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First published online 28 January 2009
doi: 10.1242/dev.027995
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1 Department of Neurobiology and Anatomy, University of Utah School of Medicine,
Salt Lake City, UT 84132, USA.
2 The Brain Institute, University of Utah School of Medicine, Salt Lake City, UT
84132, USA.
¶ Author for correspondence (e-mail: richard.dorsky{at}neuro.utah.edu)
Accepted 19 December 2008
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SUMMARY |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Key words: Zebrafish, Tcf3, Spinal progenitors, Wnt
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INTRODUCTION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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).
However, several important unanswered questions remain, including the
mechanisms underlying progenitor cell maintenance and neurogenesis.
Previous work has shown that secreted signaling molecules such as sonic
hedgehog (Shh) and bone morphogenetic protein (BMP) act to pattern the spinal
cord, instructing progenitors of their position in the dorsoventral (DV) axis
(Briscoe and Ericson, 2001;
Lee and Jessell, 1999). More
recently, it has been shown that Wnt signaling from the dorsal spinal cord
also specifies dorsal progenitor fates
(Alvarez-Medina et al., 2008;
Bonner et al., 2008;
Zechner et al., 2007).
Interestingly, the mechanisms underlying specification of intermediate spinal
cord progenitors are less clear. Neither Shh nor BMP signaling are required
for the expression of Dbx genes in intermediate progenitors
(Gribble et al., 2007), and we
and others have shown that Wnt signaling acts to prevent these genes from
being expressed in more dorsal regions
(Alvarez-Medina et al., 2008;
Bonner et al., 2008). Our
laboratory has also shown that another candidate inductive signal, retinoic
acid, is not absolutely required for Dbx expression in the spinal cord
(Gribble et al., 2007). This
leaves an open question regarding the mechanism responsible for inducing Dbx
expression, a topic of considerable interest due to the speculation that
Dbx-positive progenitors may represent a stem-cell population
(Fogarty et al., 2005).
Canonical Wnt signaling can also promote proliferation in spinal
progenitors (Bonner et al.,
2008; Ille et al.,
2007; Megason and McMahon,
2002; Zechner et al.,
2003), but the source of this signal is less clear. By contrast,
relatively little is known about the role of Wnt signaling in the process of
spinal cord neurogenesis. Manipulation of the Wnt pathway can affect the
ultimate number of spinal neurons produced
(Ille et al., 2007;
Megason and McMahon, 2002;
Zechner et al., 2003), but it
is unclear whether this is primarily a downstream result of altered progenitor
proliferation, or instead represents a direct response of progenitors to
initiate a program of neurogenesis. In other regions of the CNS, Wnt signaling
has been shown to directly regulate the expression of genes that control
neurogenesis (Lee et al.,
2006; Lie et al.,
2005; Machon et al.,
2007; Van Raay et al.,
2005), suggesting that this process is conserved in the spinal
cord.
Lef/Tcf proteins are responsible for mediating the transcriptional output
of canonical Wnt signals. These proteins can act as transcriptional activators
or repressors (Bienz, 1998;
Brantjes et al., 2002), and
have been shown to regulate specific target genes during CNS development
downstream of Wnt signaling (Lee et al.,
2006; Takemoto et al.,
2006). All vertebrates examined express Tcf7 and
Tcf3 in the spinal cord
(Alvarez-Medina et al., 2008;
Merrill et al., 2004;
Schmidt et al., 2004;
Veien et al., 2005), while
Tcf4 is expressed in chick and mouse
(Alvarez-Medina et al., 2008;
Lei et al., 2006) but not in
zebrafish spinal cord (Young et al.,
2002). We and others have described a role for Tcf7 in mediating
the dorsal patterning activity of Wnts from the roof plate
(Alvarez-Medina et al., 2008;
Bonner et al., 2008). By
contrast, Tcf3 and Tcf4 are expressed in a complementary pattern to canonical
Wnt signals (Alvarez-Medina et al.,
2008; Bonner et al.,
2008), suggesting that they may act to antagonize Wnt function.
Consistent with this model, Tcf4 appears to act primarily as a repressor in
the absence of Wnt signaling, helping to refine the dorsal boundary of the
ventral progenitor gene nkx2.2
(Lei et al., 2006). To date,
no study has examined the function of Tcf3 in the spinal cord.
All in vivo evidence thus far describes Tcf3 as a transcriptional
repressor. The Tcf3 knockout mouse exhibits defects in rostrocaudal
neural patterning and a duplicated node and notochord
(Merrill et al., 2004), which
are consistent with increased Wnt function. Mouse embryonic skin progenitors
require Tcf3 to repress terminal differentiation programs
(Nguyen et al., 2006), and in
Xenopus, depletion of Xtcf3 upregulates known Wnt target genes in the
organizer such as siamois, goosecoid, Xnr3 and chordin
(Houston et al., 2002).
Importantly, forebrain phenotypes in zebrafish tcf3a
(headless) mutants can be rescued by injection of truncated Tcf3a
fused to the Engrailed repressor domain
(Kim et al., 2000). It is
therefore likely that Tcf3 primarily functions to repress target genes in the
absence of Wnt activity.
Zebrafish have two tcf3 genes that are redundantly expressed,
required cooperatively in early zebrafish development, and encode proteins
with 82% identity (Dorsky et al.,
2003). Here we demonstrate that both tcf3 genes are
expressed in overlapping domains in the spinal cord, but are not generally
required for DV patterning. We do find, however, that Tcf3 function is
required specifically for the expression of Dbx genes in intermediate spinal
progenitors. In addition, we find that Tcf3 function prevents spinal
progenitors from prematurely undergoing neurogenesis. In the absence of Tcf3
function, proliferating progenitors ectopically express neuronal markers, but
do not produce specified neuronal subtypes. We show that Tcf3 functions as a
repressor of sox4a, a gene known to regulate neurogenesis, and that
sox4a acts downstream of Tcf3 in the spinal cord. Collectively, these
results establish a novel biological role for Tcf3 in maintaining a progenitor
state in the spinal cord.
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MATERIALS AND METHODS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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tcf-GFP)w26
(Lewis et al., 2004) zebrafish
lines. tcf3a-/-;tcf3b-/- double
mutants were produced from a heterozygous incross carrying two mutations:
tcf7l1am881 and a retroviral insertion in the first exon
of tcf3b (Znomics). Double mutants were confirmed by PCR genotyping.
All developmental stages in this study are reported in hours
post-fertilization (hpf) at 28.5°C, according to Kimmel et al.
(Kimmel et al., 1995).
Morpholino injections
The sequences of tcf3a and tcf3b translation and
splice-blocking morpholinos have been published previously
(Bonner et al., 2008;
Dorsky et al., 2003). The
sequence of the sox4a translation-blocking morpholino is:
5'-CATGCACTACAACAGTCTCAACTTT-3'. Translation-blocking morpholinos
at 1 ng/nl (tcf3a/b) or 2 ng/nl (sox4a) and splice-blocking
morpholinos at 5 ng/nl were injected into one-cell stage embryos. The
p53 morpholino (Robu et al.,
2007) was co-injected at 5 ng/nl.
RT-PCR
Total RNA from 75 embryos at 18 hpf was isolated using Trizol reagent and
was reverse transcribed by random hexamers using the SuperScript First-Strand
Synthesis System (Invitrogen). PCR was performed for 30 cycles using an
annealing temperature of 57°C (tcf3a) and 60°C
(tcf3b), and reactions were visualized on 1% agarose gels in
Tris-acetate-EDTA. The spliced product for tcf3a is 257 bp and the
unspliced product is 354 bp. The spliced product in tcf3b is 205 bp
and the unspliced product is approximately 3 kb.
In situ hybridization
Probe synthesis and in situ hybridization were performed as described
previously (Oxtoby and Jowett,
1993), using digoxigenin-labeled antisense RNA probes and BM
Purple (Roche). Double in situ hybridizations were carried out using
digoxigenin- and fluorescein-labeled antisense RNA probes
(Jowett, 2001) and visualized
using BM Purple (Roche) and Fast Red (Roche). The following probes were made
by our lab: tcf3a (tcf7l1a - Zebrafish Information Network),
tcf3b (tcf7l1b), tubb5, lhx1a, dbx1a, dbx2, sox4a,
sox11a and zic2b. We were given the following probes:
iro3 (irx3a) (Lewis et
al., 2005), pax2a
(Krauss et al., 1991),
pax3 (Seo et al.,
1998), nkx6.1
(Cheesman et al., 2004),
olig2 (Park et al.,
2002), nkx2.2 (Barth
and Wilson, 1995), pcna
(Lee and Gye, 1999),
cdkn1c (Park et al.,
2005), isl1 (Okamoto
et al., 2000), evx1
(Thaeron et al., 2000),
sox3 (Kudoh et al.,
2004) and en1b (eng1b - Zebrafish Information
Network) (Higashijima et al.,
2004). For whole-mount photography after all staining methods,
embryos were de-yolked and mounted laterally or cryosectioned.
Immunohistochemistry
Cryosections (12 µm) were incubated in Alexa Fluor 594-conjugated
(1:1000, Molecular Probes) or unconjugated BrdU (1:1000, DSHB),
phospho-histone H3 (1:1000, Upstate), and HuC/D (1:1000, Molecular Probes)
antibodies for 2 hours at room temperature. Sections were washed and incubated
in anti-mouse Alexa Fluor 488 or Cy3 secondary antibodies (1:200, Molecular
Probes) for 1 hour at room temperature. Slides were washed and mounted in
Vectashield (Vector Laboratories). Whole-mount embryos were incubated in
phospho-histone H3 antibody at 1:500 overnight at room temperature, then
incubated in anti-mouse HRP-conjugated secondary antibody (1:200, Molecular
Probes) overnight, followed by DAB staining.
BrdU labeling
Embryos at 18 or 24 hpf were incubated in 10 mM BrdU solution for 20
minutes, then fixed immediately (for determination of labeling index), or
incubated for 6 hours (for HuC/D double-labeling) then fixed. Following
cryosectioning, embryos were incubated for 1 hour in 2M HCl followed by
immunohistochemistry. For determination of labeling index, sections were
counterstained with propidium iodide.
Tcf3 heat-shock experiments
Tg(hsp70l: tcf-GFP)w26 heterozygous
males were mated with wild-type females and embryos were injected with the
tcf3 morpholinos. All embryos were heat-shocked at 18 hpf for 1 hour
at 37-39°C and fixed at 24 hpf for analysis.
mRNA injections
sox4a mRNA was synthesized from a sox4a-pCS2+ plasmid,
using the SP6 mMESSAGE mMACHINE Transcription Kit (Ambion). Approximately 1 ng
sox4a mRNA was injected into one-cell-stage wild-type embryos.
Western blotting
Dechorionated wild-type, morphant and heat-shocked
Tg(hsp70l:tcf-GFP)w26 embryos were
homogenized in 4x sample buffer, subjected to 8% SDS-PAGE, and blotted
onto polyvinylidene fluoride membrane. Affinity-purified rabbit anti-Tcf3a
serum (Open Biosystems) was applied at 1:150 dilution, and anti-rabbit IgG-HRP
(Molecular Probes) was applied at 1:10,000. The secondary antibody was
visualized with an electrogenerated chemiluminescence reaction, using standard
protocols.
Chromatin immunoprecipitation
Chromatin immunoprecipitation (ChIP) analysis was performed as described
previously (Lee et al., 2006),
using a polyclonal Tcf3a antibody (Open Biosystems). We used chromatin lysates
from whole 24 hpf embryos, and following cross-linking the DNA was sonicated
to <500 bp. Following immunoprecipitation, PCR was performed on eluted DNA,
total input chromatin, and no antibody controls, for 38-40 cycles using the
following primers: -4.8 kb fragment-L,
5'-TCCAAGAATCTATCACTTTTCTTGTTT-3'; -4.8 kb fragment-R,
5'-TCAATCCAAGGTGATGTAGCC-3'; +1.6 kb fragment-L,
5'-TGGTTGTTTTGCTTCGAGTG-3'; +1.6 kb fragment-R,
5'-AAAGCCAGCCAATTGTGTC-3'; +7.4 kb fragment-L,
5'-GCAGGCGCACTAAAACTACC-3'; and +7.4 kb fragment-R,
5'-AGTGCATGATATCGGACAAGG-3'. Each assay was performed in
triplicate to confirm positive and negative signals.
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RESULTS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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; Kim et al.,
2000). To determine the expression of tcf3 in the
developing spinal cord we performed in situ hybridization for tcf3a
and tcf3b during the period when spinal cord progenitors are
proliferating, patterned and producing postmitotic neurons. We found that at
similar axial levels tcf3a and tcf3b expression remained
consistent from 15-24 hpf (Fig.
1A-F). Both genes were expressed in the intermediate and ventral
spinal cord but were restricted from the most dorsal region until at least 30
hpf, the latest timepoint analyzed (not shown). This pattern is complementary
to expression of dorsal Wnt genes, activated β-catenin and a
Wnt-responsive reporter transgene (Bonner
et al., 2008) and suggests that Tcf3 may function as a repressor
in the absence of canonical Wnt signaling.
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), suggesting that the m881 allele may not represent
a null mutation. Injection of the tcf3b morpholino alone produced no
significant morphological phenotypes (not shown). Injection of both
tcf3a and tcf3b morpholinos resulted in embryos with minimal
brain tissue rostral to the midbrain-hindbrain boundary and a shortened
rostrocaudal axis (Fig. 1I,J).
The morphological phenotypes generated with the splice-blocking morpholinos
were similar to those generated with translation-blocking morpholinos
(Dorsky et al., 2003),
suggesting that zygotic expression of both genes is required cooperatively for
normal patterning.
Because of the overlapping spinal cord expression patterns and the compound
effect of injecting both morpholinos simultaneously, all subsequent phenotypes
were analyzed following co-injection of tcf3a and tcf3b
morpholinos, and we refer to these embryos as `tcf3 morphants'. We
also observed similar spinal cord phenotypes after co-injecting
translation-blocking morpholinos for tcf3a and tcf3b
(Dorsky et al., 2003), despite
more severe morphological defects (not shown). Furthermore, embryos doubly
homozgyous for mutations in tcf3a and tcf3b (see Materials
and methods for further details) showed similar morphological defects to
tcf3 morphants, as well as similar defects in spinal cord development
(not shown). Finally, neither tcf3a nor tcf3b splice
morpholinos produced any phenotypes in the spinal cord when injected alone
(not shown). Together, these results suggest that the spinal cord phenotypes
produced by the splice-blocking morpholinos are specific, and thus all assays
carried out in this study were performed using these reagents. We observed
that the tcf3b splice-blocking morpholino produced one non-specific
effect: widespread cell death throughout the embryos at 18 hpf and beyond (not
shown). This apoptosis was due to p53-dependent off-target effects
(Robu et al., 2007), and was
completely abolished by co-injection with a p53 morpholino. We
therefore injected both tcf3 morpholinos with a p53
morpholino for all subsequent analysis.
Tcf3 is not generally required for dorsoventral spinal cord patterning
Because previous work has shown that canonical Wnt signaling is essential
for dorsal progenitor patterning and interneuron specification
(Alvarez-Medina et al., 2008;
Bonner et al., 2008;
Ille et al., 2007;
Zechner et al., 2007), we
asked whether tcf3 morphant spinal cords were mis-patterned. In a
previous study, we concluded that Tcf3 function was not required for DV
patterning of several markers at 24 hpf
(Bonner et al., 2008). To
confirm and extend these findings, we assayed for expression of multiple genes
at 18 hpf, a time at which there are some differentiated neurons but the
majority of cells are still progenitors. By 18 hpf many well-characterized
transcription factors, which define regions of the spinal cord, are expressed,
including zic2b, pax3, iro3, nkx6.1, olig2 and nkx2.2. We
did not observe a dorsoventral shift in the expression of any of these
markers, indicating that patterning in this axis was unaffected
(Fig. 2). As each of these
markers demarcates a specific region of the spinal cord and collectively they
encompass the dorsoventral axis, we can conclude from these data that Tcf3 is
not generally required for DV patterning of spinal progenitors.
Tcf3 is required for dbx1a expression in spinal progenitors
In our previous work, we noted that although Tcf3 did not generally affect
DV patterning, it was specifically required for dbx2 expression at 24
hpf (Bonner et al., 2008). To
extend these observations, we examined the expression of dbx1a, which
normally arises earlier than dbx2
(Gribble et al., 2007). In
addition to marking a specific domain in the spinal cord, in mouse embryos
Dbx1 is expressed in a unique class of progenitors that gives rise to
neurons, astrocytes and oligodendrocytes
(Fogarty et al., 2005;
Pierani et al., 2001). We
found that tcf3 morphants have discontinuous expression of
dbx1a in spinal cord progenitors at 18 hpf throughout the
rostrocaudal axis (Fig.
3A,B'). Double in situ hybridization in wild-type embryos
shows that the dorsal limit of tcf3 expression overlaps with
dbx1a expression (Fig.
3C,D). Because dbx1a marks a specific domain of spinal
progenitors, we wanted to confirm that the cells had not adopted an
alternative positional identity in tcf3 morphants. We determined
whether the affected cells were still present by examining expression of two
adjacent genes unaffected in tcf3 morphants: pax3 and
nkx6.1 (Fig. 2). In
wild-type embryos a clear two-cell gap in expression of pax3 and
nkx6.1 was observed (Fig.
3E). In tcf3 morphants, we still observed a gap between
pax3 and nkx6.1 (Fig.
3F), suggesting that these cells are physically present and occupy
the proper DV position. In addition, we previously showed that another dorsal
marker adjacent to the Dbx domain, msxc, was unaffected in
tcf3 morphants (Bonner et al.,
2008). We conclude that intermediate progenitors in tcf3
morphants have the appropriate positional identity and specifically fail to
express multiple Dbx genes.
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;
Ille et al., 2007;
Megason and McMahon, 2002;
Zechner et al., 2003). In
addition, we previously showed that loss of Tcf3 function results in a
significant reduction in mitotic index at 24 hpf
(Bonner et al., 2008). To
determine whether progenitors lacking Tcf3 are forced to exit the cell cycle,
we performed several assays. First, we analyzed the mitotic index at 18 hpf,
when we observed a loss of dbx1a expression. Staining with the
M-phase marker phospho-histone H3 (pH3) showed a small but significant
decrease in mitotic index in tcf3 morphants
(Fig. 4A-C), indicating that
the phenotype at 18 hpf is less severe than the one we observed at 24 hpf. In
addition we determined the BrdU labeling index at 18 hpf for wild-type and
tcf3 morphants using a short (20 minute) pulse of BrdU labeling. We
found that the BrdU labeling index was also slightly but significantly
decreased in tcf3 morphants compared with controls
(Fig. 4D-F). We next performed
in situ hybridization for the proliferative marker pcna
(Lee and Gye, 1999) and
cdkn1c (p57), which is upregulated upon cell cycle exit
(Park et al., 2005). These two
assays allowed us to detect gross changes in cell cycle state. Importantly, we
did not observe loss of pcna or ectopic cdkn1c in medial
progenitor cells (Fig. 4G-J),
demonstrating that the majority of progenitors remain proliferative in
tcf3 morphants. Together, these results suggest that loss of Tcf3
results in a slower overall cell cycle time and/or arrest before the G2/M
transition.
Spinal progenitors precociously express neuronal markers in tcf3 morphants
We next asked whether Tcf3 function was required for the regulation of
neurogenesis in spinal progenitors, using both general and subtype-specific
postmitotic neuronal markers. We first examined tubulin beta 5
(tubb5), which is expressed in cells as they undergo neurogenesis,
and is the closest homolog to mammalian Tubb3 with respect to
expression pattern (Oehlmann et al.,
2004). At 18 hpf, tcf3 morphants displayed ectopic
tubb5 expression in the medial spinal cord, which normally
exclusively contains progenitor cells (Fig.
5A,B). Overall, 12/30 sections from morphants showed ectopic
tubb5 expression compared with 1/30 sections from wild-type
embryos.
Additionally, at 24 hpf we examined HuC/D protein expression, a
pan-neuronal marker (Park et al.,
2000), in embryos treated with BrdU at 18 hpf. As with
tubb5, we observed ectopic HuC/D expression in the medial spinal cord
(Fig. 5C,D). We found that
18/30 morphant sections showed medial Hu-positive cells, compared with 2/30
wild-type sections. In addition, 15.6±2.6% (s.e.m., n=30
sections) of Hu-positive cells per section were also BrdU-positive.
Significantly, we never observed cells to be double-positive for HuC/D and
BrdU in sections from wild-type embryos (n=30 sections). To confirm
that this phenotype was specific to loss of tcf3a and tcf3b
function, we analyzed embryos homozygous for mutations in both tcf3
genes. After the genotype of individual embryos was confirmed by PCR, they
were sectioned and stained for HuC/D expression. We found that 16/40 sections
from mutant embryos showed ectopic HuC/D-positive cells in the medial spinal
cord (not shown). Together, these results suggest that progenitor cells
lacking Tcf3 precociously adopt neuronal characteristics.
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; Park et al.,
2002). We found that expression of both markers was substantially
reduced in tcf3 morphants (Fig.
5E-H), suggesting that fewer specified interneurons were present.
We next examined evx1, en1b and isl1, which are expressed
earlier than lhx1a and pax2a, and mark specific classes of
zebrafish spinal neurons (Higashijima et
al., 2004; Okamoto et al.,
2000; Thaeron et al.,
2000). We found that tcf3 morphants possessed fewer
evx1+, en1b+ and
isl1+ neurons than controls
(Fig. 5I-N and
Table 1). Together, these data
suggest that although loss of Tcf3 results in precocious neurogenesis, it does
not drive progenitors into specific postmitotic fates. We therefore conclude
that Tcf3 normally acts to coordinate neurogenesis with cell cycle exit and
fate specification, and maintains cells in a progenitor state until they
receive the necessary signals for differentiation.
|
). In
zebrafish, sox4a is normally expressed very weakly in the spinal cord
at 24 hpf (Fig. 6A). By
contrast, we found that in tcf3 morphants sox4a is
ectopically expressed throughout the intermediate and ventral spinal cord
progenitor domain at 24 hpf (Fig.
6B), consistent with precocious expression of neuronal markers. To
test the function of sox4a in zebrafish neurogenesis, we injected
sox4a mRNA into embryos at the one-cell stage and analyzed HuC/D
expression at 24 hpf. We observed ectopic HuC/D-positive cells in the medial
spinal cord (Fig. 6C), similar
to the phenotype in tcf3 morphants
(Fig. 5D). We found that 11/30
sections from sox4a-injected embryos showed medial Hu-positive cells,
compared with 2/30 sections from controls. In addition, following BrdU
labeling at 18 hpf, we found that 8.2±1.5% (s.e.m., n=30
sections) of Hu-positive cells per section were also BrdU-positive at 24 hpf.
These results confirm that the function of sox4a is conserved between
zebrafish and other vertebrates, and suggest that Tcf3 acts upstream of
sox4a to prevent precocious expression of neuronal markers.
In addition, we examined the expression of the group-B Sox gene
sox3, which marks mitotic neural progenitors in the spinal cord and
is expressed reciprocally with Sox4 and Sox11 in other
vertebrates (Bergsland et al.,
2006). Consistent with the ectopic expression of sox4a,
we observed a dramatic reduction in sox3 expression in tcf3
morphants (Fig. 6D,E). We also
examined the expression of other group-C Sox genes to determine which
factors other than Sox4a might normally regulate neurogenesis in the zebrafish
spinal cord. We found that sox11a was normally expressed in both the
progenitor zone and postmitotic neurons
(Fig. 6F), but was unaffected
in tcf3 morphants (not shown). Together, these results suggest that
Tcf3 regulates the normal balance between group-B and group-C Sox
gene expression, thus preventing precocious neurogenesis.
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Tcf3 functions as a repressor upstream of sox4a and neurogenesis
To test whether Tcf3 acts as a transcriptional repressor in spinal
progenitors, we used a transgenic line,
Tg(hsp70l:tcf3-GFP)w26, which inducibly expresses a
dominant-repressor form of Tcf3 (Lewis et
al., 2004). We hypothesized that if Tcf3 functions as a repressor,
then expression of Tcf should epistatically antagonize the morphant
phenotypes. We found that induction of Tcf was able to repress the
expression of sox4a, both alone and in the presence of tcf3
morpholinos (Fig. 7A,B).
Analysis of proliferation in embryos expressing Tcf showed that this
transgene completely inhibits BrdU uptake
(Fig. 7C,D). No BrdU-positive
cells were observed in Tcf-expressing embryos at 24 hpf, following a 20
minute labeling period.
In addition, expression of Tcf inhibited the normal expression of
HuC/D throughout the spinal cord, alone and in tcf3 morphants
(Fig. 7E,F). We found that
uninjected Tcf-expressing embryos had 2.3±0.5 (s.e.m.,
n=15 sections) Hu-positive cells per section, while
Tcf-expressing embryos injected with tcf3 MO had
1.9±0.8 (s.e.m., n=15 sections) Hu-positive cells per section.
By contrast, wild-type controls had 6.2±0.9 (s.e.m., n=15
sections) Hu-positive cells per section. These results indicate that
Tcf3-mediated repression can block cell proliferation, but does not result in
premature neurogenesis. Furthermore, they suggest that Tcf3 functions as a
repressor of sox4a, and that this repressor activity is required to
prevent progenitor cells from precociously expressing neuronal markers.
To determine whether sox4a expression is sufficient to cause
ectopic neurogenesis in the presence of Tcf3-mediated repression, we
overexpressed Tcf in transgenic embryos injected with sox4a
mRNA. Because Tcf overexpression blocks proliferation, we were not able
to assess whether the absolute number of Hu-positive cells was restored
following sox4a injection. However, we did observe ectopic medial
Hu-positive cells in embryos expressing both Tcf and sox4a
(Fig. 7G). We found that 5/30
sections from sox4a-injected embryos showed medial Hu-positive cells,
compared with 0/30 sections from uninjected Tcf-expressing controls. To
test whether sox4a is required for ectopic neurogenesis in the
absence of Tcf3, we simultaneously injected tcf3 morpholinos with a
translation-blocking morpholino for sox4a. Whereas the sox4a
morpholino did not have any effect on spinal cord neurogenesis when injected
alone (not shown), we found that 0/30 sections from tcf3/sox4a
morphants contained ectopic Hu-positive cells
(Fig. 7H). Together, these data
indicate that ectopic expression of sox4a is both necessary and
sufficient to promote precocious neurogenesis in progenitors downstream of
Tcf3.
sox4a is a target of Tcf3 in vivo
Using ChIP analysis we asked whether enhancer regions of sox4a
interact with Tcf3 protein in vivo. For immunoprecipitation, we used a
polyclonal antibody against zebrafish Tcf3a that recognizes a single band of
53 kDa in lysates from wild-type 24 hpf embryos, which is absent in
tcf3 morphant embryo lysates (Fig.
8A). The Tcf3 antibody is also able to detect the Tcf3-GFP
protein in lysates from heat-shocked transgenic embryos
(Fig. 8A). We first identified
two regions of sox4a genomic sequence containing Lef/Tcf consensus
sites within evolutionarily conserved elements
(Fig. 8B), using the UCSC
Genome Browser (Kent et al.,
2002). One site was 4.8 kb upstream of the sox4a start
codon, and the other was 1.6 kb downstream of the start codon, near the
3' end of the cDNA. After sonication and immunoprecipitation of 24 hpf
chromatin extracts with the Tcf3a antibody, we performed PCR to amplify DNA
fragments near these putative binding sites, along with a negative control
fragment that did not contain any consensus sites. We were able to
immunoprecipitate fragments near both conserved sites, but not our negative
control (Fig. 8B). These
experiments indicate that Tcf3 interacts with putative regulatory regions of
sox4a in 24 hpf zebrafish embryos. Therefore, Tcf3 may be a direct
transcriptional repressor of sox4a in the spinal cord.
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|
DISCUSSION |
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SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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), and
other studies have suggested that Tcf4 regulates the dorsal limit of the
ventral progenitor gene nkx2.2
(Lei et al., 2006), our data
indicate that Tcf3 is not required for these functions. We show instead that
Tcf3 is required to inhibit progenitor differentiation in the spinal cord. Our
data indicate that in the absence of Tcf3, a gene implicated in neuronal
differentiation (sox4a) is ectopically expressed, leading to
premature expression of tubb5 and HuC/D in progenitor cells. In
addition, we find that Tcf3 is required for expression of Dbx genes in
intermediate spinal progenitors. Work by ourselves and others has shown that
Dbx expression is required for the proper specification of spinal interneurons
(Gribble et al., 2007;
Pierani et al., 2001), and
several of the cell-type-specific markers analyzed here are known to depend on
Dbx function. Thus the simultaneous loss of Dbx expression and premature
neurogenesis could lead to the production of `generic' or unspecified neurons.
Other factors may be downstream of Tcf3 in the ventral spinal cord, possibly
accounting for the decrease in primary motoneurons we observed in morphants.
Together, these findings suggest that Tcf3 normally acts to coordinate
multiple processes during neurogenesis, and maintains cells in a progenitor
state until they receive all the necessary signals for differentiation.
We found that although the majority of progenitor cells lacking Tcf3
function appeared to remain in the cell cycle, there was a small but
significant decrease in both mitotic index and BrdU labeling index. This
result could reflect either a longer overall cell cycle time, or cell cycle
arrest. Because we did not observe ectopic expression of cdkn1c, it
is unlikely that cells were exiting the cell cycle prematurely, although we
cannot conclusively rule out this possibility. Although we observed cells
double-labeled with Hu and BrdU in both tcf3 morphants and following
sox4a overexpression, these cells were labeled 6 hours before
analysis, and thus it is not clear whether they were still cycling, were
arrested, or had recently exited the cell cycle. By contrast, inhibition of
Wnt signaling by Dkk1 expression (Bonner et
al., 2008), or Tcf expression
(Fig. 7C,D), drastically
reduces cell proliferation in the spinal cord, but neither manipulation
results in increased neurogenesis. Therefore our data best fit a model in
which Tcf3 is not directly required for cell proliferation, but may affect
cell cycle time and/or progression. In this model, Tcf3-mediated repression of
sox4a coordinates neurogenesis with other signals regulating cell
cycle exit. In the absence of Tcf3 or the presence of Sox4a, neuronal markers
are expressed prematurely in progenitors that are either still in, or have
recently exited, the cell cycle.
A recent study (Bergsland et al.,
2006) determined that electroporation of Sox4 and
Sox11 into chick spinal cord induces expression of neuronal markers.
Because our experiments showed ectopic tubb5 and HuC/D expression in
tcf3 morphants, we hypothesized that group-C Sox genes may function
downstream of Tcf3 in zebrafish spinal progenitors. Our data show that Tcf3
acts as a transcriptional repressor of sox4a in the intermediate and
ventral spinal cord. Group-B Sox genes are known targets of canonical
Wnt signaling (Lee et al.,
2006; Takemoto et al.,
2006; Van Raay et al.,
2005), but we have now identified a member of the group-C Sox
family that is normally repressed by Tcf3. Through epistasis experiments we
show that ectopic sox4a expression is both necessary and sufficient
for ectopic neurogenesis in the absence of Tcf3. However, as sox4a is
normally expressed very weakly in the spinal cord at 24 hpf, and knockdown of
sox4a did not affect spinal neurogenesis (not shown), other group-C
Sox factors may play this role during normal development. Interestingly, we
found that sox11a was expressed in both progenitors and postmitotic
neurons (Fig. 6F) but was
unaffected in tcf3 morphants (not shown), suggesting that not all
group-C Sox genes are targets of Tcf3. It is possible that the normal balance
between group-B and group-C Sox factors is sufficient to keep progenitor cells
from differentiating, and the excess group-C Sox activity present in
tcf3 morphants upsets this balance, leading to precocious
differentiation.
We have also shown that Tcf3 functions as a regulator of Dbx genes in
spinal progenitors. Previous work from our laboratory and others has shown
that canonical Wnt signaling is not required for the expression of Dbx genes,
but instead acts to restrict the dorsal boundary of Dbx expression
(Alvarez-Medina et al., 2008;
Bonner et al., 2008).
Furthermore, in these studies expression of the dominant-repressor Tcf
molecule led to expansion, rather than loss, of Dbx genes. We therefore
conclude that Tcf3 acts as a repressor to allow normal Dbx
expression, presumably through an unknown intermediate transcriptional
repressor. Further examination of this regulatory mechanism is warranted, due
to the speculation that Dbx-expressing progenitors may represent a putative
stem-cell population (Fogarty et al.,
2005).
Although Lef/Tcf proteins can function in the presence and absence of Wnts
(Dorsky et al., 2002;
Labbe et al., 2000;
Travis et al., 1991;
van de Wetering et al., 1991),
the lack of Wnt activity in the intermediate and ventral spinal cord
(Bonner et al., 2008) suggests
that Tcf3 primarily acts in a Wnt-independent manner to permit Dbx expression,
as well as to repress sox4a and inhibit neurogenesis. Our data
further indicate that Tcf3 does not normally function to antagonize canonical
Wnt signals from the roof plate, but instead acts on a separate set of targets
in neural progenitors. We have no evidence that other Lef/Tcf proteins (for
example, Tcf7) are capable of activating Wnt targets in regions where Tcf3 is
present, and the phenotypes we observed do not suggest that canonical Wnt
signaling is activated in the absence of Tcf3. At this point it is unclear
whether a Wnt signal or a parallel pathway acts to antagonize Tcf3 and
initiate neurogenesis in spinal progenitors. The restriction of Tcf3 function
to specific regions of the spinal cord may reflect the fact that in zebrafish
at 24 hpf all spinal interneurons are derived from regions at or ventral to
the Dbx expression domain (Gribble et al.,
2007). It is therefore possible that neurogenesis in the dorsal
spinal cord is controlled by a separate mechanism.
It is known that Tcf3 is similarly expressed in the spinal cord of other
vertebrates (Alvarez-Medina et al.,
2008; Schmidt et al.,
2004); however, due to early lethality of Tcf3 knockout mice its
function in the spinal cord has not been studied. Importantly, because Tcf3
normally functions as a repressor, only loss-of-function experiments can be
used to determine its required role, whereas overexpression of
`dominant-negative' forms will mimic its normal function. Although
dominant-activator forms of Tcf could theoretically be used to approximate
loss of Tcf3 function, these reagents suffer from a lack of specificity, as
they may activate non-physiological targets. In chick and mouse, it is
possible that significant functional redundancy exists between Tcf3 and Tcf4,
suggesting that loss-of-function of both genes must be examined to reveal a
severe phenotype. In zebrafish, Tcf3 is the only Lef/Tcf factor expressed in
the intermediate and ventral spinal cord; therefore, our studies have
potentially revealed an evolutionarily conserved function of these proteins in
spinal cord development. Here we have provided evidence that Tcf3 is required
for preventing precocious neurogenesis in spinal progenitors, and have
identified a new transcriptional target of Tcf proteins in this process.
|
Footnotes |
|---|
* These authors contributed equally to this work 
Present address: Department of Biology, Grove City College, Grove City, PA
16127, USA
Present address: Department of Biology, Skidmore College, Saratoga Springs,
NY 12866, USA
|
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TOP
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MATERIALS AND METHODS
RESULTS
DISCUSSION
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