Howard Hughes Medical Institute, Department of Pharmacology and Center for Developmental Biology, Box 357750, University of Washington School of Medicine, Seattle, WA 98195, USA
* Author for correspondence (e-mail: rtmoon{at}u.washington.edu)
Accepted 2 February 2005
![]() |
SUMMARY |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: Sp1, Tail development, Wnt, Zebrafish
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Much of our current understanding of the role for Wnt/ß-catenin
signaling in posterior mesoderm formation in vertebrates comes from analysis
of mouse mutants. For example, mice homozygous for a null allele of
Wnt3a fail to form somites and notochord caudal to the forelimb bud,
instead making ectopic neural tissue
(Greco et al., 1996;
Takada et al., 1994
;
Yoshikawa et al., 1997
). These
embryos form only the anterior-most seven to nine somites and completely lack
a tailbud (Takada et al.,
1994
). Mice doubly homozygous for mutations in two downstream
effectors of the Wnt pathway, Lef1 and Tcf1, or homozygous
for a null allele of the Wnt co-receptor, LRP6, show a similar phenotype
(Galceran et al., 1999
;
Pinson et al., 2000
).
Studies in zebrafish have also implicated Wnt/ß-catenin signaling at
an early step in posterior mesoderm formation. Transplantation studies show
that the ventral marginal region of the zebrafish gastrula functions as a tail
organizer, able to induce the formation of an ectopic tail when transplanted
to the animal pole (Agathon et al.,
2003). In the same study, overexpression experiments indicated
that a combination of high levels of Wnt/ß-catenin, Nodal and BMP
signaling can induce the formation of ectopic tail organizers
(Agathon et al., 2003
).
wnt8 is highly expressed at the ventral margin and has been shown
to be required for specification of ventrolateral mesoderm, which contributes
to formation of the tailbud at the end of gastrulation
(Erter et al., 2001;
Lekven et al., 2001
). Strong
loss of wnt8 function, either by mutation or by antisense morpholinos
(MOs), results in tail formation defects
(Agathon et al., 2003
;
Lekven et al., 2001
). These
defects are presaged during gastrulation by a dramatic loss of expression of
ventrolateral mesoderm markers such as the T-box transcription factor
tbx6, which is strongly expressed in the ventral margin and
subsequently in the tailbud (Griffin et
al., 1998
; Hug et al.,
1997
), suggesting that wnt8 acts at a very early step of
tail formation.
While Wnt3a in the mouse and wnt8 in the zebrafish
clearly act at early steps in mesoderm induction and/or patterning that
subsequently affect tail development, whether Wnts also act later during tail
development is less clear. In zebrafish, the continued expression in the
tailbud throughout somitogenesis of both wnt8 and another Wnt gene,
wnt3a, as well as of a transgenic ß-catenin-responsive reporter,
TOPdGFP, suggests a role for continuous Wnt activity throughout tail formation
(Buckles et al., 2004;
Kelly et al., 1995
;
Lekven et al., 2001
).
Relatively little is known about the identity of downstream effectors of
Wnt/ß-catenin signaling in tail development in any species. One direct
transcriptional target of Wnt3a in the mouse is the
T(Brachyury) gene, a T-box transcription factor that is also required
for posterior development (Galceran et
al., 2001; Yamaguchi et al.,
1999
). In zebrafish, the homolog of T, no tail, is
required for tail formation (Halpern et
al., 1993
), but has not been shown to be a Wnt target. Conversely,
while tbx6 is directly regulated by Wnt signaling in zebrafish
(Szeto and Kimelman, 2004
), it
has not been demonstrated to have a functional role in tail development.
We demonstrate that Wnt/ß-catenin signaling, activated by wnt3a and wnt8, is required not only during gastrulation for specification of the tail organizer, but also during early somitogenesis for the maintenance of expression of presomitic mesoderm markers within the tailbud. We then show that the Sp1-related zinc finger transcription factor sp5-like (sp5l), is expressed in response to wnt3a and wnt8 in the tailbud and acts downstream of these Wnts to regulate tail formation. Thus, Wnt signaling is required at multiple stages of tail development, acting at least in part by activating expression of sp5l.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In situ hybridizations and antibody staining
In situ hybridizations using digoxigenin-labeled mRNA probes were performed
using standard methods (Oxtoby and Jowett,
1993). For assessing the mitotic index in the tailbud, we first
processed the embryos for tbx6 expression using a Fast Red color
reaction, which produces a fluorescent product. Subsequently, embryos were
incubated overnight in a 1:500 dilution of polyclonal rabbit
anti-phosphohistone H3 antibody (Upstate, Charlottesville, VA, USA) in PBT+10%
calf serum, washed 5 x in PBT, and incubated overnight in a 1:1000
dilution of AlexaFluor 488 anti-rabbit secondary antibody (Molecular Probes,
Eugene, OR USA). Mitotic cells within the tbx6 domain were counted
and divided by the total number of tbx6-expressing cells.
Morpholino and RNA injections
wnt3a, wnt8 ORF1, wnt8 ORF2 and sp5l/spr2
morpholinos (Gene Tools, Philomath, OR, USA) have been previously described
(Buckles et al., 2004;
Lekven et al., 2001
;
Zhao et al., 2003
). For sense
RNA injections, mRNA was synthesized using the mMessage mMachine kit (Ambion).
Both mRNA and morpholinos were diluted in Danieau's buffer prior to injection.
wnt3a MO was injected at a concentration of 2 mg/ml, except as noted
in some co-injection experiments with sp5l MO, where the
concentration was 1 mg/ml. wnt8 ORF1 and ORF2 MOs were injected at a
concentration of 0.5 mg/ml each, and sp5l MO was injected at a
concentration of 2.5 mg/ml. In all experiments, a volume of 3-5 nl was
injected into the yolk of one-cell stage embryos.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
During gastrulation, wnt8 is required for dorsoventral mesoderm
patterning and for promoting posterior neural fates in the neurectoderm
(Erter et al., 2001;
Lekven et al., 2001
). Since
wnt3a, like wnt8, is expressed in the margin during
gastrulation (data not shown), we tested whether wnt3a also plays a
role in patterning of the mesoderm and neurectoderm by co-injecting
wnt3a and wnt8 MOs and examining the expression of marker
genes. We found that while wnt3a is not required by itself for either
of these processes, wnt3a knockdown modestly enhances the effects of
partial loss of wnt8 function in both dorsoventral patterning of the
mesoderm and anterior-posterior patterning of the neurectoderm (see Fig. S1 in
supplementary material). Thus, while wnt8 is principally responsible
for these two early patterning events, wnt3a also plays a more minor,
redundant role. Consistent with this possibility, when higher doses of both
wnt3a and wnt8 morpholinos were injected, nearly all embryos
were strongly dorsalized (data not shown). Since we wanted to specifically
address a later role for wnt3a and wnt8 in tail development,
all further experiments were done using a hypomorphic dose of wnt8
MOs to minimize early mesoderm patterning defects.
Wnt signaling is required during somitogenesis for maintenance of presomitic mesoderm
To gain further insight into the mechanism by which wnt3a and
wnt8 promote tail development, we examined the expression of several
markers expressed in the tailbud at multiple time points from bud stage
through early somitogenesis (Fig.
2). In both wnt3a
(Fig. 2B,F) and wnt8
(Fig. 2C,G) morphants,
expression of ntl and fgf8 is unaffected relative to
wild-type (Fig. 2A,E) at bud
stage. tbx6 is expressed normally in wnt3a morphants
(Fig. 2J, compare with
wild-type in I), and is slightly reduced in wnt8 knockdown embryos
(Fig. 2K). Despite the much
more severe tail phenotype in wnt3a/wnt8 morphants, ntl,
fgf8 and tbx6 are all expressed at bud stage
(Fig. 2D,H,L), with
tbx6 slightly reduced, as seen in wnt8 morphants. The
relative lack of effect of the wnt3a and wnt8 MOs on the
expression of these genes was somewhat surprising, since expression of the
TOPdGFP reporter is dramatically reduced at this stage in wnt3a/wnt8
morphants (Fig. 1J), suggesting
that even low levels of Wnt signaling are sufficient for initial specification
of the tailbud. We conclude that the block in tail development apparent by
mid-somitogenesis in wnt3a/wnt8 morphants is not due to a failure to
specify early tailbud fates.
|
In the chick and mouse, Fgf signaling has been proposed to play a similar
anti-differentiation role, such that higher levels of Fgf in the caudal
tailbud inhibit the differentiation of presomitic mesoderm, and as cells move
more anteriorly within the tailbud, they escape the influence of Fgf,
differentiate, and form somites (Dubrulle
et al., 2001; Dubrulle and
Pourquie, 2004
; Mathis et al.,
2001
). Since these data support a role for Fgf signaling in
maintaining posterior cells in an undifferentiated state, we asked whether the
failure to maintain presomitic fates in wnt3a/wnt8 morphant embryos
was due to a defect in Fgf signaling. Although we had already observed normal
expression of fgf8 in the tailbud (see above), at least three
additional Fgf ligands, fgf17b, fgf3 and fgf24, are also
known to be expressed in this region (Cao
et al., 2004
; Draper et al.,
2003
; Phillips et al.,
2001
).
To test whether Fgf signaling is compromised in the tailbud of
wnt3a/wnt8 embryos, we examined expression of sprouty4
(Fig. 3), a Fgf-induced
inhibitor of the Fgf receptor that is expressed in the tailbud in response to
Fgf activity (Furthauer et al.,
2001). At both 5- and 10-somite stages, we observe robust
expression of sprouty4 in the tailbuds of wnt3a/wnt8
morphants (Fig. 3D,H). In
particular, at the 10-somite stage, when markers of presomitic mesoderm are
completely absent or severely reduced (above,
Fig. 2), sprouty4
expression, and thus Fgf signaling, is still observed. These data indicate
that a loss of Fgf signaling is not responsible for the failure to maintain
presomitic mesoderm in wnt3a/wnt8 morphants, and suggests a direct
role for Wnt/ß-catenin signaling in maintaining expression of presomitic
mesoderm markers in the tailbud.
|
wnt3a and wnt8 are required for notochord and somite formation during tail development
The mouse Wnt3a knockout results in a severe early phenotype, with
a loss of notochord and somites caudal to the forelimb bud, such that most
mutant embryos make only 7-8 somites
(Takada et al., 1994). Cells
involuting through the primitive streak that would normally contribute to
somitic tissue instead adopt a neural fate and form an ectopic neural tube
(Yoshikawa et al., 1997
).
These results in the mouse raise the question of whether Wnt loss of function
in zebrafish might result in similar fate transformations. Close examination
of wnt3a morphants at 28 hpf shows an absence of notochord near the
end of the tail (Fig. 4B,
arrow; 91% of embryos have premature truncation of the notochord,
n=253, compared with wild-type;
Fig. 4A), indicating that
wnt3a is required for late notochord development. In contrast, we
observe no notochord defects in the tail of wnt8 morphants
(Fig. 4C). While most
wnt3a/wnt8 morphants completely lack or make only a rudimentary tail,
the occasional weakly affected embryos have an even more penetrant and severe
loss of notochord in the tail than that seen in wnt3a morphants
(Fig. 4D, arrow; 100% of
embryos have a notochord phenotype, n=78). We conclude that
wnt3a and wnt8 both function to specify notochord during
tail outgrowth, with wnt3a playing the central role.
|
Since mouse Wnt3a is required for production of somites as well as
notochord (Takada et al.,
1994), we next investigated whether somites were properly formed
in the tail of wnt3a, wnt8 or wnt3a/wnt8 morphants. Since
most wnt3a/wnt8 morphants make essentially no tail, we examined more
weakly affected embryos that made a small tail. To assess the production of
somites, we stained 26 hpf embryos for
-tropomyosin
(Fig. 4I-L). While
-tropomyosin staining extends nearly to the tip of the tail in
both wild-type (Fig. 4I) and
wnt8 morphants (Fig.
4K), we observe a slight reduction of expression in wnt3a
morphants (Fig. 4J; 17/24
embryos), indicating a deficit in formation of somitic mesoderm. In
wnt3a/wnt8 MO embryos (Fig.
4L; 29/29 embryos),
-tropomyosin staining is
completely absent from the caudal tail; absence of somites and premature
termination of the notochord occur at a similar rostral/caudal level (compare
Fig. 4H,L). Thus,
wnt3a and wnt8 are also required for production of somitic
mesoderm in the tail.
In addition to the truncation of the notochord and loss of somites found in
these embryos, we also observed an apparent expansion of neural tube tissue
posterior to the end of the notochord in these more mildly affected
wnt3a/wnt8 embryos (4D, arrowhead indicates enlarged lumen of neural
tube). To confirm this with molecular markers, we stained embryos at 26 hpf
for F-spondin, which is expressed in the floor plate of the neural
tube (Fig. 4M-P) (Higashijima et al., 1997). We
observe a significant expansion of F-spondin expression in the
posterior of wnt3a/wnt8 morphants
(Fig. 4P). Expression of
collagen 2
in the floor plate is also expanded, confirming the
expansion of floor plate tissue (data not shown). In contrast, the panneural
marker ngn1 (Korzh et al.,
1998
) is not expanded in wnt3a/wnt8 morphants
(Fig. 4T), suggesting that the
loss of Wnt signaling results in an expansion only of the floor plate, and not
of other fates within the neural tube.
Lastly, we examined the expression of markers of other tail tissues,
including ventral fin epidermis (msxb)
(Akimenko et al., 1995), blood
(gata1) (Stainier et al.,
1995
), and vasculature (fli1)
(Thompson et al., 1998
), as
well as the tailbud marker (eve1)
(Joly et al., 1993
). These
data are presented in Fig. S3 in supplementary material. Briefly, while
eve1 expression in the tailbud is absent in wnt3a/wnt8
morphants, blood and vasculature is specified normally, suggesting that
wnt3a and wnt8 are not required for more lateral posterior
mesodermal fates. Also, expression of msxb in the ventral tailfin was
absent, possibly reflecting a mild dorsalization of wnt3a/wnt8
embryos. Taken together, our data show that wnt3a and wnt8
are required for the formation of notochord and somitic mesoderm in the tail,
as well as for inhibiting production of floor plate cells.
sp5l is a downstream target of Wnt signaling
To identify genes that function downstream of Wnt signaling during early
development, we performed a microarray screen for Wnt-responsive genes.
Briefly, RNA from early gastrula stage embryos that had been injected with
either wnt8 RNA or GFP RNA was used to probe a microarray chip
containing 8,000 zebrafish cDNAs from a mixed stage cDNA library. One gene,
the RNA levels of which were significantly upregulated by overexpression of
wnt8, was sp5-like (sp5l), a member of the Sp1
family of zinc-finger transcription factors. sp5l has previously been
described as spr2, and has been implicated in mesoderm induction,
acting downstream of FGF signaling (Zhao
et al., 2003).
We used several independent assays to show that sp5l is regulated
by Wnt signaling (Fig. 5).
First, we injected wnt8 RNA and examined sp5l expression at
early gastrula stage. In embryos overexpressing Wnt8, we observed ectopic
sp5l expression at the animal pole (arrowhead in
Fig. 5A, right panel),
confirming that activation of the Wnt pathway can activate sp5l
expression. Conversely, we used a dominant-negative, heat shock-inducible,
TCF GFP transgenic line to block the activation of Wnt/ß-catenin
target genes (Lewis et al.,
2004
) to test whether sp5l was down-regulated. We found
that expression of
TCF GFP can repress sp5l expression very
rapidly: 15 minutes after induction of
TCF GFP expression at tailbud
stage, sp5l RNA levels were already substantially reduced
(Fig. 5B, middle panel) and
hardly detectable after 30 minutes (Fig.
5B, right panel). This rapid downregulation suggests that
TCF GFP directly represses sp5l transcription and that
sp5l may be a direct target of Wnt/ß-catenin signaling. In
support of this, the sp5l promoter contains six consensus Tcf/Lef
binding sites within the 519 bp 5' of exon 1, which bind Lef1 protein in vitro
and are required for Wnt responsiveness in reporter assays conducted in
zebrafish embryos (Weidinger et al.,
2005
). Thus, sp5l is a direct Wnt/ß-catenin target
gene.
|
sp5l inhibition enhances loss of wnt3a function
Since sp5l expression is regulated by wnt3a and
wnt8 in the tailbud, we examined whether knockdown of sp5l
could enhance the defects seen in wnt3a or wnt8 morphants.
We used a translation blocking morpholino previously shown to specifically
inhibit sp5l function (Zhao et
al., 2003). We observed no enhancement of the wnt8
morphant phenotype when sp5l MOs were co-injected (data not shown),
perhaps because sp5l expression is already so dramatically reduced in
wnt8 morphants. In contrast, co-injection of sp5l MO
strongly enhances the phenotype of wnt3a MO embryos. While
sp5l morphants have no apparent tail defects at 48 hpf
(Fig. 6C), and wnt3a
MO embryos are only slightly shorter than wild-type embryos
(Fig. 6E, compare with wild
type in Fig. 6A),
wnt3a/sp5l morphants are dramatically shorter
(Fig. 6G; 93% of embryos,
n=104). Similarly, at the 10-somite stage, wnt3a/sp5l
morphants (Fig. 6H) have
greatly reduced expression of tbx6 relative to sp5l MO
(Fig. 6D) or wnt3a MO
(Fig. 6F) alone (32/34 embryos
with significantly reduced staining relative to either single MO injection).
Also, sp5l MO enhances the penetrance of the notochord truncation
phenotype observed in wnt3a morphants [67% of embryos injected with 1
mg/ml wnt3a MO plus control MO have truncated notochords
(n=73) vs 100% when co-injected with sp5l MO
(n=75)]. Lastly, like wnt3a/wnt8 morphants,
wnt3a/sp5l embryos lack somites posterior to the truncated notochord
(data not shown). These data indicate that sp5l functions redundantly
with wnt3a in tail development, both in regulation of presomitic
mesoderm markers such as tbx6 and in promoting mesodermal fates in
the caudal tail.
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Alternatively, as Wnt3a has recently been shown to regulate
oscillation of the segmentation clock (via regulation of axin2) in
mouse presomitic mesoderm (Aulehla et al.,
2003), it is also possible that a defect in segmentation underlies
the failure of tail formation observed in wnt3a/wnt8 morphants. We do
not favor this hypothesis for two main reasons. First, the data from the mouse
experiments indicates that repression of Wnt signaling in the presomitic
mesoderm leads to larger somites being formed, while in our
wnt3a/wnt8 embryos, the posterior somites appear somewhat smaller
than normal (see Fig. 4L).
Secondly, in zebrafish segmentation mutants, as well as in the mouse
Wnt3a mutant, vestigial tail, defects in segmentation are
associated with a failure of somite boundary formation, leading to diffuse,
unsegmented expression of somitic markers such as myoD
(Aulehla et al., 2003
;
Holley et al., 2000
). We do
not observe this phenotype in wnt3a/wnt8 morphants (see
Fig. 1K,L;
Fig. 4L), suggesting that
segmentation is grossly normal. A detailed analysis of the expression of
oscillating clock genes, such as her1, will be necessary to
definitively address this issue, although it is noteworthy that axin2
expression is not reported to cycle in zebrafish as it does in the mouse
(Aerne and Ish-Horowicz, 2004
),
raising the possibility that regulation of segmentation in these two
vertebrates may not be strictly orthologous.
Wnt signaling is required for production of both somitic mesoderm and notochord in the caudal tail
Zebrafish wnt3a is required for formation of caudal notochord,
which is missing in nearly all wnt3a MO embryos. Since Wnt3a
directly activates the transcription of T in the mouse
(Yamaguchi et al., 1999), and
the zebrafish T homolog, ntl, is required for notochord
formation (Halpern et al.,
1993
; Schulte-Merker et al.,
1994
), it is tempting to speculate that the notochord defect in
wnt3a morphants is due to a loss of expression of ntl.
Although ntl continues to be expressed in the tailbud of
wnt3a morphants (Fig.
3), ntl expression at the caudal end of the notochord
the chordoneural hinge is lost, beginning at approximately the
20-somite stage (C.J.T., unpublished results). Fate mapping studies have shown
that cells in this region can adopt a notochord or floor plate fate, and
ntl has been shown to be important in promoting notochord fates;
ntl mutants lack notochord and produce ectopic floor plate cells
(Halpern et al., 1997
).
Although we observe a clear loss of notochord in the caudal tail of
wnt3a morphants, we do not observe any increase in floor plate,
suggesting that notochord progenitors may not be adopting floor plate
identity. In the mouse Wnt3a mutant, the ectopic neural tissue
observed in the absence of notochord and somites is derived from prospective
somitic mesodermal cells (Takada et al.,
1994
; Yoshikawa et al.,
1997
). Our observation that ectopic floor plate is not produced
when only notochord is missing (in the wnt3a morphant), but only when
somitic mesoderm is also lacking (the wnt3a/wnt8 double morphant),
suggests that the same fate transformation could be occurring in the
zebrafish.
Careful fate mapping within the tailbud to determine the origin of the ectopic floor plate will be required to directly address this possibility.
sp5l is a downstream target of Wnt signaling
Sp1-related transcription factors are characterized by having multiple
zinc-finger DNA binding domains related to the kruppel gene from
Drosophila (Pieler and
Bellefroid, 1994). Sp1 proteins bind to GC-rich promoter regions,
and while some members of this protein family are expressed ubiquitously and
are thought to be required generally as enhancers of transcription, others,
including members of the Sp5 subfamily, are expressed in restricted domains
and are thought to participate in specific processes during development
(Bell et al., 2003
;
Briggs et al., 1986
;
Harrison et al., 2000
;
Tallafuss et al., 2001
;
Treichel et al., 2001
;
Treichel et al., 2003
).
Our data strongly suggest that sp5l functions downstream of Wnts
in zebrafish tail development. What role might sp5l play in this
process? One function could be to bind the promoters of downstream Wnt targets
and enhance their activation. For example, in the mouse T promoter,
both Tcf/Lef sites and regions containing Sp1 binding sites are important for
normal expression of T in the primitive streak and tailbud
(Clements et al., 1996;
Yamaguchi et al., 1999
), and
mutations in the Sp1 family member Sp5 enhance the tail truncation
phenotype of heterozygous T/+ mice
(Harrison et al., 2000
),
suggesting that Sp5 could be functioning with Wnt3a to
activate T transcription. Also, in vitro analysis indicates that
LEF-1 can activate transcription from the HIV-1 promoter only when an
Sp1-containing fraction, or purified Sp1 protein, is added to the
transcription reaction (Sheridan et al.,
1995
).
The synergistic loss of tbx6 expression observed when both
sp5l and wnt3a are inhibited suggests that full activation
of the tbx6 promoter may also require the binding of both
ß-catenin/Tcf complexes to Tcf/Lef sites
(Szeto and Kimelman, 2004) and
Sp5l to putative Sp1 binding sites found in the tbx6 promoter
(C.J.T., unpublished). Additional deletion analysis of the tbx6
promoter will be required to more directly address a requirement for
sp5l in its activation.
Since the GC-rich promoter elements bound by Sp1 proteins are found
upstream of many genes, it is conceivable that activation of multiple Wnt
targets could be potentiated by sp5l. Since sp5l morphants,
like the mouse Sp5 knockout, have no discernable tail phenotype, this
function may not be essential, or may be redundantly encoded. The latter is a
distinct possibility, as in both mouse and zebrafish, another Sp5 homolog is
largely co-expressed in the same tissues
(Harrison et al., 2000;
Tallafuss et al., 2001
).
Interestingly, the other identified zebrafish Sp5 homolog, called
buttonhead/Sp-related 1(bts1), is also a target of Wnt signaling
(Harrison et al., 2000
;
Tallafuss et al., 2001
;
Weidinger et al., 2005
). Also,
a recent report suggesting that a mouse buttonhead homolog, mBtd,
also called SP8, may play a role in maintaining expression of Wnt targets in
the limb bud (Bell et al.,
2003
; Treichel et al.,
2003
) is an additional link between Sp1 family members and Wnt
signaling during vertebrate development.
We suggest that in addition to a previously characterized role during gastrulation for specification of future tail fates, Wnt/ß-catenin signaling is also required during somitogenesis for maintenance of presomitic mesoderm in the tailbud, and also to promote mesodermal fates and inhibit floor plate formation in subsequent tail outgrowth (Fig. 8). The early functions in dorsal-ventral patterning and specification of the tail organizer are principally carried out by wnt8. During early somitogenesis, both wnt3a and wnt8 function to maintain presomitic mesoderm fates in the tailbud, while the later mesoderm/floor plate distinction is more sensitive to loss of wnt3a function. Thus, Wnt/ß-catenin signaling, functioning in part through sp5l, is required throughout tail development to properly specify, pattern and maintain a precursor population in the tailbud. Further examination of the defects in mesoderm formation in Wnt-inhibited embryos and identification of additional Wnt/ß-catenin targets will be important for a more complete understanding of tail development.
|
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
Footnotes |
---|
Supplementary material for this article is available at http://dev.biologists.org/cgi/content/full/132/8/1763/DC1
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Aerne, B. and Ish-Horowicz, D. (2004). Receptor
tyrosine phosphatase psi is required for Delta/Notch signalling and cyclic
gene expression in the presomitic mesoderm.
Development 131,3391
-3399.
Agathon, A., Thisse, C. and Thisse, B. (2003). The molecular nature of the zebrafish tail organizer. Nature 424,448 -452.[CrossRef][Medline]
Akimenko, M. A., Johnson, S. L., Westerfield, M. and Ekker,
M. (1995). Differential induction of four msx homeobox genes
during fin development and regeneration in zebrafish.
Development 121,347
-357.
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.[Medline]
Aulehla, A., Wehrle, C., Brand-Saberi, B., Kemler, R., Gossler, A., Kanzler, B. and Herrmann, B. G. (2003). Wnt3a plays a major role in the segmentation clock controlling somitogenesis. Dev. Cell 4,395 -406.[CrossRef][Medline]
Bell, S. M., Schreiner, C. M., Waclaw, R. R., Campbell, K.,
Potter, S. S. and Scott, W. J. (2003). Sp8 is crucial for
limb outgrowth and neuropore closure. Proc. Natl. Acad. Sci.
USA 100,12195
-12200.
Briggs, M. R., Kadonaga, J. T., Bell, S. P. and Tjian, R. (1986). Purification and biochemical characterization of the promoter-specific transcription factor, Sp1. Science 234, 47-52.[Medline]
Buckles, G. R., Thorpe, C. J., Ramel, M. C. and Lekven, A. C. (2004). Combinatorial Wnt control of zebrafish midbrain-hindbrain boundary formation. Mech. Dev. 121,437 -447.[CrossRef][Medline]
Cambray, N. and Wilson, V. (2002). Axial progenitors with extensive potency are localised to the mouse chordoneural hinge. Development 129,4855 -4866.[Medline]
Cao, Y., Zhao, J., Sun, Z., Zhao, Z., Postlethwait, J. and Meng, A. (2004). fgf17b, a novel member of Fgf family, helps patterning zebrafish embryos. Dev. Biol. 271,130 -143.[CrossRef][Medline]
Charrier, J. B., Teillet, M. A., Lapointe, F. and le Douarin, N.
M. (1999). Defining subregions of Hensen's node essential for
caudalward movement, midline development and cell survival.
Development 126,4771
-4783.
Clements, D., Taylor, H. C., Herrmann, B. G. and Stott, D. (1996). Distinct regulatory control of the Brachyury gene in axial and non-axial mesoderm suggests separation of mesoderm lineages early in mouse gastrulation. Mech. Dev. 56,139 -149.[CrossRef][Medline]
Dorsky, R. I., Sheldahl, L. C. and Moon, R. T. (2002). A transgenic Lef1/beta-catenin-dependent reporter is expressed in spatially restricted domains throughout zebrafish development. Dev. Biol. 241,229 -237.[CrossRef][Medline]
Draper, B. W., Stock, D. W. and Kimmel, C. B.
(2003). Zebrafish fgf24 functions with fgf8 to promote posterior
mesodermal development. Development
130,4639
-4654.
Dubrulle, J., McGrew, M. J. and Pourquie, O. (2001). FGF signaling controls somite boundary position and regulates segmentation clock control of spatiotemporal Hox gene activation. Cell 106,219 -232.[CrossRef][Medline]
Dubrulle, J. and Pourquie, O. (2004). fgf8 mRNA decay establishes a gradient that couples axial elongation to patterning in the vertebrate embryo. Nature 427,419 -422.[CrossRef][Medline]
Erter, C. E., Wilm, T. P., Basler, N., Wright, C. V. and Solnica-Krezel, L. (2001). Wnt8 is required in lateral mesendodermal precursors for neural posteriorization in vivo. Development 128,3571 -3583.[Medline]
Furthauer, M., Reifers, F., Brand, M., Thisse, B. and Thisse, C. (2001). sprouty4 acts in vivo as a feedback-induced antagonist of FGF signaling in zebrafish. Development 128,2175 -2186.[Medline]
Galceran, J., Farinas, I., Depew, M. J., Clevers, H. and
Grosschedl, R. (1999). Wnt3a-/like phenotype and limb
deficiency in Lef1(/)Tcf1(/) mice.
Genes Dev. 13,709
-717.
Galceran, J., Hsu, S. C. and Grosschedl, R.
(2001). Rescue of a Wnt mutation by an activated form of LEF-1:
regulation of maintenance but not initiation of Brachyury expression.
Proc. Natl. Acad. Sci. USA
98,8668
-8673.
Gont, L. K., Steinbeisser, H., Blumberg, B. and de Robertis, E.
M. (1993). Tail formation as a continuation of gastrulation:
the multiple cell populations of the Xenopus tailbud derive from the
late blastopore lip. Development
119,991
-1004.
Greco, T. L., Takada, S., Newhouse, M. M., McMahon, J. A., McMahon, A. P. and Camper, S. A. (1996). Analysis of the vestigial tail mutation demonstrates that Wnt-3a gene dosage regulates mouse axial development. Genes Dev. 10,313 -324.[Abstract]
Griffin, K. J., Amacher, S. L., Kimmel, C. B. and Kimelman,
D. (1998). Molecular identification of spadetail: regulation
of zebrafish trunk and tail mesoderm formation by T-box genes.
Development 125,3379
-3388.
Griffin, K. J. and Kimelman, D. (2002). One-Eyed Pinhead and Spadetail are essential for heart and somite formation. Nat. Cell Biol. 4,821 -825.[CrossRef][Medline]
Griffin, K. J. and Kimelman, D. (2003). Interplay between FGF, one-eyed pinhead, and T-box transcription factors during zebrafish posterior development. Dev. Biol. 264,456 -466.[CrossRef][Medline]
Halpern, M. E., Hatta, K., Amacher, S. L., Talbot, W. S., Yan, Y. L., Thisse, B., Thisse, C., Postlethwait, J. H. and Kimmel, C. B. (1997). Genetic interactions in zebrafish midline development. Dev. Biol. 187,154 -170.[CrossRef][Medline]
Halpern, M. E., Ho, R. K., Walker, C. and Kimmel, C. B. (1993). Induction of muscle pioneers and floor plate is distinguished by the zebrafish no tail mutation. Cell 75, 99-111.[CrossRef][Medline]
Hammerschmidt, M., Pelegri, F., Mullins, M. C., Kane, D. A.,
Brand, M., van Eeden, F. J., Furutani-Seiki, M., Granato, M., Haffter, P.,
Heisenberg, C. P. et al. (1996). Mutations affecting
morphogenesis during gastrulation and tail formation in the zebrafish,
Danio rerio. Development
123,143
-151.
Harrison, S. M., Houzelstein, D., Dunwoodie, S. L. and Beddington, R. S. (2000). Sp5, a new member of the Sp1 family, is dynamically expressed during development and genetically interacts with Brachyury. Dev. Biol. 227,358 -372.[CrossRef][Medline]
Higashijima, S., Nose, A., Eguchi, G., Hotta, Y. and Okamoto, H. (1997). Mindin/F-spondin family: novel ECM proteins expressed in the zebrafish embryonic axis. Dev. Biol. 192,211 -227.[CrossRef][Medline]
Holley, S. A., Geisler, R. and Nusslein-Volhard, C.
(2000). Control of her1 expression during zebrafish somitogenesis
by a delta-dependent oscillator and an independent wave-front activity.
Genes Dev. 14,1678
-1690.
Hug, B., Walter, V. and Grunwald, D. J. (1997). tbx6, a Brachyury-related gene expressed by ventral mesendodermal precursors in the zebrafish embryo. Dev. Biol. 183, 61-73.[CrossRef][Medline]
Joly, J. S., Maury, M., Joly, C., Boulekbache, H. and Condamine, H. (1993). [Ventral and posterior expression of the homeo box gene eve1 in zebrafish (Brachydanio rerio) is repressed in dorsalized embryos]. C R Seances Soc. Biol. Fil. 187,356 -363.[Medline]
Kelly, G. M., Greenstein, P., Erezyilmaz, D. F. and Moon, R.
T. (1995). Zebrafish wnt8 and wnt8b share a common activity
but are involved in distinct developmental pathways.
Development 121,1787
-1799.
Korzh, V., Sleptsova, I., Liao, J., He, J. and Gong, Z. (1998). Expression of zebrafish bHLH genes ngn1 and nrd defines distinct stages of neural differentiation. Dev. Dyn. 213,92 -104.[CrossRef][Medline]
Krauss, S., Korzh, V., Fjose, A. and Johansen, T.
(1992). Expression of four zebrafish wnt-related genes
during embryogenesis. Development
116,249
-259.
Lekven, A. C., Thorpe, C. J., Waxman, J. S. and Moon, R. T. (2001). Zebrafish wnt8 encodes two wnt8 proteins on a bicistronic transcript and is required for mesoderm and neurectoderm patterning. Dev. Cell 1,103 -114.[CrossRef][Medline]
Lewis, J. L., Bonner, J., Modrell, M., Ragland, J. W., Moon, R.
T., Dorsky, R. I. and Raible, D. W. (2004). Reiterated Wnt
signaling during zebrafish neural crest development.
Development 131,1299
-1308.
Mathieu, J., Griffin, K., Herbomel, P., Dickmeis, T., Strahle,
U., Kimelman, D., Rosa, F. M. and Peyrieras, N. (2004). Nodal
and Fgf pathways interact through a positive regulatory loop and synergize to
maintain mesodermal cell populations. Development
131,629
-641.
Mathis, L., Kulesa, P. M. and Fraser, S. E. (2001). FGF receptor signalling is required to maintain neural progenitors during Hensen's node progression. Nat. Cell Biol. 3,559 -566.[CrossRef][Medline]
Muller, M., v Weizsacker, E. and Campos-Ortega, J. A.
(1996). Expression domains of a zebrafish homologue of the
Drosophila pair-rule gene hairy correspond to primordia of
alternating somites. Development
122,2071
-2078.
Oxtoby, E. and Jowett, T. (1993). Cloning of the zebrafish krox-20 gene (krx-20) and its expression during hindbrain development. Nucleic Acids Res. 21,1087 -1095.[Abstract]
Phillips, B. T., Bolding, K. and Riley, B. B. (2001). Zebrafish fgf3 and fgf8 encode redundant functions required for otic placode induction. Dev. Biol. 235,351 -365.[CrossRef][Medline]
Pieler, T. and Bellefroid, E. (1994). Perspectives on zinc finger protein function and evolution an update. Mol. Biol. Rep. 20,1 -8.[CrossRef][Medline]
Pinson, K. I., Brennan, J., Monkley, S., Avery, B. J. and Skarnes, W. C. (2000). An LDL-receptor-related protein mediates Wnt signalling in mice. Nature 407,535 -538.[CrossRef][Medline]
Rauch, G. J., Hammerschmidt, M., Blader, P., Schauerte, H. E., Strahle, U., Ingham, P. W., McMahon, A. P. and Haffter, P. (1997). Wnt5 is required for tail formation in the zebrafish embryo. Cold Spring Harb. Symp. Quant. Biol. 62,227 -234.[Medline]
Schulte-Merker, S., van Eeden, F. J., Halpern, M. E., Kimmel, C.
B. and Nusslein-Volhard, C. (1994). no tail
(ntl) is the zebrafish homologue of the mouse T
(Brachyury) gene. Development
120,1009
-1015.
Sheridan, P. L., Sheline, C. T., Cannon, K., Voz, M. L., Pazin, M. J., Kadonaga, J. T. and Jones, K. A. (1995). Activation of the HIV-1 enhancer by the LEF-1 HMG protein on nucleosome-assembled DNA in vitro. Genes Dev. 9,2090 -2104.[Abstract]
Stainier, D. Y., Weinstein, B. M., Detrich, H. W., 3rd, Zon, L.
I. and Fishman, M. C. (1995). cloche, an early
acting zebrafish gene, is required by both the endothelial and hematopoietic
lineages. Development
121,3141
-3150.
Szeto, D. P. and Kimelman, D. (2004).
Combinatorial gene regulation by Bmp and Wnt in zebrafish posterior mesoderm
formation. Development
131,3751
-3760.
Takada, S., Stark, K. L., Shea, M. J., Vassileva, G., McMahon, J. A. and McMahon, A. P. (1994). Wnt-3a regulates somite and tailbud formation in the mouse embryo. Genes Dev. 8, 174-189.[Abstract]
Tallafuss, A., Wilm, T. P., Crozatier, M., Pfeffer, P., Wassef, M. and Bally-Cuif, L. (2001). The zebrafish buttonhead-like factor Bts1 is an early regulator of pax2.1 expression during mid-hindbrain development. Development 128,4021 -4034.[Medline]
Thompson, M. A., Ransom, D. G., Pratt, S. J., MacLennan, H., Kieran, M. W., Detrich, H. W., 3rd, Vail, B., Huber, T. L., Paw, B., Brownlie, A. J. et al. (1998). The cloche and spadetail genes differentially affect hematopoiesis and vasculogenesis. Dev. Biol. 197,248 -269.[CrossRef][Medline]
Treichel, D., Becker, M. B. and Gruss, P. (2001). The novel transcription factor gene Sp5 exhibits a dynamic and highly restricted expression pattern during mouse embryogenesis. Mech. Dev. 101,175 -179.[CrossRef][Medline]
Treichel, D., Schock, F., Jackle, H., Gruss, P. and Mansouri,
A. (2003). mBtd is required to maintain signaling during
murine limb development. Genes Dev.
17,2630
-2635.
Vasiliauskas, D. and Stern, C. D. (2001). Patterning the embryonic axis: FGF signaling and how vertebrate embryos measure time. Cell 106,133 -136.[CrossRef][Medline]
Weidinger, G., Wuennenberg-Stapleton, K., Thorpe, C. J., Ngai, J. and Moon, R. T. (2005). Wnt/ß-catenin regulation of the Sp1-related transcription factor sp51 is required for mesoderm and neurectoderm patterning. Curr. Biol. (in press).
Yamaguchi, T. P., Takada, S., Yoshikawa, Y., Wu, N. and McMahon,
A. P. (1999). T (Brachyury) is a direct target of Wnt3a
during paraxial mesoderm specification. Genes Dev.
13,3185
-3190.
Yoshikawa, Y., Fujimori, T., McMahon, A. P. and Takada, S. (1997). Evidence that absence of Wnt-3a signaling promotes neuralization instead of paraxial mesoderm development in the mouse. Dev. Biol. 183,234 -242.[CrossRef][Medline]
Zhao, J., Cao, Y., Zhao, C., Postlethwait, J. and Meng, A.
(2003). An SP1-like transcription factor Spr2 acts downstream of
Fgf signaling to mediate mesoderm induction. EMBO J.
22,6078
-6088.
|