AER precursor cells in the surface ectoderm that will
migrate toward the dorsoventral boundary and form the AER. These cells start
to express fibroblast growth factor 8 (Fgf8), a member of the Fgf
superfamily that acts as an essential signaling molecule involved in
vertebrate limb outgrowth (Crossley et
al., 1996b; Lewandoski et al.,
2000
; Moon and Capecchi,
2000
; Vogel et al.,
1996
).
(2) Maturation of the AER that results in formation of the characteristic,
thickened structure (Loomis et al.,
1998). The initial induction of AER precursor cells depends on the
activity of Fgf10 emanating from the lateral plate mesoderm
(Min et al., 1998
;
Ohuchi et al., 1997
;
Sekine et al., 1999
). In the
absence of Fgf10, no Fgf8 expression is detected, indicating
that induction of the AER precursor cells does not take place, and the AER is
not formed.
In addition, ectodermal Wnt/ß-catenin signaling and Bmp signaling are
essential for induction of Fgf8 expression in AER precursors
(Ahn et al., 2001;
Barrow et al., 2003
;
Kengaku et al., 1998
;
Pizette et al., 2001
;
Soshnikova et al., 2003
). The
AER precursors migrate to the dorsoventral boundary, and form a structurally
distinguishable AER by apical compaction
(Loomis et al., 1998
).
Secreted from the AER, Fgf8 signals to the underlying mesenchyme, and Fgf10
produced by the distal mesenchyme in turn signals to the AER, resulting in
establishment of the reciprocal positive feedback loop that maintains the
expression of each one (Ohuchi et al.,
1997
; Xu et al.,
1998
). This positive-feedback loop, mediated by different splicing
isoforms of Fgfr2 (Arman et al.,
1999
; Ornitz et al.,
1996
), maintains the AER and limb outgrowth. Further studies have
shown that Wnt/ß-catenin activity is also required for maintaining
Fgf8 expression in the AER (Barrow
et al., 2003
; Kawakami et al.,
2001
; Soshnikova et al.,
2003
). Our current understanding of AER development implies that
the concerted signaling of these and possibly other growth factors leads to
the activation of different transcription factors that in turn elicit the
instructions that permit proper limb development.
Among the different transcription factors involved in these processes, it
was recently shown, by gene targeting analysis, that Sp8, a
buttonhead (btd)-like zinc (Zn) finger transcription factor
is required for maintaining, but not for initial induction of Fgf8
expression in AER precursor cells (Bell et
al., 2003; Treichel et al.,
2003
). The Sp family of transcription factors is united by a
particular combination of a C2H2 type Zn-finger DNA-binding domain and
btd domain (Bouwman and
Philipsen, 2002
; Philipsen and
Suske, 1999
). Eight Sp members have been identified in mouse and
human. Although the necessity of Sp8 for mouse limb outgrowth has
been reported, it is largely unknown how Sp8 interacts with the
previously mentioned signaling pathways to maintain limb outgrowth. For
example, although Fgf10, Wnt/ß-catenin and Sp8 are factors that are
required for AER maintenance and subsequent limb outgrowth, it is not clear
whether they are sufficient or if other factors are also required.
To gain further insights into the role of Sp genes during vertebrate limb development, we have performed several experiments. They include the isolation of a novel btd-like Zn-finger transcription factor, Sp9. Sp9 contains a btd domain and Zn-finger domain that are highly homologous to those of Sp8. Using mouse, chick and zebrafish embryos, we have studied the embryonic expression pattern, regulation and role of Sp8 and Sp9 during limb development. Both Sp8 and Sp9 are expressed in the AER, but regulated differently by Wnt and Fgf signaling. Loss- and gain-of-function approaches revealed that both Sp8 and Sp9 positively regulate Fgf8 expression in the AER and contribute to limb outgrowth in vertebrate embryos.
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Materials and methods |
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In situ hybridization and cartilage staining
Chick Sp8 and Sp9 probes were derived from a partial open
reading frame covering 3' to the Zn-finger domain and 3'UTR in
order to avoid cross hybridization. The mouse Sp8 probe contains 1 kb
of the 3'UTR and the Sp9 probe contains 750 bp covering the
5'UTR and partial ORF, 5' to the btd domain. Zebrafish
sp8 and sp9 probes are derived from 3'UTR sequences.
Chick Fgf8 and zebrafish fgf8 probes have been described
previously (Ng et al., 2002;
Vogel et al., 1996
). Zebrafish
prx1 was cloned by RT-PCR and confirmed by nucleotide sequencing
(Accession Number BC053228).
Chick and mouse embryos were examined by whole-mount in situ hybridization
and Alcian green cartilage staining as described
(Vogel et al., 1996).
Zebrafish embryos were examined by whole-mount in situ hybridization as
described (Ng et al.,
2002
).
Mutant mice and zebrafish
Mouse embryos deficient for Fgf10
(Min et al., 1998;
Sekine et al., 1999
),
Dkk1 (Mukhopadhyay et al.,
2001
) and Lrp6
(Pinson et al., 2000
) were
used. Zebrafish mutants, heartstrings (hst)
(Garrity et al., 2002
;
Ng et al., 2002
),
neckless (nkl) (Begemann
et al., 2001
) and dackel (dak)
(Grandel et al., 2000
) have
been described previously.
Viral production and injection into chick embryos
The full-length mouse Sp8 and Sp9 were subcloned into
RCAS BP(A) vector. In order to construct dominant-active and
-negative forms of Sp8 and Sp9, a part of the open reading
frame (from the btd domain to the termination codon; N-Sp8 and
N-Sp9) was fused to a VP16 activation domain (VP16-) or an
Engrailed-repressor domain (EnR-), and subcloned into the RCAS BP(A)
vector. The dominant-active ß-catenin clone has been described previously
(Capdevila et al., 1998
).
Retroviral production was performed as previously described
(Vogel et al., 1996
). Staging
of chick embryos was according to Hamburger and Hamilton (HH;
Hamburger and Hamilton, 1951
).
Prospective limb fields of chick embryos at HH stage 9-11 were infected with
the viruses. An RCAS BP(A)-alkaline phosphatase virus was used as a
control and no phenotypic changes in gene expression or limb morphology were
observed. The injected embryos were developed until desired stages and fixed
for analysis.
Bead and cell-pellet implantation
Heparin beads were soaked in Fgf10 (1 mg/ml) or Fgf8 (1 mg/ml). AG-X beads
were soaked in the Fgf receptor kinase inhibitor SU5402 (Calbiochem), at 2
mg/ml in DMSO. The beads were implanted into stage 19-21 developing chick limb
buds as described previously (Kawakami et
al., 2003). Chick embryonic fibroblasts were infected with
RCAS BP(A)-Wnt3a
(Kengaku et al., 1998
), and
implanted into limb buds as described previously
(Wada et al., 1999
). Control
beads soaked in PBS or DMSO and chick embryonic fibroblasts with RCAS
BP(A)-alkaline phosphatase were used at the same stage and no change in gene
expression was observed. The manipulated embryos were incubated for desired
periods and processed for in situ hybridization analysis.
Morpholino injections
Morpholino oligo nucleotides were designed by and obtained from GeneTools
LLC (Eugene, OR). The zebrafish sp8 morpholino lies from nucleotide
position 1 to +24, relative to the translation start site:
5'-TTTGTTACACGTCGCAGCCAACATG-3'.
The zebrafish sp9 morpholino sequence lies from nucleotide position 14 to +11, relative to the translation start site: 5'-CTATAAAACATAGCTGGCTTGTGTG-3'.
The standard control oligonucleotide available from GeneTools was used. The
morpholinos were solubilized in 1xDanieu's solution and injected into
one-cell stage zebrafish embryos at a range of 5-15 ng/embryo
(Ng et al., 2002).
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Results |
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The highly homologous Zn finger domain made it difficult to compare amino acid sequences of Sp8 and Sp9 with other Sp family members within the domain (Fig. 1B). Phylogenetic analysis of deduced amino acid sequences of mouse Sp1-Sp9 together with two Drosophila Sp family members, btd and Sp1 revealed that mouse Sp8 and Sp9 are closely related to Drosophila Sp1 (Fig. 1A). Sp8 contains characteristic Ser-rich, Ala-rich and Gly-rich stretches in its N-terminal domain. The zebrafish Sp8 is slightly different from the other Sp8 sequences in its N-terminal region, and is shorter than the others (Fig. 1C). The Sp9 sequence contains Ser-rich and Ala-rich domains in its N-terminal region; however, it does not contain the Gly-rich domain found in Sp8. Both Sp8 and Sp9 contain a Gly-rich sequence in the region 3' to the Zn-finger domain. In all of the Sp8 and Sp9 sequences analyzed in this study, the amino acid sequences from the btd domain to the Zn-finger domain are identical except for one amino acid (Fig. 1B). The high degree of conservation of amino acid sequences among different species hints at their importance in vertebrate evolution.
Expression pattern of Sp8 in chick, mouse and zebrafish embryos
In order to understand the roles of Sp8 and Sp9, we first
decided to analyze their embryonic expression pattern in chick, mouse and
zebrafish embryos by in situ hybridization.
Sp8 expression was detected in early stages of chick embryos as an
oval and 2 stripes at HH stage 5 (Fig.
2A), which became two lateral stripes at HH stage 7, running along
the anteroposterior body axis from the head ectoderm to the anterior region of
the primitive streak (Fig. 2B).
At HH stage 8, strong expression in the anterior neuroectoderm, which forms
the central nervous system, was observed
(Fig. 2C), and at HH stage 9,
Sp8 is expressed in the most anterior region of the forebrain,
midbrain, neural groove and Hensen's node
(Fig. 2D). The expression in
the midbrain was later confined to the midbrain/hindbrain boundary, a
signaling center that controls midbrain development
(Fig. 2G)
(Chi et al., 2003;
Crossley et al., 1996a
;
Lee et al., 1997
). At HH stage
15-16, the expression of Sp8 was broadly observed in the surface
ectoderm of the limb-forming fields (Fig.
2E,F, arrowheads), in addition to the neural tube. The signal in
the neural tube marks proliferating neural cells, and is excluded from the
dorsal and ventral region (Fig.
2F). The expression in the limb field became confined to the
distal region of the limb at HH stage 16 and 17, with still scattered signal
visible in the surface ectoderm of the limb bud
(Fig. 2H,I). Sp8 is
strongly expressed in the AER and weakly in the ectoderm in the developing
limb bud at HH stage 21 (Fig.
2J,K). At this stage, the neural tube expression was restricted to
proliferating interneurons with a reversed triangle shape, and excluded from
the dorsal-most and ventral-most regions
(Fig. 2L). The expression in
the AER was detected throughout later stages of limb development
(Fig. 2M).
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Overall, the expression pattern of Sp8 is conserved among the different vertebrate species studied. In particular, the expression in the limb buds, forebrain, midbrain/hindbrain boundary and neural tube suggests that Sp8 has a conserved role in the development of these structures during mouse, chick and zebrafish embryogenesis.
Expression pattern of Sp9 in chick, mouse and zebrafish embryos
Sp9 expression was not detected in early stages of chick
development (HH stage 3-7). A strong signal was detected at HH stage 8 in the
neural groove and anterior part of the regressing Hensen's node
(Fig. 3A). Sp9
expression showed a restricted pattern in the nervous system, like
Sp8, but is excluded from the forebrain
(Fig. 3B-D). The expression was
detected in the anterior hindbrain (Fig.
3B,C), which will be confined to the midbrain/hindbrain boundary
at HH stage 13 (Fig. 3D). In
the developing limb, it is expressed in the AER and weakly in the distal
surface ectoderm (Fig. 3E-G).
Unlike Sp8, Sp9 is expressed in a small area in the anterior border
at HH stage 27 and later (Fig.
3H). After HH stage 28, expression in the AER disappears, and the
transcripts start to be detected in the anterior and posterior edges of the
autopod, but are excluded from the distal edge
(Fig. 3I).
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Mutant analysis indicates that expression of Sp8 and Sp9 correlates with proper limb outgrowth
It has recently been reported that Sp8 has a role in maintaining
Fgf8 expression and limb outgrowth in mice
(Bell et al., 2003;
Treichel et al., 2003
). In
order to examine whether Sp9 also has a role in limb development, as
well as to examine possible molecular and genetic interactions of Sp8
and Sp9 with known signaling pathways involved in limb development,
we made use of zebrafish pectoral fin mutants. We initially analyzed
hst mutants, animals that bear a point mutation in the
tbx5-coding sequence that results in a loss of function mutation
(Garrity et al., 2002
;
Ng et al., 2002
). Tbx5 is a
mesenchymal factor required for limb bud initiation and outgrowth as an
upstream regulator of fgf10 in the pectoral fin field
(Ahn et al., 2002
;
Ng et al., 2002
). We observed
significant downregulation of sp8 and sp9 expression in
pectoral fin buds of hst mutants at 40 hpf
(Fig. 4B,E), when compared with
wild-type embryos (Fig. 4A,D).
This result places sp8 and sp9 downstream of tbx5
in limb development, and suggests that sp9 as well as sp8
must play a role in normal limb outgrowth. Similar results were obtained by
using nkl mutant embryos (data not shown), which carry a mutation in
the raldh2 gene (Begemann et al.,
2001
). Given that nkl mutation lies upstream of tbx5
function in the pectoral fin formation, it further confirms that sp8
and sp9 are ectodermal factors downstream of mesenchymal signals.
|
These analyses show that expression of sp8 and sp9 correlates with fin/limb development in zebrafish, and defects in fin/limb development are associated with their downregulation. The specific mutations in hst and nkl indicates that both sp8 and sp9 are ectodermal factors downstream of tbx5, a mesenchymal factor required for proper limb outgrowth.
Fgf10 signaling regulates both Sp8 and Sp9 expression
The Wnt and Fgf signaling pathways are two of the major pathways that
positively control Fgf8 expression in the AER (reviewed by
Kato and Sekine, 1999;
Martin, 1998
;
Tickle and Munsterberg, 2001
;
Yang, 2003
). We therefore
wanted to investigate whether the expression of Sp8 and Sp9
is regulated by these two signaling pathways.
Fgf10 is essential for inducing and maintaining Fgf8 expression and AER formation during limb development. In order to analyze whether Fgf signaling also regulates expression of Sp8 and Sp9, we first analyzed their expression in Fgf10/ mouse embryos. We observed downregulation of Sp8 and Sp9 in embryos lacking Fgf10 (Fig. 5A-D). This result provides genetic evidence that initial induction of Sp8 and Sp9 in the ectoderm depends on Fgf10 signaling from the lateral plate mesoderm, and further confirms the result obtained by zebrafish pectoral fin mutant analysis (Fig. 4).
|
Wnt signaling regulates Sp8, but not Sp9 expression
Wnt3a, which signals through the ß-catenin pathway, is
expressed in the AER precursors and the established AER in the developing
chick limb, and has been demonstrated to regulate Fgf8 expression
(Kawakami et al., 2001;
Kengaku et al., 1998
). In
order to examine the role of the Wnt/ß-catenin signaling pathway on
Sp8 and Sp9 expression, we implanted cells expressing
Wnt3a into the anterior distal tip of the chick limb bud at HH stage
19-21. This manipulation resulted in induction of ectopic expression of
Sp8 in the limb ectoderm in close proximity to the implanted cell
pellet, at 6 and 12 hours post-implantation (n=2/6 at 6 hours, and
n=5/6 at 12 hours; Fig.
6A,B). Moreover, retrovirus-mediated overexpression of
dominant-active ß-catenin resulted in ectopic induction of Sp8
in a broad region of the chick limb ectoderm (n=5/5;
Fig. 6C), which was associated
with the formation of ectopic ridge-like structures
(Fig. 6D). Contrary to what was
observed with Sp8, we did not observe significant changes in the
expression of Sp9 in the limb ectoderm after the same experiments
were performed (data not shown).
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Sp8 and Sp9 regulates Fgf8 expression and limb outgrowth in vertebrates
To further investigate the roles of Sp8 and Sp9 during
limb development, we used gain- and loss-of-function approaches by using
viral-mediated expression in chick embryos. First, we overexpressed
full-length Sp8 and Sp9 throughout the entire limb bud, and
observed expansion of the AER marked by an expanded Fgf8-expressing
domain (Fig. 7A,B). This
phenotype was observed after overexpressing both Sp8 and Sp9
(n=3/17 and 10/60, for Sp8 and Sp9, respectively).
This result indicates that Sp8 and Sp9 can positively
regulate Fgf8 expression and AER morphology.
|
Next, we tried to examine the roles of Sp8 and Sp9 by a
loss-of-function approach. Injection of
RCAS-EnR-N-Sp8 and
RCAS-EnR-
N-Sp9 produced similar phenotypes, resulting
in significant downregulation of Fgf8 expression in the AER
(n=3/15 and n=11/28 for EnR-
N-Sp8
and EnR-
N-Sp9, respectively;
Fig. 7E,F). This downregulation
of Fgf8 could lead to a partial loss of the AER, which was associated
with an indentation phenotype in
RCAS-EnR-
N-Sp8-injected embryos
(Fig. 7G,H). Correlating with
hypoplasia of the AER, we observed a variety of skeletal defects at later
stages in RCAS-EnR-
N-Sp8 or
RCAS-EnR-
N-Sp9 injected embryos. In the most severe
cases, we observed a small projection (Fig.
7K). Where limbs should have formed, small cartilaginous rudiments
were observed. The shoulder griddle, which is not formed from the limb bud,
appears normal. In the milder phenotypes, even though the limb was formed, it
lacked several skeletal elements. Consistent with an indentation phenotype
(Fig. 7H),
Fig. 7L shows a phenotype
lacking the radius and digit II. These results strongly support that both
Sp8 and Sp9 may act to maintain the expression of key
signaling molecules such as Fgf8, and the AER during limb
development.
Because the frequency of phenotype is not very high (10-39%), we carefully examined virus infection using an RCAS probe in embryos injected with the aforementioned viruses. In 54-64% of embryos analyzed (n=28-44), we observed widely spread infection of RCAS viruses in the ectoderm, and widely spread strong mesenchymal infection was observed in 25-36% of the embryos. Some embryos showed a phenotype with only infection to the ectoderm (data not shown). This analysis, together with lack of phenotype produced by control RCAS virus (e.g. RCAS-alkaline phosphatase, n=60), indicates that the phenotype observed was specific for each virus injection experiment.
The high degree of amino acid sequence conservation between Sp8
and Sp9 raised the possibility that the VP16- and EnR-fusion
constructs used, may act redundantly, and the phenotype observed could be not
specific for Sp8 or Sp9. To address this possibility, and
also to avoid redundancy between Sp8 and Sp9, we performed
knock-down experiments in the zebrafish using morpholinos. The effects of
sp8 and sp9 morpholinos on the development of the pectoral
fin were analyzed using the fgf8 and prx1 markers.
prx1 is known to be expressed broadly in the chick limb bud
mesenchyme in an AER-independent manner
(Nohno et al., 1993), and was
used to visualize the morphology of the developing pectoral fin in this study.
Injection of either sp8 morpholino or sp9 morpholino
resulted in downregulation of fgf8 expression in the apical fold
(Fig. 7M-O). As summarized in
Table 1, we observed complete
loss of fgf8 expression in 10% of sp9 morphants and very
faint fgf8 signal in 55% of morphants, resulting in fgf8
downregulation in a total of 65% of sp9 morphants at 36 hpf.
sp8 morphants showed milder phenotypes, and fgf8 expression
was downregulated in 33% of the sp8 morphants.
Fig. 7 shows the typical `faint
signal' phenotype, in which the signal is nearly invisible
(Fig. 7M-O). Consistent with
downregulation of fgf8 expression, injection of either sp8
morpholino or sp9 morpholino resulted in interfering with pectoral
fin outgrowth as visualized by expression of prx1
(Fig. 7P-R). Moreover, we
observed a synergistic effect of sp8 morpholino and sp9
morpholino on the expression of fgf8. Co-injection of sp8
morpholino and sp9 morpholino at a lower dose (5 ng each) resulted in
downregulation of fgf8 expression in
80% of morphants.
Increasing the amount of morpholinos (10 ng each) produced higher efficiency
of downregulating fgf8 expression, and fgf8 expression was
completely abolished in more than half of morphants.
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Discussion |
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Evolution of Sp gene family
As previously demonstrated, the release of the mouse and human genome
sequences revealed that Sp genes are arranged in a paired manner on
chromosomes; Sp1-Sp7, Sp2-Sp6 and
Sp4-Sp8. Our analysis identified Sp9 in close
proximity to and in an opposite direction to Sp3. Based upon the
arrangement of Sp genes on chromosomes, it is likely that a single primordial
gene underwent a tandem duplication event and produced progenitor genes for
the Sp1-Sp4 subfamily and Sp6-Sp9 subfamily. Then
whole-cluster duplication(s) might have taken place to generate four Sp
clusters. This is also supported by the proposal that the diversity of the
amino acid sequences outside the Zn-finger domain were created by gene
duplication (Kolell and Crawford,
2002). A similar scenario to gene evolution has been proposed for
the Tbx2 subfamily genes, Tbx2-Tbx5
(Agulnik et al., 1996
). Our
finding that Sp5 is not linked to other Sp genes supports a
previously proposed evolutionary mechanism of Sp genes, in which Sp5
might be an evolutionary link between the Sp family and KLF family,
another Zn finger factor family whose primary structure of the Zn finger
domain is related to that of Sp family but lacks the btd domain
(Ravasi et al., 2003
;
Treichel et al., 2001
).
Sp8 and Sp9 are differentially regulated by Fgf and Wnt signaling
While necessity of Sp8 for maintaining Fgf8 expression in
mice has recently been demonstrated, its placement in the genetic cascade that
permits normal limb outgrowth and possible additional roles remained unknown.
Furthermore, our identification of Sp9, a novel btd-like
gene generated a new question: does Sp9 have a similar or distinct
role from Sp8? In order to try to address these issues and to gain
insights into the mechanisms of Sp8 and Sp9 action, we
analyzed their possible regulation by different signaling pathways known to
play a key role during vertebrate limb development.
Genetic and embryological analyses have revealed that Fgf10-Fgfr2b in
tandem is pivotal for inducing the expression of Fgf8 in the AER
precursor cells, migration of AER precursors from the surface ectoderm to the
dorsoventral boundary, and formation of the AER
(Gorivodsky and Lonai, 2003;
Min et al., 1998
;
Ohuchi et al., 1997
;
Sekine et al., 1999
). This
also maintains expression of Fgf8 in the AER in the established limb
(Arman et al., 1999
;
Xu et al., 1998
). Our analysis
using Fgf10/ embryos, as well as the
manipulation of chick embryos, revealed that both Sp8 and
Sp9 are ectodermal targets of Fgf10 signaling emanating from the
mesenchyme during initiation and outgrowth of the limb bud
(Fig. 5). This is supported by
zebrafish mutant analysis, where retinoic acid and tbx5 lie upstream
of the fgf10 signaling cascade in the mesenchyme
(Begemann et al., 2001
;
Garrity et al., 2002
;
Ng et al., 2002
). In these
mutant embryos, sp8 and sp9 are significantly downregulated
(Fig. 4), which further
supports the fact that the regulation of sp8 and sp9 by
mesenchymal signals is a conserved feature during vertebrate evolution.
In the ectoderm, Wnt/ß-catenin signaling is known to be a crucial
factor for induction and maintenance of Fgf8 (reviewed by
Yang, 2003). Unlike Fgf10,
however, we observed differential regulation of Sp8 and Sp9
by Wnt/ß-catenin signaling in both chick and mouse embryos
(Fig. 6). Although we did not
observe alteration of Sp9 expression, Sp8 expression was
positively regulated by Wnt/ß-catenin signaling. The fact that Fgf10
signaling and Wnt/ß-catenin signaling can induce Fgf8 in the
ectoderm raised the possibility that activation of Sp8 might be
mediated through Fgf8 protein (Barrow et
al., 2003
; Kawakami et al.,
2001
; Kengaku et al.,
1998
; Ohuchi et al.,
1997
). This, however, does not seem to be the case, as exogenously
applied Fgf8 could not induce Sp8 and Sp9, while Fgf10
could. Our molecular and genetic analysis positions Sp8 as a
downstream factor of Fgf10 and Wnt/ß-catenin, while Sp9 is
placed downstream of Fgf10, but independent of Wnt/ß-catenin.
Sp8 and Sp9 regulate Fgf8 expression in the limb development
Our gain- and loss-of-function analyses have unveiled a role of
Sp9 and a new role of Sp8 as positive regulators for
Fgf8 expression and AER formation. Our results with viral constructs
of full length and VP16-fused forms of Sp8 and Sp9 strongly
suggest that both genes are able to activate Fgf8 expression as
transcriptional activators (Fig.
7). This is also supported by the complementary results obtained
using EnR fusion constructs and morpholinos. The fact that the proximal region
of the Fgf8 gene is GC-rich and includes several copies of consensus
Sp1-recognition sequences (Brondani et
al., 2002) further suggests that Sp8 and Sp9
directly regulate Fgf8 expression in the limb.
During mouse embryogenesis, the precursors of the AER originate in the
ventral ectoderm (Kimmel et al.,
2000; Loomis et al.,
1998
). In the chick, these cells are distributed in the wide range
of the surface ectoderm, both dorsally and ventrally
(Altabef et al., 1997
;
Michaud et al., 1997
).
Interestingly, the distribution of Sp8 transcripts at the time of
Fgf8 induction correlates with the appearance of the AER precursors.
Expression of Sp8 is detected in the AER precursors with a ventrally
biased manner in mouse embryos (Fig.
2P,Q) (Bell et al.,
2003
; Treichel et al.,
2003
), and in a wide region of the surface ectoderm in chick
(Fig. 2E,F). It has been
demonstrated that Fgfr2b, the high-affinity receptor for Fgf10, is
expressed widely in the surface ectoderm
(Arman et al., 1999
). The
expression pattern of Sp8, together with the ability of Sp8
and Sp9 to induce Fgf8 expression suggests that Sp8
contributes to the initial induction of Fgf8 in mouse and chick.
Consistent with this, lower levels of Fgf8 expression in the
limb-forming area were observed in Sp8/
embryos at E9.5, when Fgf8 expression becomes evident in the
limb-forming field (Treichel et al.,
2003
). The initial expression of Fgf8 was not abolished
completely in Sp8/ embryos; however, this
could be due to the redundant activity of Sp9. It will therefore be
interesting to analyze double mutants of Sp8 and Sp9.
Our data not only support the previously shown requirement of Sp8 in the expression of Fgf8 and limb outgrowth, but also demonstrate that Sp9 is required for Fgf8 expression and limb outgrowth. Our experiment with sequence-specific morpholinos excludes the possibility of redundant activity of the EnR fusion constructs (Fig. 7M-R). Therefore, the fact that downregulation of either Sp8 or Sp9 is sufficient to downregulate Fgf8 expression and limb outgrowth, as well as the synergistic effects produced by co-injection of sp8 morpholino and sp9 morpholino on fgf8 expression in zebrafish, strongly support the argument that these Sp factors may cooperate during normal limb outgrowth. A similar scenario could take place in other regions of the embryo where both genes are co-expressed, such as the midbrain/hindbrain boundary.
Conclusion
Vertebrate limb outgrowth requires proper activity of the AER. As such,
induction and maintenance of the AER is a central issue for limb outgrowth and
morphogenesis. Molecular and genetic approaches have revealed that Fgf10,
Wnt/ß-catenin and Sp8 are crucial factors for these
processes. Our studies have identified a novel btd-like transcription
factor Sp9. Both Sp8 and Sp9 are regulated by
Fgf10, and Sp8 is additionally regulated by Wnt/ß-catenin
signaling. Furthermore, our results indicate that Sp8 and
Sp9 mediate the induction and maintenance of Fgf8 expression
in the AER precursors and in the established AER, allowing proper limb/fin
outgrowth in mouse, chick and zebrafish.
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ACKNOWLEDGMENTS |
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