1 The University of Iowa, Department of Biological Sciences, 257 BB, Iowa City,
IA 52246-1324, USA
2 Cincinnati Children's Hospital Medical Center, Division of Developmental
Biology MLC 7007, 3333 Burnet Avenue, Cincinnati, OH 45229-3039, USA
* Author for correspondence (email: douglas-houston{at}uiowa.edu)
Accepted 26 August 2005
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SUMMARY |
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Key words: Zic2, Nodal, Xnr, VegT, Holoprosencephaly (HPE), Forebrain, Gastrulation, Maternal mRNA
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Introduction |
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Experiments in Xenopus have demonstrated that the expression of
Nodal-related genes (Xnrs) is initiated and modulated by maternally inherited
factors. Maternal VegT mRNA, which encodes a T-domain transcription
factor, is required for the initiation of Xnr1, Xnr2, Xnr4, Xnr5 and
Xnr6, and the subsequent specification of the mesoderm and endoderm
germ layers (Kofron et al.,
1999; Zhang et al.,
1998
). In vegetal cells, VegT is thought to directly initiate
Xnr5 and Xnr6 expression immediately upon the onset of
zygotic transcription (Hilton et al.,
2003
; Takahashi et al.,
2000
; Xanthos et al.,
2002
). VegT also cooperates with the maternal ß-catenin
pathway, which is required for dorsal axis specification, to initiate Xnr
expression dorsally early in gastrulation
(Agius et al., 2000
;
Lee et al., 2001
;
Xanthos et al., 2002
). VegT is
the only factor known to be necessary and sufficient for the induction of
Xnr5/6 expression (Rex et al.,
2002
; Takahashi et al.,
2000
); however, loss-of-function experiments have demonstrated
roles for several transcription factors in repressing the degree of
Xnr5/6 expression. Depletion of maternal Xtcf3, FoxH1 and
Sox3 mRNAs (Houston et al.,
2002
; Kofron et al.,
2004a
; Zhang et al.,
2003
), and morpholino oligo-induced depletion of Mixer protein
(Kofron et al., 2004b
) all
result in increased expression of Xnr5/6. Sox3 and Xtcf3 are likely
to mediate repression in the absence of stabilized ß-catenin; however,
the roles of FoxH1 and Mixer are surprising, as these proteins have been
identified as mediators of gene activation downstream of Nodal signaling
(Chen et al., 1996b
;
Germain et al., 2000
).
During a functional screen of maternal genes, we identified a cDNA encoding
a truncated Zic2 protein that altered head formation when overexpressed in
embryos. The zinc-finger proteins of the cerebellum (Zic) genes are a
conserved family related to Drosophila Odd-Paired
(Aruga et al., 1996) and encode
proteins of
500 amino acids in length. The functions of Zic proteins in
early development are mostly unknown, and have been characterized as having
both transcriptional activator and repressor functions
(Brewster et al., 1998
;
Salero et al., 2001
).
Zic2, the focus of this work, is expressed at high levels maternally
and during gastrulation in Xenopus. Later expression is evident in
the dorsal neural tube, somites, optic vesicle and neural crest
(Brewster et al., 1998
;
Nakata et al., 1998
).
Published overexpression studies in Xenopus found that Zic2
induced neural crest genes and inhibited neurogenesis
(Brewster et al., 1998
;
Nakata et al., 1998
), possibly
by acting as a transcriptional repressor
(Brewster et al., 1998
). In the
mouse, Zic2 is also expressed during the gastrula stages
(Elms et al., 2004
) and
loss-of-function studies in mice indicate important roles in neural/neural
crest development (Elms et al.,
2003
; Nagai et al.,
2000
) and axon pathfinding
(Herrera et al., 2003
).
Mutations in human ZIC2 result in holoprosencephaly (HPE), a
severe malformation of the developing brain in which the forebrain fails to
form separate left and right hemispheres
(Brown et al., 2001;
Brown et al., 1998
;
Orioli et al., 2001
). However,
the exact developmental role of ZIC2 in brain development and in HPE
remains undefined. Here we show, through maternal mRNA depletion, that
Xenopus Zic2 has important roles in regulating anteroposterior
patterning in early development through its regulation of Xnr gene expression.
We show that the formation of head structures and forebrain is abnormal in
embryos depleted of maternal Zic2. We also show that
Zic2-depleted embryos exhibit elevated and sustained levels of Xnr
gene expression and activity, and that this excess Xnr signaling has a causal
role in the head defects observed. Furthermore, we show that Zic2 is required
to indirectly attenuate the levels of Xnr genes induced by VegT.
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Materials and methods |
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Oocytes and embryos
Oocytes were manually defolliculated and cultured in oocyte culture medium
(OCM) at 18°C. Oocytes were injected with antisense oligos against
Zic2 and cultured for 24-48 hours prior to being stimulated to mature
with 2 mM progesterone. Matured oocytes were colored with vital dyes,
implanted into females and fertilized using the host-transfer technique as
described previously (Zuck et al.,
1998). For rescue experiments, mRNAs were injected into depleted
oocytes 24 hours after injection of the oligo. This allowed sufficient time
for the oligos to degrade so that rescue of the Zic2 depletion could
be accomplished by replacement of Zic2 and not by competition for
oligo binding. Alternatively, mRNAs were injected into control or depleted
embryos following recovery from the female and fertilization.
Eggs were recovered from laying females, fertilized using a sperm suspension and maintained in 0.1xMMR. For injections of mRNAs after fertilization, embryos were dejellied and transferred to 2% Ficoll/0.5xMMR at the one-cell stage. For explant assays, stage 9 embryos were dissected in 1xMMR on 2% agarose-coated dishes using sharp forceps or tungsten needles. The explants were cultured in OCM until sibling embryos reached the desired stage.
Antisense oligos
The antisense oligodeoxynucleotides (oligos) used were HPLC purified
phosphorothioate-phosphodiester chimeric oligos (IDT) with the sequences
5'-C*T*A*CCGCTGCATGGT*G*A*T-3' (Zic2-5MP) and
5'-T*G*T*CCGTGCGACTGTGC*C*C*A-3' (Zic2-10MP). Asterisks (*)
represent phosphorothioate bonds.
A morpholino oligo (MO) against VegT was obtained from Gene-Tools:
VegT-MO, 5'-CCCGACAGCAGTTTCTCATTCCAGC-3'
(Heasman et al., 2001).
Oligos and morpholinos were re-suspended in sterile, filtered water and injected in doses as described in the text.
Plasmids and mRNAs
The full-length Zic2-coding region was isolated by RT-PCR and
inserted into the vector pCRII-TOPO using TOPO-TA cloning (Invitrogen).
Zic2 was then subcloned into the EcoRI site of the vector
pCS2+ and linearized with NotI for SP6 in vitro transcription.
Zic2 was also cloned into pRN3 as a ClaI/XbaI
fragment and linearized with SfiI. Capped Zic2 mRNA was
synthesized using the SP6 or T3 mMessage mMachine kits (Ambion), respectively.
VegT and CerS, both in pCS2+, were digested with
NotI and transcribed with SP6. RNAs were precipitated with lithium
chloride, washed thoroughly in 70% ethanol and then re-suspended in sterile
distilled water for injection. In vitro translation was carried out using a
rabbit reticulocyte-lysate coupled transcription-translation system (TNT SP6
kit, Promega).
Luciferase assays
Reporter plasmids A3-luciferase, containing three tandem copies of the
Mix.2 activin-response element (ARE) driving firefly luciferase, and
pRLTK (25ng/embryo), containing a ubiquitous thymidine kinase promoter driving
Renilla luciferase, were co-injected vegetally into either control or
Zic2-depleted embryos. Embryos were frozen in triplicate during the
gastrula stages and analyzed for luciferase activities according the protocol
of the Luciferase Assay System (Promega).
RT-PCR
Analysis of gene expression was performed either by semi-quantitative,
real-time RT-PCR using the LightCyclerTM System (Roche) as described by
Houston et al. (Houston et al., 2003), or by gel electrophoresis. For this
latter method, 500 ng purified RNA was used for random hexamer-primed cDNA
synthesis using MMLV (100 U/reaction; Invitrogen). cDNA reactions were diluted
to 180 µl with TE [3 mM Tris (pH 8.0)/0.2 mM EDTA] and 1/20th of the
diluted cDNA was used for RT-PCR. Reactions were performed on a PTC-200 (MJ
Research/BioRad) with a dual 48-well Alpha block. Cycling conditions were:
94°C (2 minutes, 1 cycle), 94°C, 54°C, 72°C (10 seconds each,
27 cycles), 72°C, 10 minutes. Samples were then run on 2% agarose gels
containing SYBR-Safe dye (Molecular Probes). The primer sequences used and
real-time PCR conditions are available upon request. Figures of real-time PCR
presented show a representative result of each experiment, not averages of
experiments, although each experiment was repeated at least twice. Real-time
data were quantified against a standard curve of diluted, uninjected embryo
cDNA. Undiluted control cDNA was set to 100%, thus values above 100% may not
be accurately quantified as they lie outside the range of the standard
curve.
Whole-mount in situ hybridization
Whole-mount in situ hybridization was performed essentially as described
(Sive et al., 2000).
Digoxigenin-labeled antisense probes against Zic2 (CDS in pCRII-TOPO)
were prepared by digestion with SpeI and transcription with T7.
Xnr5 (a gift from Dr M. Asashima) was linearized with NotI
and transcribed with T7. Embryos were post-fixed after staining and bleached
prior to imaging.
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Results |
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Embryos injected vegetally with tZic2 mRNA (500 pg) developed normally through early gastrulation, but were subsequently delayed in its completion. tZic2-injected embryos developed a shortened dorsal axis with reduced head and eye development (Fig. 1C). Analysis of histological sections showed that tZic2-injected embryos had severely reduced forebrains and either cyclopic, reduced or absent eyes (Fig. 1D). These results demonstrate that overexpression of tZic2 protein, lacking the N terminus, could disrupt normal axial and head development.
Nakata et al. (Nakata et al.,
2000) and Kitaguchi et al.
(Kitaguchi et al., 2000
)
showed that deletion of the N-terminal region of Xenopus Zic5 and
Zic3, respectively, could generate dominant-negative proteins. Owing
to the similarity of tZic2 to these constructs, we tested the
hypothesis that the tZic2 cDNA could interfere with full-length
Zic2 function. Co-expression of tZic2 with full-length
Zic2 in animal caps showed that tZic2 efficiently blocked
Zic2-mediated induction of neural crest markers, whereas
tZic2 alone had no such activity
(Fig. 1E). These results
suggest that the neural crest-inducing activity of Zic2 lies in the N
terminus, and that tZic2 may inhibit endogenous Zic2 to cause microcephaly and
cyclopia. However, because of the potential for cross-interference of tZic2
protein with other Zic proteins, and because tZic2 overexpression does not
discriminate between maternal and zygotic roles for Zic2, we used an mRNA
depletion, loss-of-function approach to specifically examine the function of
maternal Zic2 in early development.
|
We obtained embryos from Zic2-depleted or uninjected oocytes via
the host-transfer method; Zic2-depleted embryos developed normally
from fertilization and initiated gastrulation at the same time as control
embryos. During gastrulation, embryos lacking Zic2 were delayed in
blastopore closure and 30% of these underwent exogastrulation
(Table 1). The remainder
exhibited deep involution of the marginal zone and went on to develop reduced
heads, wrinkled epidermis and a stunted dorsal axis
(Fig. 2C,D). These embryos
formed cement glands, suggesting that gross anteroposterior patterning was
unaffected (Fig. 2D). In
addition, depletion of maternal Zic2 did not eliminate zygotic
Zic2 expression. Zygotic Zic2 mRNA was expressed correctly
at the neural plate border and accumulated to normal levels by late
gastrulation in maternal Zic2-depleted embryos
(Fig. 2E,F). However, overall
neural plate development appeared delayed
(Fig. 2F) in these embryos.
Histological analysis confirmed the lack of forebrain tissue in
Zic2-depleted embryos (Fig.
2G, upper panel). In addition, neural tube morphology was
disrupted and somite structure was abnormal. Notochords formed in
Zic2-depleted embryos, but appeared larger in diameter than controls;
in some sections, a duplicated notochord was evident. Interestingly, we did
not observe cyclopia in Zic2-depleted embryos as was seen in those
injected with tZic2, suggesting that tZic2 may also
interfere with zygotic Zic2, or possibly with other Zic proteins. Injection of
Zic2 mRNA into Zic2-depleted embryos rescued normal
gastrulation and head development in a majority of cases
(Table 1), demonstrating that
these phenotypes are due to specific depletion of endogenous
Zic2.
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Zic2 negatively regulates the expression of Xenopus nodal-related (Xnr) genes
Head formation in vertebrates requires the simultaneous repression of Wnt,
BMP and Nodal signaling pathways (Piccolo
et al., 1999). Nodal proteins also have roles in regulating
morphogenesis, and excess Nodal signaling results in altered gastrulation
movements (reviewed by Solnica-Krezel,
2003
). Some Zic2-depleted embryos underwent
exogastrulation and developed reduced head structures, we therefore tested the
hypothesis that Zic2 regulates Xnr expression or signaling.
We initially examined the expression of Xnr1, Xnr3 and
Xnr5 in controls and in embryos injected with a dose range of
tZic2 mRNA to approximate Zic2 loss-of-function. At two
stages during gastrulation, Xnr5 mRNA levels were elevated in
tZic2-injected embryos in a dose-dependent manner
(Fig. 3A). Similar results were
also obtained for Xnr1 and Xnr3 (data not shown). In
addition, overexpression of full-length Zic2 could also weakly
repress Xnr5 expression, but only to 70% of control levels (data
not shown).
We next assayed the expression of Xnr genes and mesendodermal marker genes in control and Zic2-depleted embryos. Embryos were obtained by the host-transfer method, frozen at mid-gastrula stages (stage 10.25 and 10.5) and assayed for gene expression by real-time, semi-quantitative RT-PCR. We found that Zic2-depleted embryos expressed higher levels of Xnr mRNAs compared with sibling embryos (Fig. 3B). This was the case for all of the Xnr genes, with the exception of Xnr4, which was expressed at similar levels in Zic2-depleted and control embryos (data not shown). The degree of increase in Xnr expression appeared greater at the later stage, suggesting persistence, as well as upregulation, of Xnr expression.
In the mesoderm, expression of Goosecoid (Gsc), a target
of Nodal-like signaling, was elevated in Zic2-depleted embryos,
whereas expression of the pan-mesoderm marker Xbra was reduced, a
pattern consistent with elevated Nodal-like signaling. Chordin
expression was unaffected in Zic2-depleted embryos, suggesting that
while the expression of some organizer genes is elevated, this may not reflect
increased organizer formation. In the endoderm, we found an initial decrease
in endoderm gene expression at stage 10.25, followed by elevated levels for
Xsox17 and Xhex at stage 10.5, suggesting a delay in
their expression. Interestingly, Cerberus expression was only
slightly affected, suggesting again that some, but not all, dorsoanterior
markers are increased in Zic2-depleted embryos. To provide additional
evidence that Zic2-depleted embryos exhibit increased Nodal-like
signaling, we injected a Nodal-responsive reporter plasmid (A3-luciferse) into
control embryos and siblings deficient in maternal Zic2. Analysis of
luciferase activity at the mid-gastrula stage showed that activity of the
reporter construct was higher in Zic2-depleted embryos
(Fig. 3C). Activity at the
early gastrula stage was equivalent to or slightly lower than control levels.
These data show that Zic2-depleted embryos exhibit high levels of Xnr
expression that is reflected by increased downstream signaling activity.
Zic2 mRNA expression rescues Xnr expression in Zic2-depleted embryos
In addition to phenotypic analysis, we assessed the specificity of
Zic2-depletion by molecular marker analysis by real-time RT-PCR. We
analyzed the levels of Xnr1, Xnr3 and Xnr5 in control
uninjected embryos, Zic2-depleted embryos and Zic2-depleted
embryos injected with Zic2 mRNA over three stages during
gastrulation. Consistent with the phenotypic rescue, we found that injection
of Zic2 mRNA restored roughly normal levels of Xnr genes by stage 11
(Fig. 4A), although the degree
of rescue was less pronounced at the earlier stages. We also saw at least
partially restored levels of Nodal target genes Gsc and
Xhex, whereas Chordin was unaffected by the absence of
endogenous Zic2 or the presence of injected Zic2
(Fig. 4B). In a second
experiment, we saw a slightly higher degree of rescue for Xnr5
(Fig. 4C). It is likely that
varying penetrance of phenotype and differing rescue efficiency, as well as
differences in the peak expression times of Xnr genes, contributes to
variations in the amount of elevated expression or rescue that we observe.
Additional sources of variability in the levels of increased Xnr expression
across experiments include slight gastrulation delays in oligo-injected
embryos and inherent errors in quantifying values greater than the 100%
standard. Overall, these data show that injected Zic2 mRNA can
reduce, to some extent, excess Xnr expression caused by
Zic2-depletion and demonstrates the specificity of this
depletion.
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We next examined Xnr1, Xnr3 and Xnr5 expression in isolated dorsal and ventral halves of control and Zic2-depleted embryos. In these experiments, we found that expression of Xnr1 was elevated in both dorsal and ventral halves of Zic2-depleted embryos compared with control halves, although the degree of elevation was more pronounced on the ventral side (Fig. 5B). Xnr3 and Xnr5 were also increased ventrally and either unchanged, or slightly elevated in dorsal cells. Nodal target genes Gsc and Xhex were also ectopically expressed ventrally in Zic2-depleted embryos (Fig. 5B).
To determine to what extent Zic2 regulates the expression of Xnr
genes and their target genes in different germ layers, we dissected animal
caps, equatorial regions and vegetal bases from control and
Zic2-depleted embryos at the late blastula stage. These explants were
cultured to the early gastrula stage and analyzed for expression of Xnr genes
and mesendoderm markers. None of the markers analyzed showed any ectopic germ
layer expression in Zic2-depleted animal caps (data not shown). Xnr5
was expressed only in vegetal explants of control embryos
(Fig. 5C). In
Zic2-depleted embryos, consistent with the in situ data,
Xnr5 was expressed at higher levels in the bases and was not
expressed in equators. Xnr1 was also elevated only in vegetal
explants, as were endoderm markers Xhex and Xsox17.
Gsc was elevated in both equators and bases, and Xbra was
slightly decreased in equators. These results suggest that Zic2 is required to
regulate the extent of Xnr expression primarily in vegetal cells.
Nodal antagonism rescues head and axial defects in Zic2-depleted embryos
The anterior truncations and gastrulation defect phenotypes of
Zic2-depleted embryos correlate with the effects of excess Nodal
signaling in Xenopus (Branford and
Yost, 2002). To determine the causal role of Nodal signaling in
these defects, we expressed a Nodal antagonist, Cerberus-short
(CerS) (Piccolo et al.,
1999
), in Zic2-depleted embryos. We generated
Zic2-depleted and control embryos via the host-transfer method and
then injected CerS mRNA (50 pg) at the two-cell stage. Embryos were
collected at the gastrula stage for RT-PCR, or reared to the tailbud stage for
phenotypic analysis. At the dose used, CerS-injected control embryos
developed anterior truncations (microcephaly) in a number of cases; however,
this dose was sufficient to reduce the incidence of head and axial defects in
Zic2-depleted embryos (Fig.
6A,C,C'). CerS injection alone did not result in
axial truncations. In control gastrula stage embryos, CerS injection
severely reduced Xnr1 expression, but caused an elevation of
Xnr5 and Xnr6 (Fig.
6B), consistent with previous reports
(Kofron et al., 2004a
). In
Zic2-depleted embryos, Xnr1 expression was elevated, as was
Xnr5, consistent with our previous experiments. Injection of
CerS into Zic2 depleted embryos rescued Xnr1
expression slightly but had no effect on Xnr5 or Xnr6
expression. Interestingly, Xnr5 and Xnr6 abundance was
similar in embryos both depleted of Zic2 and expressing
CerS, indicating lack of an additive effect
(Fig. 6B). These results
suggest that Zic2 acts primarily at the level of Xnr5/6, and only
secondarily regulates Xnr1.
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Discussion |
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Zic2 function in Xenopus development
Xenopus Zic2 was initially described as having both neural/neural
crest-inducing activity and anti-neurogenic activity in Xenopus
embryos (Brewster et al., 1998;
Nakata et al., 1998
). The
molecular mechanisms behind these activities have not been explored in depth.
Analysis of hypomorphic and potential null alleles in mice have shown that
Zic2 regulates the timing and extent of neurulation and neural crest
formation, as well as the patterning of the forebrain and hindbrain
(Elms et al., 2003
;
Nagai et al., 2000
). In
Xenopus, Zic2 is expressed maternally and during the gastrula stages,
suggesting a possible role in early developmental events. Other Zic genes are
not maternally expressed (Fig.
2), thus the role of Zic2 can be studied in this context
without compensation from other Zic proteins.
Depletion of maternal Zic2 results in alterations of gastrulation
movements and in abnormal development of the forebrain, neural tube and
notochord. These late stage effects are likely to represent embryos mildly
affected (or embryos with less Zic2 depletion) as 30% of
Zic2-depleted embryos undergo exogastrulation. Elevated Nodal
signaling, which is produced by the loss of Nodal antagonists, triggers
exogastrulation in frog and fish embryos
(Branford and Yost, 2002
;
Feldman et al., 2002
), and
abnormal gastrulation movements in the mouse
(Iratni et al., 2002
;
Perea-Gomez et al., 2002
).
This same mechanism may also be responsible for exogastrulation in
Zic2-depleted embryos given the increased expression and activity of
Xnr genes in these embryos. The loss of Zic2 produces a milder
phenotype in this regard, most likely because Nodal antagonists are still
expressed in Zic2-depleted embryos (data not shown). In mouse
embryos, Zic2 is also expressed prior to gastrulation
(Elms et al., 2004
); however,
functional studies have not yet identified a role for this early phase of
Zic2 expression. It will be interesting to discover if mammalian
Zic2 regulates Nodal expression and gastrulation movements
in a manner analogous to maternal Zic2 in frogs.
Zic2 regulation of Xnr gene expression
Several lines of evidence presented here suggest that maternal
Zic2 negatively regulates the expression of Xenopus
nodal-related (Xnr) genes, primarily that of Xnr5. It is likely
that maternal Zic2 serves to limit the extent of the initial wave of
Xnr5 expression, which would normally be required for proper spatial
and temporal regulation of Xnr1, Xnr2, Xnr3 and Nodal target genes.
Among these targets are endoderm markers, which show an initial delay in
expression, possibly owing to repression by high levels of Nodal signaling
(Yasuo and Lemaire, 1999). It
was surprising to find that Xnr4 was not upregulated in
Zic2-depleted embryos, as would be expected from its expression
pattern (Joseph and Melton,
1997
). It is possible that Xnr4 has lost the regulatory
sequences needed for autoregulation owing to functional redundancy of
Xnr1 and Xnr2. Interestingly, Xnr3 which lacks
typical Nodal activity, appears to have retained such regulation, as shown by
our results (Fig. 7) and by the
upregulation of Xnr3 in Xlefty-depleted embryos
(Branford and Yost, 2002
). It
is unclear to what extent elevated Xnr3 is a major contributor to the
Zic2-depleted embryo phenotype. These embryos do not exhibit
hallmarks of Xnr3 overexpression, such as finger-like projections,
and CerS, which does not inhibit Xnr3, can reduce the
severity of defects.
The mechanisms by which Zic2 regulates Xnr5, and probably
Xnr6, expression remain to be determined. Zic2 is dependent on VegT
to regulate Xnr5; however, we found that Zic2 expression does not
inhibit VegT in animal cap experiments, and Zic2 is a poor Nodal
inhibitor in general. These results suggest an indirect mechanism, or the
presence of other factors not present in animal caps. One likely candidate is
Nodal signaling itself. We found that depletion of Zic2 and
antagonism of Nodal signaling (by CerS injection) did not produce an
additive effect on Xnr5 and Xnr6 expression. These results
suggest a model is which Zic2 could associate with active Smad2 complexes to
mediate negative feedback inhibition of Xnr5. A number of other
transcription factors, including XTcf3, Sox3, Mixer and FoxH1 have been
recently shown to have roles in repressing Xnr5
(Houston et al., 2002;
Kofron et al., 2004a
;
Kofron et al., 2004b
;
Zhang et al., 2003
).
Interestingly, Mixer and FoxH1 are known to associate with activated Smad2
(Chen et al., 1996b
;
Germain et al., 2000
), but do
not posses intrinsic transcriptional activation activity. Intracellular
TGFß-negative feedback loops have been described in other model systems
but have not been well studied in embryos.
Zic2 in holoprosencephaly
Hemizygosity of ZIC2 underlies cases of human HPE; however, the
mechanism(s) by which loss of ZIC2 causes HPE remain uncertain. In this work,
we show that depletion of Zic2 results in increased Xnr expression at
the gastrula stages. The majority of identified human HPE mutations are in the
sonic hedgehog (SHH) pathway (Duborg et al., 2004). However,
experiments in mouse and fish have also implicated Nodal loss-of-function in
HPE, possibly through the induction of Shh expression in the
prechordal mesoderm (Lowe et al.,
2001; Rohr et al.,
2001
). NODAL mutations have not yet been identified in
human HPE cases, although mutations in TDGF1 (CRIPTO), a
NODAL co-receptor, have been found in individuals with HPE-like, but not
definitive, HPE (de la Cruz et al.,
2002
). Mutations in human TGIF, an activated-SMAD
associated, homeodomain co-repressor
(Wotton et al., 1999
), have
been reported (Gripp et al.,
2000
) in HPE, suggesting a role for increased NODAL activity. Our
results presented here reinforce the idea that increased NODAL signaling may
be a factor in HPE. Further clarification of the mechanism of Zic2 in the
control of Xnr expression may be useful in the identification of additional
HPE candidate genes.
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ACKNOWLEDGMENTS |
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