1 Department of Neurobiology and Anatomy, University of Utah, Salt Lake City, UT
84132, USA
2 Laboratory of Molecular Genetics, NICHD, NIH, Bethesda, MD 20892, USA
3 Howard Hughes Medical Institute/Department of Pharmacology and Center for
Developmental Biology, University of Washington, Seattle, WA 98195, USA
Authors for correspondence (e-mail:
richard.dorsky{at}hsc.utah.edu and chitnisa@mail.nih.gov)
Accepted 17 January 2003
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SUMMARY |
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Key words: Tcf3b, Headless, Zebrafish, Wnt, Neural patterning, Morphogen gradient
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INTRODUCTION |
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Analysis of zebrafish maternal-zygotic headless (hereafter simply
called hdl) mutants has suggested that caudalizing factors, in
particular Wnts, operate in the context of basal repression provided by this
Tcf3 homolog (Kim et al.,
2000). Canonical Wnt signaling facilitates the expression of
downstream target genes through ß-catenin, which associates with Lef/Tcf
proteins that bind to DNA regulatory elements
(Barker et al., 2000
). When
ß-catenin levels are low, Lef/Tcf proteins maintain target genes in a
repressed state (Brannon et al.,
1997
). Although Lef/Tcf transcription factors can have dual roles
in activation or repression of target genes, it appears that in vivo Lef1 has
a primary role in activation, whereas Tcf3 has a primary role as a repressor
(Kengaku et al., 1998
;
Houston et al., 2002
).
Several studies have converged to provide evidence for the role of
Wnt/ß-catenin activity in defining discrete domains of gene expression
along the rostral-caudal axis of the neural plate and in the subsequent
establishment of rostral-caudal compartments of the vertebrate neural tube
(Domingos et al., 2001;
Erter et al., 2001
;
Hashimoto et al., 2000
;
Lekven et al., 2001
;
McGrew et al., 1995
;
Nordstrom et al., 2002
;
Shinya et al., 2000
;
van de Water et al., 2001
;
Houart et al., 2002
;
Kiecker and Niehrs, 2001
;
Kim et al., 2002
). These
studies have shown that exaggerated Wnt signaling leads to loss of rostral
neural domains and expansion of more caudal neural domains, whereas reduced
Wnt signaling leads to expansion of rostral neural domains and loss of more
caudal domains. However, in some contexts ß-catenin is not primarily
required for activation of target genes but rather for antagonizing repression
mediated by Tcf homologs (Chan and Struhl,
2002
; Houston et al.,
2002
). The primary role for Wnt/ß-catenin signaling in
rostral-caudal patterning thus remains unclear.
Consistent with the role of hdl in repressing genes that define relatively caudal domains, hdl mutants are characterized by expanded expression of genes that define the MHB domain. At the same time, the forebrain, whose specification is most dependent on the repression of caudal genes, is lost in hdl mutants. Interestingly, patterning defects are restricted to the rostral neurectoderm, leaving the hindbrain and spinal cord relatively unaffected. Many zygotic hdl mutants survive to adulthood, suggesting that other lef/tcf genes may limit the severity of phenotypes observed in these fish.
Previously, we identified a partial cDNA clone of a second zebrafish
tcf3 gene, which we named tcf3b
(Dorsky et al., 1999). We
report here the full-length sequence of tcf3b, and show that although
both hdl and tcf3b are expressed maternally and throughout
development, there are important differences in their expression patterns,
most notably during early gastrulation. By examining loss-of-function
phenotypes and performing mRNA rescue experiments, we determine that both
genes have unique and cooperative roles in early zebrafish development. By
comparing the abilities of tcf3b and lef1 to suppress the
caudalization in hdl mutants, we reveal functional differences
between these lef/tcf family members in repressing caudal target
genes. In addition, we show that Wnt8 function is primarily required in the
neurectoderm for de-repression of caudal genes rather than for their
activation. Finally, by analyzing changes in the shape of gene expression
domains caused by reduction of tcf3b function in hdl
mutants, we make specific predictions about the shape of the caudalizing
activity gradient in the neurectoderm.
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MATERIALS AND METHODS |
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RT-PCR
PCR was performed on cDNA from various developmental stages, using the
following primers and an annealing temperature of 50°C for 30 cycles.
Products were run on a 2% agarose gel and stained with ethidium bromide.
In situ hybridization
In situ hybridization with digoxigenin-labeled mRNA probes was performed as
described previously (Oxtoby and Jowett,
1993). Probes for hdl, tcf3b, lef1 and wnt1 were
made from full-length cDNAs. Digital images were processed with Adobe
Photoshop software.
Other plasmids used to make in situ probes have been published previously:
opl (Grinblat et al.,
1998), pax2.1 (Krauss
et al., 1991a
), pax6
(Krauss et al., 1991b
),
gbx1 (Itoh et al.,
2002
), gsc (Stachel
et al., 1993
), krox20
(Oxtoby and Jowett, 1993
),
en2 (Ekker et al.,
1992
), isl1 (Inoue et
al., 1994
), and mar
(Popperl et al., 2000
). Double
in situs using digoxigenin- and fluorescein-labeled RNA probes were performed
as described (Jowett,
2001
).
Phalloidin staining
Fixed embryos were soaked in 0.1 mg/ml AlexaFluor 594 phalloidin (Molecular
Probes) for one hour at room temperature and rinsed in PBS. Embryos were
mounted in glass coverslips and imaged on a Nikon PCM2000 confocal
microscope.
TNT reactions
We added 200 ng of hdl and tcf3b cDNAs in pCS2+
(Turner and Weintraub, 1994)
to TNT Quick Coupled reactions (Promega, Madison, WI). Morpholinos (MOs) were
added to the reactions as indicated, and reactions were labeled with
35S Methionine. Following incubation, reactions were run on 10%
acrylamide gels, dried and exposed overnight for autoradiography.
Zebrafish maintenance and hdl mutant embryos
Zebrafish were raised and maintained under standard conditions. To collect
maternal zygotic headlessm881 mutant embryos, heterozygous
males and homozygous females were crossed
(Kim et al., 2000).
MO and mRNA injections
MO antisense oligonucleotides were designed by and purchased from Gene
Tools (Philomath, OR). The MO sequences are as follows:
For both MOs, doses ranging from 500 pg-5 ng were injected. After examining
phenotypes and embryo survival, 1 ng was chosen as the optimal dose for
producing specific phenotypes. wnt8 MOs were kindly provided by Arne
Lekven (Lekven et al.,
2001).
For mRNA injections, transcripts were synthesized using the mMessage
mMachine kit (Ambion). Expression constructs were made by inserting
full-length cDNAs into pCS2+ (Turner and
Weintraub, 1994). For rescue experiments, we injected
approximately 1 ng MO with 10 pg hdl, tcf3b,
tcf3b, lef1 or
lef1 mRNA and 100 pg wnt1 mRNA.
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RESULTS |
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We next examined the phenotype following injection of one-cell embryos with 1 ng of a MO targeted against tcf3b. In contrast to hdl, we observed no gross morphological abnormalities through 72 hours post-fertilization (h.p.f.), except that the brain appeared slightly smaller and there was minor cardiac edema (Fig. 3D).
We then co-injected embryos with 1 ng each of hdl and tcf3b MOs. The size of the brain in many of these embryos was substantially smaller than with either MO alone, especially when examined from the ear to the rostral limit of the brain (Fig. 3E). These results suggest that the two genes may have cooperative roles in these tissues.
The hdl phenotype can be rescued by tcf3b
overexpression in a wnt1-reversible manner
To determine whether hdl and tcf3b encode proteins with
similar functions, we attempted to rescue the hdl phenotype by
overexpressing tcf3b. We first titrated the dose of tcf3b
mRNA to find a concentration that was insufficient to produce a phenotype when
overexpressed. When 100 pg of hdl or tcf3b mRNAs were
injected at the one-cell stage, we observed identical phenotypes that included
cyclopia, short tails and somite defects (not shown). Because Wnt signaling
can regulate the activity of Tcf3, it is difficult to interpret such
overexpression phenotypes. Nevertheless, we hypothesized that at a gross
level, the two genes may encode proteins with equivalent functions. At a dose
of 10 pg, we observed no obvious defects in injected embryos, so we chose this
amount for our rescue experiments.
Injecting 10 pg of tcf3b mRNA with 1 ng of hdl MO resulted in rescue of the hdl phenotype. Expression of pax2.1, a marker for the MHB, was expanded rostrally in 89% (25/28) of embryos injected with the hdl MO alone (Fig. 3F,G). Co-injection of tcf3b mRNA eliminated the expansion of pax2.1 in 78% (25/32) of embryos examined (Fig. 3H). Injection of tcf3b was also able to rescue hdl morphology at 24 h.p.f. (Fig. 3I). Although only 8% of embryos injected with the hdl MO had eyes, this fraction increased to 91% when tcf3b mRNA was co-injected (Table 1). To further demonstrate that tcf3b mRNA suppressed the defects caused by loss of hdl function, we injected tcf3b mRNA into hdl mutant embryos. Again, injection of tcf3b mRNA increased the number of embryos with eyes from 0% to 91% (57/64). We therefore conclude that ectopic expression of tcf3b can functionally replace hdl in patterning the embryonic brain.
|
Different Lef/Tcf factors are known to play distinct roles in development
(Roel et al., 2002). To
further characterize the functions of these proteins in activating and
repressing target genes, we attempted to suppress the hdl MO
phenotype with a third family member, lef1. In our experiments,
neither lef1 (3% with eyes) nor
lef1, a form lacking
the ß-catenin binding domain, (5% with eyes) could compensate for the
loss of hdl function (Table
1). The failure of lef1 to suppress the hdl MO
phenotype is consistent with its suggested primary role in activating rather
than repressing target genes (Kengaku et
al., 1998
; Merrill et al.,
2001
).
Cooperative functions of hdl and tcf3b in early
brain patterning
Analysis of hdl mutant embryos showed that this gene plays an
essential role in forebrain specification. However, hdl and
tcf3b double MO injections resulted in a more severe phenotype than
that produced by the hdl MO alone
(Fig. 3E), suggesting a
cooperative role for hdl and tcf3b in early development.
Furthermore, tcf3b is expressed both maternally and zygotically,
indicating that it may function in the early embryo. We therefore explored the
possibility that the two tcf3 genes contribute to a common function
during embryogenesis.
To examine rostral-caudal neural patterning, we compared the expression of pax6, pax2.1 and gbx1, which respectively mark the future eye field and dorsal diencephalon, the MHB and the rostral hindbrain (Fig. 4). In hdl mutants, the size of the rostral pax6 expression domain was reduced in 83% (20/24) of the embryos (Fig. 4A2). Our observations of pax6 expression were restricted to the rostral neurectoderm, because mechanisms that determine its expression in the caudal neurectoderm remain poorly defined at this stage. There was also, as described earlier, a rostral expansion of the MHB domain marked by pax2.1 expression in 26% (7/27) of the embryos (Fig. 4B2). There was no obvious change, however, in the size of the gbx1 expression domain in hdl mutants (Fig. 4C2).
|
A striking feature of caudalized embryos is the systematic manner in which
expression of caudal genes expands rostrally to resemble the wild-type gene
expression in these compartments. For example, pax2.1 expression
expands in hdl mutants from its normal domain at the MHB to a rostral
domain that resembles wild-type pax6 expression (compare
Fig. 4A1 and
Fig. 4B2). At tailbud stage,
the diencephalic marker pax6 is expressed in a compartment that
extends rostrally to enclose an unlabelled area
(Fig. 4D). At the same time,
pax2.1 and gbx1 are expressed in more caudal compartments
where they define the MHB domain and rostral hindbrain, respectively
(Fig. 4D). In caudalized
embryos, pax6 expression is lost and pax2.1 expands
rostrally within an oval domain that is surrounded by gbx1 expression
(Fig. 4E, also compare
Fig. 4B3 and
Fig. 4C3). As described
earlier, in many caudalized embryos pax2.1 is most prominently
expressed in a rostral crescent within this oval domain
(Fig. 4B4,
Fig. 4E), resembling the
wild-type expression of genes such as emx1 that define the
prospective telencephalon (Houart et al.,
2002).
Loss of Hdl and Tcf3 function leads to changes in patterning that are
evident by the shield stage
We showed that although tcf3b can functionally replace
hdl, a third family member, lef1, which is more likely to
have a role in gene activation, cannot. The respective roles of hdl
and lef1 in repressing and activating target genes correlates with
their complementary expression in the blastoderm at the shield stage
(Fig. 5A,B). lef1 is
expressed in a domain that overlaps with wnt8 at the ventrolateral
blastoderm margin and where Wnt/ß-catenin signaling is expected to be
high. In contrast, lef1 expression is excluded from the prospective
rostral neurectoderm, where hdl is expressed and where
Wnt/ß-catenin signaling is expected to be low. In embryos injected with
hdl and tcf3b MOs, lef1 expression expands to cover
most of the blastoderm at the shield stage
(Fig. 5C). This indicates that
caudalization of neurectoderm following loss of Tcf3 function is preceded by
expanded lef1 expression at early gastrulation. Furthermore, it
indicates that one role of Tcf3 is to restrict lef1 expression to the
blastoderm margin during normal development. It is important to note that
hdl and tcf3b MOs do not cause increased activation of a
ß-catenin-dependent reporter transgene
(Dorsky et al., 2002) (data
not shown). This suggests that Wnt/ß-catenin signaling may be able to
antagonize repression by Tcf3, but it may not play a role in the direct
activation of caudal genes.
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Analysis of the late tcf3b MO phenotype
The analysis of embryos lacking both hdl and tcf3b
function revealed the cooperative roles of these genes in rostral-caudal
neural patterning. To determine the unique function of tcf3b we
examined molecular markers for brain development following tcf3b MO
injection. At the earliest stages of brain development, the basic patterning
of injected embryos appeared normal. We examined the expression of
pax2.1 at bud stage and found no changes compared to uninjected
embryos (not shown), indicating that rostral-caudal patterning is unaffected
by the tcf3b MO. In addition, other MHB markers (wnt1 and
en2) and dorsal/ventral patterning markers (pax2.1 and
axial) appear normal at 18 somites (not shown). In fact, we were
unable to detect any effects on brain development until 24 h.p.f., when we
examined hindbrain and MHB morphology.
We observed a severe loss of hindbrain rhombomere segmentation in tcf3b MO-injected embryos at 24 h.p.f. This phenotype was never observed in either hdl mutant or MO-injected embryos, suggesting a unique function for tcf3b in hindbrain development. Analysis of hindbrain morphology by phalloidin staining revealed a lack of physical boundaries (Fig. 6A-D). At the molecular level, wnt1 is normally expressed at inter-rhombomere boundaries along the dorsolateral edge of the hindbrain (Fig. 6E). However, in injected embryos, wnt1 expression is noticeably absent from boundary areas and appears uniform or patchy along the hindbrain margin (Fig. 6F). The mariposa gene is expressed ventrally in the hindbrain, again localized to rhombomere boundaries (Fig. 6G). Following tcf3b MO injection, there is a uniform level of expression throughout the hindbrain (Fig. 6H). We also observed a defect in the closure of the dorsal MHB (Fig. 6I,J), although the position and identity of the MHB appear normal as marked by the expression of en2 (Fig. 6K,L). Rhombomere identity also does not appear to be affected in these embryos, because patterning markers such as krox20 (rhombomeres 3 and 5) are expressed normally (Fig. 6M,N). Neurogenesis is also normal, as indicated by isl1, which marks ventral neurons of the hindbrain (Fig. 6O,P). These phenotypes indicate defects in brain morphogenesis, rather than in patterning or differentiation. We attempted to rescue these hindbrain defects by co-injection of both hdl and tcf3b mRNA with the MO, but were unable to restore normal morphology because of the fact that both mRNAs produced similar defects when overexpressed (not shown).
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DISCUSSION |
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Both tcf3 genes are required for rostral-caudal brain
patterning
Reduction of tcf3b function does not significantly affect early
patterning in wild-type embryos, suggesting that hdl plays a more
prominent role in this process. However, tcf3b can rescue the
hdl mutant phenotype and reduction of tcf3b function leads
to further caudalization of hdl mutant embryos. This suggests that
low levels of Tcf3b present in the embryo during early gastrulation help limit
the degree of caudalization caused by loss of Hdl function. Indeed, in zygotic
hdl mutants, the loss of rostral neural structures is minimal and
persistence of maternal hdl and tcf3b transcripts permits
some mutants to be grown up to adulthood.
Hdl and Tcf3b function in the context of Wnt signaling
The essential function of Hdl protein revealed in zebrafish
(Kim et al., 2000) and
Xenopus (Brannon et al.,
1997
; Houston et al.,
2002
) is as a repressor. In contrast, Lef1 appears to be a more
effective activator of target genes in vivo
(Kengaku et al., 1998
;
DasGupta and Fuchs, 1999
).
Complementary roles of Hdl/Tcf3b and Lef1 in mediating repression and
activation of target genes, respectively, are consistent with their
complementary expression during early zebrafish development
(Fig. 5A,B)
(Dorsky et al., 2002
), as well
as their complementary roles in mouse and Xenopus development
(Merrill et al., 2001
;
Roel et al., 2002
).
Our experiments support a mechanism in which genes that are Hdl and Tcf3b
targets in the neurectoderm do not require Wnt/ß-catenin signaling for
either their repression or their endogenous activation. Furthermore, in the
absence of Tcf3 function we observe an increase in lef1 expression
without a corresponding increase in expression of a ß-catenin-responsive
reporter (Dorsky et al.,
2002). Other Lef/Tcf factors have been shown to activate targets
in lymphocytes (Travis et al.,
1991
; van de Wetering et al.,
1991
), and Xenopus embryos
(Labbe et al., 2000
) in a
Wnt/ß-catenin-independent manner. Through this evidence, it is possible
to conclude that Hdl and Tcf3b function in a Wnt-independent manner as well.
However, data from our studies indicates that the developmental roles of these
factors are closely linked to Wnt signaling in the embryo. First, we show that
rescue of hdl mutants is reversible by Wnt signaling in a manner that
requires ß-catenin binding. Second, loss of Wnt8 function results in
expansion of the same rostral genes that require Tcf3 function for their
expression. Although Wnt8 may act through Lef1 to activate target genes in the
ventrolateral mesoderm, we conclude that it primarily antagonizes Tcf3
function in the neurectoderm.
Wnt signals are only a part of the system that determines gene expression
along the rostral-caudal axis. Multiple caudalizing factors, including Wnts,
FGFs and Activin/Nodal-related factors, contribute to rostral-caudal
patterning (McGrew et al.,
1997; Altmann and Brivanlou,
2001
; Thisse et al.,
2000
). Wnt and TGFß signals can operate synergistically
through Lef1 to activate target genes
(Nishita et al., 2000
;
Riese et al., 1997
). In
addition, the Wnt and MAPK pathways work synergistically to reduce repression
of target genes mediated by Lef/Tcf homologs
(Behrens, 2000
;
Meneghini et al., 1999
;
Rocheleau et al., 1999
).
Together these studies suggest a broad role for Lef/Tcf family members in
coordinating the response to multiple signaling pathways.
Opposing gradients of Tcf3-mediated repression and caudalizing
activity in the neurectoderm
Our results are consistent with the emerging view that a gradient of
Wnt/ß-catenin activity helps define discrete domains of gene expression
in the neural plate. As mentioned above, we propose that Tcf3 represses
targets of caudalizing factors, and ß-catenin prevents Tcf3 from being
effective as a repressor, resulting in a rostral-caudal gradient of effective
Tcf3 repression (Fig. 7A,
broken lines). This effect of the Tcf3 repression gradient could be
represented by lowering the rostral end of a caudalizing gradient
(Fig. 7B, broken purple line).
Loss of basal Tcf3 function would thus raise the low end of the caudalizing
gradient, decreasing its slope (Fig.
7C,D). In the context of this gradient, specific thresholds of
caudalizing activity define discrete windows of gene expression along the
rostral-caudal axis (Fig.
7B-D). Progressive loss of Tcf3 function would cause loss of
rostral and expansion of caudal gene expression domains
(Fig. 7C,D).
|
The gbx1 gene is consistently expressed just caudal to pax2.1, defining a compartment expected to depend on a slightly higher window of caudalizing activity. Expansion of the gbx1 domain around the pax2.1 domain in severely caudalized embryos suggests that pax2.1 represents the low end of the caudalizing gradient in these embryos and that the surrounding gbx1 expression reflects a slightly higher caudalizing activity. Indeed, the arc-like rostral expansion of gbx1 in caudalized embryos resembles the arc-like expression of pax6 in wild-type embryos. These observations imply that the low end of the caudalizing gradient is not at the rostral edge of the neural plate, but rather in a slightly caudal domain that is surrounded by pax6 in wild-type embryos and surrounded by gbx1 in severely caudalized embryos.
We have shown here and in previous analysis of hdl mutants
(Kim et al., 2000) that loss
of Tcf3 function leads to changes in patterning that become evident by early
gastrulation. Embryos treated with lithium chloride soon after the shield
stage are caudalized in a manner similar to hdl mutants
(van de Water et al., 2001
;
Kim et al., 2002
). At shield
stage the ventrolateral blastoderm margin in zebrafish is the source of
caudalizing factors and corresponds with the highest ß-catenin activity
(Woo and Fraser, 1997
;
Dorsky et al., 2002
). If
caudalizing factors at the ventrolateral margin help establish the gradient of
caudalizing activity, the low end of the gradient should be located at the
furthest distance from the source, slightly dorsal to the animal pole
(Fig. 7E). At the same time,
BMP antagonists secreted at the dorsal margin define the prospective
neurectoderm (Grinblat et al.,
1998
) (Fig. 7F).
Different levels of caudalizing activity in the prospective neurectoderm are
expected to define discrete domains of gene expression
(Fig. 7G,H). The yellow, green
and blue compartments defined by different thresholds of caudalizing activity
illustrate how expression of pax6, pax2.1 and gbx1,
respectively, might be determined in wild-type embryos. As described above,
loss of Tcf3-mediated repression is expected to alter the shape of the
caudalizing gradient (Fig.
7I,K) and thus alter pax6, pax2.1 and gbx1
expression domains (Fig. 7J,L).
This model provides a potential explanation for the arc-shaped early
expression of pax6 in wild-type embryos and illustrates why
gbx1 expression would expand rostrally around an oval pax2.1
expression domain in severely caudalized embryos.
The shape of the caudalizing gradient can also be influenced by factors
that inhibit function of Wnts. As gastrulation proceeds, the prechordal plate,
which is the source of at least one secreted Wnt antagonist, Dkk1
(Hashimoto et al., 2000;
Shinya et al., 2000
), might
help define the low point of the Wnt activity gradient in the overlying
rostral neurectoderm. Furthermore, during gastrulation the anterior neural
ridge also becomes a source of a Wnt antagonist, Tlc
(Houart et al., 2002
), and it
probably contributes to the pattern of Wnt-mediated derepression as
gastrulation is completed. Clearly, other factors such as cell and tissue
movements contribute to patterning of the neurectoderm throughout this
process. However, for simplicity their contribution is not emphasized in our
model, which represents a static view at the beginning of gastrulation.
tcf3b is uniquely required for rhombomere boundary
formation
Following injection of the tcf3b MO, we observed a marked defect
in morphogenesis of the MHB and hindbrain rhombomeres. Although we were unable
to rescue this phenotype by overexpressing either gene, we believe it is
specific to tcf3b because we never observed hindbrain defects in
other MO-injected embryos. Our data predict that Tcf3b might affect the
expression of genes involved in hindbrain morphogenesis. One obvious target
for further investigation would be the Ephrin/Eph family of receptor tyrosine
kinases and ligands, which have been demonstrated to play a role in cell
sorting and boundary formation in the hindbrain
(Cooke et al., 2001;
Lumsden, 1999
).
Redundant and unique functions of hdl and
tcf3b
The hdl gene plays a unique role in forebrain patterning during
development. Likewise, injection of the tcf3b MO produced unique
phenotypes in hindbrain and MHB morphogenesis. Because our rescue experiments
indicate that hdl and tcf3b encode proteins that can
function identically, some of these unique roles can be explained by
nonoverlapping expression patterns of the two genes. This may be true in the
hindbrain and MHB as well, where we observed subtle differences in the
expression patterns of hdl and tcf3b
(Fig. 2K,L). Alternatively, the
two genes may encode proteins with different DNA targets or transcriptional
cofactors in the hindbrain and MHB, and the function encoded by hdl
may be dispensable. Our inability to rescue the tcf3b MO phenotype
with either gene leaves these possibilities open.
In some tissues in which either one or both genes are expressed, we observed no phenotype in our MO injections. For example, both hdl and tcf3b are expressed in the notochord, but no obvious notochord defects were seen in MO-injected embryos. The function of hdl in the tailbud and paraxial mesoderm is unclear as well, as neither MO-injected embryos nor hdl mutants exhibit patterning defects in these tissues. Loss of hdl and tcf3b function prior to gastrulation resulted in minimal effects on initial dorsal-ventral patterning. The most probable explanation for these results is that in zebrafish, other genes are able to compensate for hdl and tcf3b in these regions.
In this study, we have demonstrated specific and overlapping developmental roles for two zebrafish tcf3 genes. Our results suggest regions in the embryo where Tcf3 function may be important for patterning and morphogenesis. It will now be important to identify the transcriptional targets of Hdl and Tcf3b in these regions so the cellular responses to this pathway become clear. In addition, the biochemical differences between Lef/Tcf proteins must be further investigated, so that both their redundancies and distinct functions can be better understood.
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ACKNOWLEDGMENTS |
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Footnotes |
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REFERENCES |
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---|
Altmann, C. R. and Brivanlou, A. H. (2001). Neural patterning in the vertebrate embryo. Int. Rev. Cytol. 203,447 -482.[Medline]
Barker, N., Morin, P. J. and Clevers, H. (2000). The Yin-Yang of TCF/betacatenin signaling. Adv. Cancer Res. 77,1 -24.[Medline]
Behrens, J. (2000). Cross-regulation of the Wnt
signalling pathway: a role of MAP kinases. J. Cell
Sci. 113,911
-919.
Brannon, M., Gomperts, M., Sumoy, L., Moon, R. T. and Kimelman,
D. (1997). A beta-catenin/XTcf-3 complex binds to the
siamois promoter to regulate dorsal axis specification in
Xenopus. Genes Dev. 11,2359
-2370.
Chan, S. K. and Struhl, G. (2002). Evidence that armadillo transduces wingless by mediating nuclear export or cytosolic activation of pangolin. Cell 111,265 -280.[Medline]
Cooke, J., Moens, C., Roth, L., Durbin, L., Shiomi, K., Brennan,
C., Kimmel, C., Wilson, S. and Holder, N. (2001). Eph
signalling functions downstream of Val to regulate cell sorting and boundary
formation in the caudal hindbrain. Development
128,571
-580.
DasGupta, R. and Fuchs, E. (1999). Multiple
roles for activated LEF/TCF transcription complexes during hair follicle
development and differentiation. Development
126,4557
-4568.
Domingos, P. M., Itasaki, N., Jones, C. M., Mercurio, S., Sargent, M. G., Smith, J. C. and Krumlauf, R. (2001). The Wnt/beta-catenin pathway posteriorizes neural tissue in Xenopus by an indirect mechanism requiring FGF signalling. Dev. Biol. 239,148 -160.[CrossRef][Medline]
Dorsky, R. I., Moon, R. T. and Raible, D. W. (1998). Control of neural crest cell fate by the Wnt signalling pathway. Nature 396,370 -373.[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]
Dorsky, R. I., Snyder, A., Cretekos, C. J., Grunwald, D. J., Geisler, R., Haffter, P., Moon, R. T. and Raible, D. W. (1999). Maternal and embryonic expression of zebrafish lef1.Mech. Dev. 86,147 -150.[CrossRef][Medline]
Ekker, M., Wegner, J., Akimenko, M. A. and Westerfield, M.
(1992). Coordinate embryonic expression of three zebrafish
engrailed genes. Development
116,1001
-1010.
Eroshkin, F., Kazanskaya, O., Martynova, N. and Zaraisky, A. (2002). Characterization of cis-regulatory elements of the homeobox gene Xanf-1. Gene 285,279 -286.[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]
Grinblat, Y., Gamse, J., Patel, M. and Sive, H.
(1998). Determination of the zebrafish forebrain: induction and
patterning. Development
125,4403
-4416.
Hashimoto, H., Itoh, M., Yamanaka, Y., Yamashita, S., Shimizu, T., Solnica-Krezel, L., Hibi, M. and Hirano, T. (2000). Zebrafish Dkk1 functions in forebrain specification and axial mesendoderm formation. Dev. Biol. 217,138 -152.[CrossRef][Medline]
Heasman, J., Kofron, M. and Wylie, C. (2000). Beta-catenin signaling activity dissected in the early Xenopus embryo: a novel antisense approach. Dev. Biol. 222,124 -134.[CrossRef][Medline]
Houart, C., Caneparo, L., Heisenberg, C., Barth, K., Take-Uchi, M. and Wilson, S. (2002). Establishment of the telencephalon during gastrulation by local antagonism of Wnt signaling. Neuron 35,255 -265.[Medline]
Houston, D. W., Kofron, M., Resnik, E., Langland, R., Destree,
O., Wylie, C. and Heasman, J. (2002). Repression of organizer
genes in dorsal and ventral Xenopus cells mediated by maternal XTcf3.
Development 129,4015
-4025.
Inoue, A., Takahashi, M., Hatta, K., Hotta, Y. and Okamoto, H. (1994). Developmental regulation of islet-1 mRNA expression during neuronal differentiation in embryonic zebrafish. Dev. Dyn. 199,1 -11.[Medline]
Itoh, M., Kudoh, T., Dedekian, M., Kim, C. H. and Chitnis, A. B. (2002). A role for iro1 and iro7 in the establishment of an anteroposterior compartment of the ectoderm adjacent to the midbrain-hindbrain boundary. Development 129,2317 -2327.[Medline]
Jowett, T. (2001). Double in situ hybridization techniques in zebrafish. Methods 23,345 -358.[CrossRef][Medline]
Kengaku, M., Capdevila, J., Rodriguez-Esteban, C., De La Pena,
J., Johnson, R. L., Belmonte, J. C. I. and Tabin, C. J.
(1998). Distinct WNT pathways regulating AER formation and
dorsoventral polarity in the chick limb bud. Science
280,1274
-1277.
Kiecker, C. and Niehrs, C. (2001). A morphogen
gradient of Wnt/beta-catenin signalling regulates anteroposterior neural
patterning in Xenopus. Development
128,4189
-4201.
Kim, C. H., Oda, T., Itoh, M., Jiang, D., Artinger, K. B., Chandrasekharappa, S. C., Driever, W. and Chitnis, A. B. (2000). Repressor activity of Headless/Tcf3 is essential for vertebrate head formation. Nature 407,913 -916.[CrossRef][Medline]
Kim, S. H., Shin, J., Park, H. C., Yeo, S. Y., Hong, S. K., Han,
S., Rhee, M., Kim, C. H., Chitnis, A. B. and Huh, T. L.
(2002). Specification of an anterior neuroectoderm patterning by
Frizzled8a-mediated Wnt8b signalling during late gastrulation in zebrafish.
Development 129,4443
-4455.
Krauss, S., Johansen, T., Korzh, V. and Fjose, A. (1991a). Expression of the zebrafish paired box gene pax[zf-b] during early neurogenesis. Development 113,1193 -1206.[Abstract]
Krauss, S., Johansen, T., Korzh, V., Moens, U., Ericson, J. U. and Fjose, A. (1991b). Zebrafish pax[zf-a]: a paired box-containing gene expressed in the neural tube. EMBO J. 10,3609 -3619.[Abstract]
Labbe, E., Letamendia, A. and Attisano, L.
(2000). Association of Smads with lymphoid enhancer binding
factor 1/T cell-specific factor mediates cooperative signaling by the
transforming growth factor-beta and wnt pathways. Proc. Natl. Acad.
Sci. USA 97,8358
-8363.
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.[Medline]
Lumsden, A. (1999). Closing in on rhombomere boundaries. Nat. Cell Biol. 1, E83-85.[CrossRef][Medline]
McGrew, L. L., Hoppler, S. and Moon, R. T. (1997). Wnt and FGF pathways cooperatively pattern anteroposterior neural ectoderm in Xenopus. Mech. Dev. 69, 1-2.
McGrew, L. L., Lai, C. J. and Moon, R. T. (1995). Specification of the anteroposterior neural axis through synergistic interaction of the Wnt signaling cascade with noggin and follistatin. Dev. Biol. 172,337 -342.[CrossRef][Medline]
Meneghini, M. D., Ishitani, T., Carter, J. C., Hisamoto, N., Ninomiya-Tsuji, J., Thorpe, C. J., Hamill, D. R., Matsumoto, K. and Bowerman, B. (1999). MAP kinase and Wnt pathways converge to downregulate an HMG-domain repressor in Caenorhabditis elegans.Nature 399,793 -797.[CrossRef][Medline]
Merrill, B. J., Gat, U., DasGupta, R. and Fuchs, E.
(2001). Tcf3 and Lef1 regulate lineage differentiation of
multipotent stem cells in skin. Genes Dev.
15,1688
-1705.
Nasevicius, A. and Ekker, S. C. (2000). Effective targeted gene `knockdown' in zebrafish. Nat. Genet. 26,216 -220.[CrossRef][Medline]
Nishita, M., Hashimoto, M. K., Ogata, S., Laurent, M. N., Ueno, N., Shibuya, H. and Cho, K. W. (2000). Interaction between Wnt and TGF-beta signalling pathways during formation of Spemann's organizer. Nature 403,781 -785.[CrossRef][Medline]
Nordstrom, U., Jessell, T. M. and Edlund, T. (2002). Progressive induction of caudal neural character by graded Wnt signaling. Nat. Neurosci. 5, 525-532.[CrossRef][Medline]
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]
Popperl, H., Rikhof, H., Chang, H., Haffter, P., Kimmel, C. B. and Moens, C. B. (2000). lazarus is a novel pbx gene that globally mediates hox gene function in zebrafish. Mol. Cell 6,255 -267.[Medline]
Riese, J., Yu, X., Munnerlyn, A., Eresh, S., Hsu, S. C., Grosschedl, R. and Bienz, M. (1997). LEF-1, a nuclear factor coordinating signaling inputs from wingless and decapentaplegic. Cell 88,777 -787.[Medline]
Rocheleau, C. E., Yasuda, J., Shin, T. H., Lin, R., Sawa, H., Okano, H., Priess, J. R., Davis, R. J. and Mello, C. C. (1999). WRM-1 activates the LIT-1 protein kinase to transduce anterior/posterior polarity signals in C. elegans.Cell 97,717 -726.[Medline]
Roel, G., Hamilton, F. S., Gent, Y., Bain, A. A., Destree, O. and Hoppler, S. (2002). Lef-1 and Tcf-3 transcription factors mediate tissue-specific Wnt signaling during Xenopus development. Curr. Biol. 12,1941 -1945.[CrossRef][Medline]
Schreiber-Agus, N., Horner, J., Torres, R., Chiu, F. C. and DePinho, R. A. (1993). Zebrafish myc family and max genes: differential expression and oncogenic activity throughout vertebrate evolution. Mol. Cell. Biol. 13,2765 -2775.[Abstract]
Shinya, M., Eschbach, C., Clark, M., Lehrach, H. and Furutani-Seiki, M. (2000). Zebrafish Dkk1, induced by the pre-MBT Wnt signaling, is secreted from the prechordal plate and patterns the anterior neural plate. Mech. Dev. 98, 3-17.[CrossRef][Medline]
Stachel, S. E., Grunwald, D. J. and Myers, P. Z.
(1993). Lithium perturbation and goosecoid expression
identify a dorsal specification pathway in the pregastrula zebrafish.
Development 117,1261
-1274.
Thisse, B., Wright, C. V. and Thisse, C. (2000). Activin- and nodal-related factors control antero-posterior patterning of the zebrafish embryo. Nature 403,425 -428.[CrossRef][Medline]
Travis, A., Amsterdam, A., Belanger, C. and Grosschedl, R. (1991). LEF-1, a gene encoding a lymphoid-specific protein with an HMG domain, regulates T-cell receptor alpha enhancer function. Genes Dev. 5,880 -894.[Abstract]
Turner, D. L. and Weintraub, H. (1994). Expression of achaete-scute homolog 3 in Xenopus embryos converts ectodermal cells to a neural fate. Genes Dev. 8,1434 -1447.[Abstract]
van de Water, S., van de Wetering, M., Joore, J., Esseling, J., Bink, R., Clevers, H. and Zivkovic, D. (2001). Ectopic Wnt signal determines the eyeless phenotype of zebrafish masterblind mutant. Development 128,3877 -3888.[Medline]
van de Wetering, M., Oosterwegel, M., Dooijes, D. and Clevers, H. (1991). Identification and cloning of TCF-1, a T lymphocyte-specific transcription factor containing a sequence-specific HMG box. EMBO J. 10,123 -132.[Abstract]
Woo, K. and Fraser, S. E. (1997). Specification
of the zebrafish nervous system by nonaxial signals.
Science 277,254
-257.