RESEARCH ARTICLE
735
Development 134, 735-746 (2007) doi:10.1242/dev.02756
The zebrafish zic2a-zic5 gene pair acts downstream of
canonical Wnt signaling to control cell proliferation in the
developing tectum
Molly K. Nyholm1, Shan-Fu Wu2, Richard I. Dorsky2 and Yevgenya Grinblat1,*
Wnt growth factors acting through the canonical intracellular signaling cascade play fundamental roles during vertebrate brain
development. In particular, canonical Wnt signaling is crucial for normal development of the dorsal midbrain, the future optic
tectum. Wnts act both as patterning signals and as regulators of cell growth. In the developing tectum, Wnt signaling is mitogenic;
however, the mechanism of Wnt function is not known. As a step towards better understanding this mechanism, we have identified
two new Wnt targets, the closely linked zic2a and zic5 genes. Using a combination of in vivo assays, we show that zic2a and zic5
transcription is activated by Tcf/Lef transcription factors in the dorsal midbrain. Zic2a and Zic5, in turn, have essential, cooperative
roles in promoting cell proliferation in the tectum, but lack obvious patterning functions. Collectively these findings suggest that
Wnts control midbrain proliferation, at least in part, through regulation of two novel target genes, the zic2a-zic5 gene pair.
KEY WORDS: Zic, Wnt, Proliferation, Tectum, Zebrafish
1
Departments of Zoology and Anatomy, University of Wisconsin, Madison, WI,
53706, USA. 2Department of Neurobiology and Anatomy, University of Utah, Salt
Lake City, UT 84132, USA.
*Author for correspondence (e-mail: [email protected])
Accepted 23 November 2006
Zic genes (Aruga, 2004; Aruga et al., 1994) are candidate targets
of Wnt signaling based on their functions and expression patterns.
Zic genes, which are vertebrate homologs of Drosophila odd paired
(Benedyk et al., 1994), encode zinc-finger transcription factors
(TFs). Five mammalian (Aruga, 2004) and seven zebrafish zic genes
(Grinblat et al., 1998; Grinblat and Sive, 2001; Parinov et al., 2004;
Toyama et al., 2004) have been described and are commonly
arranged in closely linked gene pairs. In humans, partial loss-offunction of ZIC2 is associated with holoprosencephaly and neural
tube closure defects (Grinberg and Millen, 2005). Mutant analysis
of mouse Zic2 and Zic5, a linked gene pair, showed that these genes
are required for neural tube closure along the entire anteroposterior
(AP) axis, including the midbrain (Elms et al., 2003; Inoue et al.,
2004; Nagai et al., 2000), and for midbrain-derived neural crest
formation (Elms et al., 2003; Inoue et al., 2004). Several zic genes
regulate neural precursor proliferation (Aruga et al., 2002b; Brewster
et al., 1998; Ebert et al., 2003), but no brain-specific regulators and
few transcriptional targets of zic genes have been identified (Ebert
et al., 2003).
In this study, we use genetic and transgenic methods, combined
with chromatin-binding assays, to show that Tcf/Lef factors can
activate zic2a and zic5 transcription in the tectum, and that this
activation is likely to be direct. Using morpholino oligo (MO)
knockdown assays, we demonstrate a requirement for zic2a and zic5
in midbrain proliferation. Collectively, these data place zebrafish
zic2a and zic5 either directly or very proximally downstream of Wnt
signaling, and document an important role for these genes in
regulating midbrain growth.
MATERIALS AND METHODS
Zebrafish strains and embryo culture
Adult zebrafish were maintained according to established methods
(Westerfield, 1995). Embryos were obtained from natural matings and
staged according to Kimmel et al. (Kimmel et al., 1995). For transient
analysis, wild-type embryos were injected with 25-50 pg of plasmid DNA
or PCR product at the one-cell stage. Deletion constructs were PCR
amplified from zic2aD5:gfp (green fluorescent protein) plasmid.
Zic2aD5:gfp stable transgenics were produced using established methods
(Stuart et al., 1990).
DEVELOPMENT
INTRODUCTION
In the developing embryo, cells acquire distinct positional identities,
or pattern, while simultaneously undergoing rapid division. Precise
coordination of pattern formation and proliferation is clearly
essential for normal embryogenesis, yet the molecular mechanisms
responsible for this coordination are not well understood for any
embryonic tissue. The midbrain, one of the fastest growing
embryonic regions, is an attractive model system for dissecting these
mechanisms. In adults, the midbrain is located within the brainstem
and is divided into the tectum dorsally and tegmentum ventrally. The
tectum controls visual reflexes and coordinated muscle movements
and relays auditory information, whereas the tegmentum contains
nerve tracts that send information between the spinal cord and
forebrain. These essential functions require patterns to be correctly
formed during development, while the midbrain tissue is rapidly
growing.
Canonical Wnt signaling plays a major role during midbrain
formation, as illustrated by midbrain deletion in Wnt1 mouse
mutants (McMahon and Bradley, 1990; Thomas and Capecchi,
1990). Both patterning and proliferation-promoting roles have been
ascribed to Wnt signaling during neural development. Wnt signaling
plays a mitogenic role in the spinal cord (Chesnutt et al., 2004;
Dickinson et al., 1994; Megason and McMahon, 2002), midbrain
and hindbrain of higher vertebrates (Ikeya et al., 1997; Panhuysen et
al., 2004), and in zebrafish midbrain (Lekven et al., 2003). Whether
Wnts are patterning molecules, trophic factors or both (Buckles et
al., 2004) in the forming midbrain is still unclear. However,
canonical Wnt signaling is likely to have a central role in the genetic
network that links midbrain patterning and proliferation.
Discovering genes regulated downstream of Wnt signaling is a
necessary step toward elucidating this network.
RESEARCH ARTICLE
Sequence analysis and plasmid construction
A zic2a-zic5-containing BAC clone was identified by high-stringency
hybridization to the CHORI-211 library (BACAP Resources Center,
Oakland, CA), and sequenced. Sequence was also obtained from the Danio
rerio Sequencing Group at the Sanger Institute. Conserved regions were
identified using Pipmaker (Schwartz et al., 2000). To generate the
zic2aD5:gfp transgene, a portion of the zic2a-zic5 BAC clone was subcloned
with gfp into pBluescript KS (Stratagene).
In situ hybridization (ISH) and histology
Antisense RNA probes were transcribed from the following plasmid
templates: zic2a (Grinblat and Sive, 2001), zic5 (Toyama et al., 2004), dlx3
(dlx3b – Zebrafish Information Network) (Akimenko et al., 1994), pax7 (Seo
et al., 1998), wnt1 (Molven et al., 1991), wnt3a (wnt3l – Zebrafish
Information Network) (Buckles et al., 2004), shh (Krauss et al., 1993), hlx1
(Fjose et al., 1994), and krox-20 (egr2b – Zebrafish Information Network)
(Oxtoby and Jowett, 1993). ISH was performed as previously described
(Gillhouse et al., 2004). Stained embryos were embedded in Eponate 12
medium (Ted Pella), and sections (5 ␮m) were cut with a steel blade on an
American Optical Company microtome. Nuclei were counterstained with
Methyl Red.
Heat-shock induction of Tg(hs:GFP⌬tcf)
Heterozygous Tg(hs:Gfp⌬tcf) embryos (Lewis et al., 2004) were heat
shocked at 37°C for 30 or 45 minutes, then incubated at 29°C and fixed for
ISH. Embryos expressing Gfp⌬Tcf were identified using a Leica MZFLIII
stereoscope.
Ectopic activation of Wnt signaling
To ectopically activate Wnt signaling, 15-25 pg of hs:␤-catenin-gfp plasmid
(a gift from A. Lekven, Texas A&M, College Station, TX) was injected at
the one-cell stage. Injected embryos were heat shocked at shield-stage
(37°C, 30 minutes) and fixed for ISH at 90-100% epiboly. GR-lef⌬N-␤-cat
plasmid was used as previously described (Ramel and Lekven, 2004).
Following in vitro transcription (mMessage Machine, Ambion), GR-lef⌬N␤-cat RNA was injected at 100 pg per embryo at the one-cell stage. For
treatments, injected embryos were incubated in 10 mM dexamethasone
(DEX, Sigma) and/or 20 mM cycloheximide (CHX, Sigma) in E3 embryo
medium for 2 hours, starting at 70% epiboly, until fixation at 90-100%
epiboly. Control embryos were treated with equivalent concentrations of
vehicle (ethanol or DMSO) in E3.
Chromatin immunoprecipitation (ChIP) assays
ChIP was performed as described previously (Lee, 2006; Weinmann et al.,
2001) using an anti-Gfp antibody (Molecular Probes). The following PCR
primers were used:
ngn1, 5⬘-GGGCTCATTGGAGCAAGTTTGATT-3⬘ and 5⬘-CGCGGTAGCCTACATTACTGCACA-3⬘;
ngn1 negative, 5⬘-GTGACAGTTTTGTTGACCACGACG-3⬘ and 5⬘CTGGAGATTCGGCGTGGGGTTGGG-3⬘;
nacre, 5⬘-GCAATTACCAAAGGCCCATCAGAC-3⬘ and 5⬘-ACTGGCTTACGGCTAACTAACGTT-3⬘;
zic2a-zic5 intergenic, 5⬘-TTGAACGGAGAAAATCACAACTAA-3⬘ and
5⬘-TATTACGAATCGATGTAACGGAGA-3⬘;
zic5 3⬘ enhancer, 5⬘-CTGCTTTGATGATTGACCATTTAG-3⬘ and 5⬘-ACTTTATGAAAAGCGGAATAGCAC-3⬘;
zic5 intron, 5⬘-GGTGATGTGCATATTTAACTGGAA-3⬘ and 5⬘-ACGTTGTACAAATGCTATGTTGCT-3⬘.
PCR products were visualized on ethidium bromide-stained agarose
gels. Each ChIP was repeated two to six times, except for hs:Gfp controls
which were performed once.
MO knockdown assays
The following antisense MOs were purchased from GeneTools:
zic2a translation-blocking 2MOA, 5⬘-CGATGAAGTTCAATCCCCGCTCACA-3⬘ and 2MOB, 5⬘-CTCTTTCAAGCAGTCTATTCACGGC-3⬘;
zic2a intron 1/exon 2 splice-blocking 2MOC, 5⬘-CTCACCTGAGAAGGAAAACATCATA-3⬘;
Development 134 (4)
zic5 intron 1/exon 2 splice-blocking 5MOC, 5⬘-GGCTTCTCACCTGTCAAATGTAAAA-3⬘;
tcf3a translation-inhibiting MO, 5⬘-TTTTTTGCTTACTTCGGAGTCTGATG-3⬘;
tcf3b splice-blocking MO, 5⬘-CGCCTCCGTTAAGCGGCATGTT-3⬘;
tcf7 splice-blocking MO, 5⬘-AGCTGCGGCATGATCCAAACTTTCT-3⬘;
lef1 exon 7/intron 7 splice-blocking MO, 5⬘-ACTGCCTGGATGAAACACTTACATG-3⬘; and
standard control conMO, 5⬘-CCTCTTACCTCAGTTACAATTTATA-3⬘.
MOs were diluted in 1⫻ Danieau buffer (Nasevicius and Ekker, 2000) to
2 ng/nl (2MOC, 5MOC, lef1MO, or 2MOC and 5MOC combined), 6.6
ng/nl (2MOA and 2MOB combined), or 7-8 ng/nl (conMO). 1 nl of MO per
embryo was injected at the one- to two-cell stage.
Immunohistochemistry
Embryos were fixed in 4% formaldehyde in PBS and stained using the
following antibodies: anti-phosphohistone H3 (PH3; Upstate Biotechnology,
1:500), anti-Pax7 (1 ␮l/ml, Developmental Studies Hybridoma Bank,
University of Iowa, developed by Atushi Kawakami, NICHB), Alexa488conjugated goat anti-rabbit secondary (Molecular Probes, 1:500) and
Alexa564-conjugated goat anti-rabbit secondary (Molecular Probes, 1:500).
For double immunostaining, fixed embryos were treated with 1% hydrogen
peroxide.
Cell proliferation analysis
PH3-stained embryos were incubated in 0.1 ␮M ToPro3 (Molecular Probes),
rinsed and mounted in DABCO antifade reagent (Sigma). A confocal z-series
was generated at 40⫻ using an Axiovert 100M (Carl Zeiss MicroImaging,
Inc.) with Lasersharp Confocal Package (model 1024, Bio-Rad). Midbrain
was located relative to the retinae. Mitotic index (MI) was calculated by
dividing PH3+ cell number by total cell number (ToPro3+). This was repeated
at nine dorsoventral (DV) positions for each embryo. Mean MIs were
converted using the arc sin square root calculation to homogenize variances
between morphants and controls (Snedecor and Cochran, 1980). T2, a
generalization of the standard t-test for data with multivariate responses, was
used to compare combined MIs (all DV positions). MIs at individual DV
points were compared using a standard independent-sample t-test. For
PH3+Pax7-stained embryos, confocal z-sections were processed using ImageJ
and Adobe Photoshop to generate three z-stacks: the dorsal half of the Pax7
domain included MI points 1 and 2; the ventral half of the Pax7 domain
included dorsal point 3 and hinge point; and the Pax7-negative domain
included all ventral points. TUNEL staining was performed using the In Situ
Cell Death Detection Kit, POD (Cole and Ross, 2001).
RESULTS
Zebrafish zic2a and zic5 are linked and coexpressed
A linked, divergently transcribed genomic arrangement is
characteristic of zic family members (Aruga, 2004; Grinberg and
Millen, 2005). Zebrafish zic2a and zic5 are paired (Fig. 1A) and their
transcription start sites (Grinblat and Sive, 2001; Toyama et al.,
2004) are separated by 4.6 kb of intergenic sequence, suggesting that
zic2a and zic5 may share regulatory elements. The expression
patterns of zic2a and zic5 have been described previously (Grinblat
and Sive, 2001; Toyama et al., 2004), but not compared side-by-side.
At midgastrula stages, zic2a was expressed throughout the
neurectoderm (Fig. 1B), but zic5 was not expressed. By late gastrula
stages, zic2a and zic5 were co-expressed at the neural–non-neural
border, the presumptive dorsal neural tube (Fig. 1C and data not
shown). During somitogenesis, zic2a and zic5 were co-expressed in
the dorsal brain, including the forming tectum (Fig. 1D,E,
arrowheads). By 24 hours post-fertilization (hpf), tectal expression
was restricted to the dorsal midline (Fig. 1F,G). Their close genomic
proximity and similar expression patterns suggest that zic2a and zic5
are likely to share regulatory elements and to have overlapping
functions during development.
DEVELOPMENT
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zic2a and zic5 regulate tectal proliferation
RESEARCH ARTICLE
737
Fig. 1. Zic2a and zic5 are linked and co-expressed in presumptive dorsal brain. (A) Genomic arrangement of zic2a and zic5. Coding regions
are shown as gray boxes, non-coding transcribed regions as white boxes, and introns as lines. (B-G) Embryos stained by ISH for zic2a or zic5
(purple), and dlx3 (orange). (B) At midgastrula stages, zic2a is expressed throughout neurectoderm, whereas dlx3 marks the adjacent non-neural
ectoderm. (C) By late gastrula stages, zic2a is restricted to the lateral neural plate and forms a sharp border with dlx3. (D,E) During somitogenesis,
zic2a and zic5 are similarly expressed in the dorsal neural tube, ventral diencephalon and optic stalks. (F,G) At 24 hpf, zic2a and zic5 are coexpressed in the optic stalk, telencephalon and dorsal diencephalon, and weakly in the tectum. All views are lateral, anterior to the left, except in
B,C where anterior (a) is at the top. Arrowheads mark anterior and posterior borders of presumptive midbrain. d, dorsal; m, midbrain; h, hindbrain;
os, optic stalk; tel, telencephalon.
the 12- to 13-somite stage (15.5 hpf). Reporter expression
recapitulated endogenous zic gene expression in the midbrain and
posterior hindbrain at midsomitogenesis (Fig. 3, compare A,B,C)
and late somitogenesis (Fig. 3D,E), whereas endogenous zic2a is
expressed throughout the dorsal brain (Fig. 3F). At 24 hpf, reporter
expression (Fig. 3G,H) was identical to endogenous expression (Fig.
3I) in the midbrain and posterior hindbrain. Collectively, this
analysis demonstrated that enhancers located in the D5 region direct
dorsal midbrain and hindbrain expression, and that additional
enhancers must control zic gene expression in other tissues.
Tcf/Lefs regulate zic2a and zic5 transcription and
directly bind zic2a-zic5 genomic DNA
The zic2a-zic5 locus contains several consensus binding sites for
Tcf/Lefs, effectors of canonical Wnt signaling (Dorsky et al., 2000;
Eastman and Grosschedl, 1999; Nusse, 1999; Ryu et al., 2001). Six
consensus sites were found in the region (D5) that contains
functional midbrain and hindbrain enhancers (see Fig. S1 in the
supplementary material). In addition, expression from zic2aD5:gfp
overlaps expression from Tg(TOP:GFP), a Wnt-signaling reporter
(Dorsky, 2002) (see Fig. S2 in the supplementary material). These
observations led us to ask if Wnt signaling regulates zic2a and zic5.
We used hs:gfp⌬tcf transgenics to inhibit Tcf/Lef-mediated
transcriptional activation (Lewis et al., 2004). Upon heat shock,
Tg(hs:gfp⌬tcf) embryos produce a Gfp⌬Tcf fusion protein that is
unable to bind ␤-catenin and acts as a dominant repressor of Wnt
target genes. Tg(hs:gfp⌬tcf) embryos were heat shocked during
somitogenesis, allowed to recover and accumulate Gfp⌬Tcf protein,
and assayed for zic expression (Fig. 4A). The result was a strong
reduction in zic2a RNA only 20 minutes after Gfp⌬Tcf protein
DEVELOPMENT
The zic2a-zic5 locus contains midbrain
transcriptional enhancers
To identify potential regulatory regions, we compared the zic2a-zic5
locus to the orthologous region in Fugu rubripes. Four conserved
regions were identified (thick lines in Fig. 2A): the proximal
promoters of zic2a (P2) and zic5 (P5), part of the intergenic region
(IG), and a region 3⬘ to zic5 (D5) (see Fig. S1 in the supplementary
material). We designed several reporter constructs to test the
enhancer activities of the conserved regions in vivo. The first two,
zic5D5:gfp and zic2aD5:gfp, contained the IG and D5 regions, but
differed in placement of gfp (Fig. 2B). In transient assays, Gfp was
expressed strongly from both constructs, and Gfp-positive cells were
concentrated in the dorsal midbrain and hindbrain (Fig. 2C-E).
We next tested three deletions of zic2aD5:gfp: zic2aD5⌬IG:gfp,
which lacks IG; zic2aIG⌬D5, which lacks D5; and zic2a:gfp,
which lacks both IG and D5 (Fig. 2B). Zic2aD5⌬IG:gfp was
expressed strongly and distributed similarly to the full-length
construct (Fig. 2F). Zic2a:gfp was expressed in a few randomly
distributed cells (Fig. 2G). The latter pattern is characteristic of
reporter constructs that contain only proximal promoter sequences
and no enhancers. Zic2aIG⌬D5:gfp was not expressed in the
midbrain and hindbrain, but was expressed strongly in the
telencephalon (data not shown). Based on these data, we conclude
that D5 contains transcriptional enhancers that, independently of
their genomic orientation, are sufficient to activate midbrain and
hindbrain transcription.
Restricted expression of zic2aD5:gfp in the midbrain and
hindbrain was confirmed in a stable transgenic line, Tg(zic2aD5:gfp)
(Fig. 3). Gfp RNA in Tg(zic2aD5:gfp) embryos was first detected at
the 10-somite stage (14 hpf), and Gfp fluorescence was detected at
738
RESEARCH ARTICLE
Development 134 (4)
Fig. 2. Transient transgenic analysis of zic2a-zic5 genomic
regions. (A) Conserved DNA regions (thick lines) at the zic2a-zic5
locus. P2 and P5, each approximately 300 nt, are 70% identical with
Fugu. IG (840 nt) is 49% identical to Fugu, and D5 (535 nt) is 60%
identical to Fugu. Eight consensus Tcf/Lef-binding sites were found in IG
and six in D5 (asterisks). (B) Transgenic constructs tested in transient
assays. (C,D) Dorsal midbrain and hindbrain expression of Gfp in
representative zic5D5:gfp and zic2aD5:gfp transient transgenics. Insets
are dorsal views of the embryos shown. Asterisks mark mid-hindbrain
organizer. (E-G) gfp RNA in representative transient transgenics. In
embryos injected with 25 pg of zic2aD5:gfp (E) or zic2aD5⌬IG:gfp (F),
expression was concentrated in dorsal midbrain and hindbrain. Embryos
injected with 25, 50 or 70pg of zic2a:gfp (G) did not show enhanced
expression. All views are lateral, except for insets.
began to accumulate (Fig. 4B-D, and see Table S1 in the
supplementary material). zic2a continued to be inhibited for at least
two hours post-heat-shock (Fig. 4E,F), as was zic5 (Fig. 4G,H).
We also tested the effect of Gfp⌬Tcf on zic2aD5:gfp transgene
expression. zic2aD5:gfp contains enhanced gfp with several
modified codons, whereas hs:gfp⌬tcf encodes unmodified Gfp. The
two gfp variants could therefore be distinguished at the RNA level.
Embryos carrying both hs:gfp⌬tcf and zic2aD5:gfp were heat
shocked and assayed for expression of zic2aD5:gfp RNA. The result
was complete loss of zic2aD5:gfp expression (Fig. 4I,J, and see
Table S1 in the supplementary material). An overlapping dorsal
␤-catenin and Tcf activate ectopic zic2a
transcription
Having demonstrated a role for Tcf/Lefs in regulating zic genes, we
asked if the zic genes could be activated by ␤-catenin, a key
component of the Wnt signal transduction pathway that interacts
with Tcf/Lefs to activate target genes (reviewed by Stadeli et al.,
2006). We used hs:␤-catenin-gfp to transiently overexpress a ␤catenin-Gfp fusion protein during gastrulation. At the end of
gastrulation, when zic2a expression is tightly restricted at the neural
border (Fig. 5A-C), ␤-catenin failed to induce zic2a in the medial
neural plate (Fig. 5A). However, it induced robust ectopic zic2a
expression in the lateral ectoderm (Fig. 5B,C). Ectopic zic2a
(purple) overlapped ␤-catenin-gfp expression (orange), consistent
DEVELOPMENT
neural tube marker, wnt3a, was expressed normally in the presence
of Gfp⌬Tcf (Fig. 4K,L, and see Table S1 in the supplementary
material), indicating that Gfp⌬Tcf specifically repressed zic gene
transcription.
Next we asked whether the ⌬Tcf protein bound directly to the
conserved IG and D5 regions using ChIP (Weinmann et al., 2001).
Tg(hs:gfp⌬tcf) embryos were heat shocked and subjected to ChIP
with an anti-Gfp antibody to isolate DNA bound to Gfp⌬Tcf.
Precipitated DNA was tested by PCR for the presence of known direct
targets of canonical Wnt signaling – nacre (mitfa – Zebrafish
Information Network) (Dorsky et al., 2000) and ngn1 (neurog1 –
Zebrafish Information Network) (Hirabayashi et al., 2004). For both
genes, promoter DNA was amplified after ChIP with anti-Gfp
antibody, but not after control ChIP (Fig. 4M, compare lane 1 with
lanes 2-4). An upstream ngn1 region, which contains one
nonconserved putative Tcf/Lef-binding site, failed to amplify (Fig.
4M, lane 5). These controls verified the assay could efficiently and
specifically identify direct targets of canonical Wnt signaling in
zebrafish embryos.
When the products of Tg(hs:gfp⌬tcf)–anti-Gfp ChIP were
subsequently assayed for IG and D5, both were amplified (Fig. 4N).
The zic5 intron contained no conserved consensus Tcf/Lef sites and
was not amplified (Fig. 4N). To confirm that IG and D5 were bound
to the Tcf portion of the fusion protein, ChIP was applied to
Tg(hs:gfp) embryos (Halloran et al., 2000), which express
unmodified Gfp protein after heat shock. Neither IG nor D5 was
detected in this experiment (Fig. 4N, lane 5). The results from ChIP
analysis showed that transgenic Tcf protein was bound directly to D5
and IG portions of the zic2a-zic5 DNA and demonstrated the
presence of Tcf/Lef-binding sites in these regions, thereby suggesting
that zic2a and zic5 are direct targets of the canonical Wnt pathway.
zic2a and zic5 regulate tectal proliferation
RESEARCH ARTICLE
739
with ␤-catenin acting cell-autonomously to activate zic gene
transcription. In control embryos, Gfp protein produced by transient
expression of hs:gfp failed to induce zic2a (Fig. 5D).
We next asked if the ␤-catenin-Tcf complex could activate zic
transcription directly (i.e. without synthesis of an intermediate
transcriptional activator), using a hormone-inducible ␤-catenin-Lef
fusion protein, GR-Lef⌬N-␤-cat (GR-Lef) (Domingos et al., 2001).
GR-Lef contains the Tcf DNA-binding domain, the ␤-catenin
transactivation domain, and the glucocorticoid-receptor hormonebinding domain (GR), which allows it to become fully active when
bound to dexamethasone. This construct has been demonstrated to
activate Wnt target genes in zebrafish (Ramel and Lekven, 2004).
By activating GR-Lef in the presence of cycloheximide, an inhibitor
of protein synthesis, it is possible to determine whether GR-Lef
functions independently of translation.
To test whether GR-Lef could induce zic gene expression, GRLef-injected zic2aD5:gfp embryos were assayed for gfp
expression (Fig. 5E-I). This analysis was performed at late
gastrula stage, prior to the normal onset of zic2aD5:gfp
expression (Fig. 3); therefore, any gfp expression at this stage is
ectopic. GR-Lef alone induced some ectopic transgene expression
(Fig. 5E). However, the ability of GR-Lef to activate gfp
expression was enhanced by addition of DEX (Fig. 5F), even
when translation was inhibited with CHX (Fig. 5G, and see Fig.
S3 in the supplementary material). Expression of zic2aD5:gfp was
never observed in uninjected, vehicle-treated controls (Fig. 5H).
Endogenous zic2a, normally expressed in late gastrula
neurectoderm, was also ectopically induced by GR-Lef
independently of translation (not shown). Collectively, these data
demonstrate that zic2a can be activated directly downstream of
Wnt signaling in the zebrafish neurectoderm and suggest that the
zic2aD5:gfp transgene contains a Wnt-responsive enhancer.
Multiple endogenous Tcf/Lefs regulate zic gene
transcription
The data reported thus far provide strong evidence that the canonical
Wnt pathway directly regulates zic2a and zic5. We next used
morpholino analysis to ascertain which endogenous Tcf/Lefs
mediate this regulation. Two zebrafish tcf3 homologs, tcf3 (hdl) and
tcf3b (tcf7l1a and tcf7l1b, respectively – Zebrafish Information
Network) are ubiquitously expressed and function redundantly
(Dorsky et al., 2003). Endogenous zic2a was reduced in strongly
affected tcf3+tcf3b morphants (Fig. 6D, and see Fig. S4 in the
supplementary material), but still expressed in two discrete domains
in the midbrain and hindbrain (asterisks in Fig. 6D). Zic2aD5:gfp,
which is normally transcribed in these domains, was also expressed
in Tcf3-depleted embryos (Fig. 6C, and see Fig. S4 in the
supplementary material). Although midbrain size changes due to
caudalization in Tcf3 morphants and mutants (Dorsky et al., 2003;
Kim et al., 2000) (see Fig. S4 in the supplementary material), wnt3a
was strongly expressed in tcf3 morphants, suggesting that the dorsal
midbrain was not substantially reduced (see Fig. S4 in the
supplementary material, compare C with O). Altogether, these data
suggest that Tcf3 and Tcf3b are required to activate zic2a
transcription outside the midbrain and hindbrain, and are partially
responsible for its activation in the midbrain and posterior hindbrain.
We next asked if midbrain-restricted tcf/lef genes, lef1 and tcf7
(Dorsky et al., 1999; Lee et al., 2006), regulate the zic genes. In lef1
morphants, zic2aD5:gfp expression in the tectum was strongly
reduced compared with controls (Fig. 6E, and see Table S2 in the
supplementary material). By contrast, endogenous zic2a expression
was largely unaffected. Zic2aD5:gfp expression was mildly affected
in tcf7 morphants (Fig. 6G), and endogenous expression was not
affected (Fig. 6H). In double lef1+tcf7 morphants, zic2aD5:gfp
expression was strongly reduced or eliminated (Fig. 6I). Expression
DEVELOPMENT
Fig. 3. Stable zic2aD5:gfp expression
recapitulates endogenous midbrain and
posterior hindbrain expression. Gfp
expression in Tg(zic2aD5:gfp) embryos
detected by ISH (A,D,G,H), or by fluorescence
(B,E). (C,F,I) Endogenous zic2a expression
detected by ISH. (A-C) gfp RNA at the 11- to
12-somite stage (A) and Gfp protein at 14somites (B) are restricted to dorsal midbrain
and posterior hindbrain. Endogenous zic2a is
transcribed in the dorsal diencephalon,
midbrain and hindbrain at 11-somites (C). (FH) Transgenic (D,E) and endogenous (F)
expression overlap in the midbrain and
posterior hindbrain at the 20-somite stage.
At 24 hpf, transgenic expression (G,H)
overlaps endogenous expression (I) in the
dorsal midbrain. All views are lateral, anterior
to the left, except H,I which are dorsal views.
Arrowheads mark anterior and posterior
limits of tectum. m, midbrain; h, hindbrain.
RESEARCH ARTICLE
Development 134 (4)
Fig. 4. Tcf/Lefs are required for zic gene expression. (A) The method used to disrupt Tcf/Lef signaling at controlled times. (B) Temporal
correlation between Gfp⌬Tcf induction (by fluorescence) and zic2a RNA (by ISH). (C-L) Representative embryos after ISH. Stage of heat-shock and
recovery time are indicated at upper right. Zic2a expression is normal in ⌬Tcf-negative heat-shocked controls (C,E), and reduced or absent in ⌬Tcfpositive siblings (D,F). Zic5 expression was normal in ⌬Tcf-negative (G) embryos, and absent in ⌬Tcf-positive embryos (H). Zic2aD5:gfp expression
was normal in ⌬Tcf-negative embryos (I) and absent in ⌬Tcf-positive embryos (J). Wnt3a expression was normal in heat-shocked controls (K) and in
⌬Tcf-positive siblings (L). All views are lateral, anterior to the left. Arrowheads mark anterior and posterior tectal borders. (M,N) Gfp ChIP analysis of
heat-shocked Tg(hs:gfp⌬tcf) and Tg(hs:gfp) embryos. Genotype of embryos and PCR templates are shown above, as follows: GFP Ab, chromatin
after anti-Gfp IP; input, total chromatin before IP; no Ab, no-antibody ChIP; mock, no chromatin. Regions amplified are labeled next to the
corresponding bands. (M) Promoters of known Wnt targets, ngn1 and nacre. An upstream region of ngn1, lacking functional Tcf/Lef-binding sites
(lane 5). (N) IG and D5 regions of zic2a-zic5 and zic5 intron.
of zic2a was reduced, but only mildly (Fig. 6J). Taken together, these
data indicate that several endogenous Tcf/Lefs regulate zic2a
expression: Lef1 and Tcf7 in the midbrain, acting through the
midbrain enhancer in zic2aD5:gfp; and Tcf3 and Tcf3b throughout
the endogenous zic2a expression domain.
Antisense MOs effectively knockdown zic2a and
zic5 and disrupt formation of the dorsal brain
As Wnt signaling targets, zic2a and zic5 are likely to mediate some
of its functions. To analyze the functions of zebrafish zic2a and zic5,
MOs were used to knockdown zic2a and zic5 separately or in
DEVELOPMENT
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zic2a and zic5 regulate tectal proliferation
RESEARCH ARTICLE
741
Fig. 5. Ectopic activation of the canonical Wnt pathway induces zic gene transcription. (A-C) Embryos were injected with hs:␤Cat-gfp DNA,
heat shocked at shield-stage, and assayed for zic2a (purple) and gfp (orange) at late-gastrula. (A) ␤Cat-gfp is expressed in the medial neural plate;
the zic2a pattern is normal (no ectopic expression; n=10, two experiments). (B,C) ␤Cat-gfp is expressed in lateral ectoderm; zic2a is ectopically
expressed (arrows; n=8, 8 ectopic, two experiments). (D) Control embryo expressing hs:gfp in lateral ectoderm showing no ectopic zic2a expression
(n=18, 0 ectopic, one experiment). a, anterior. (E-G) Zic2aD5:gfp embryos injected with GR-Lef RNA and assayed for gfp RNA at late-gastrula
(purple). (E) GR-Lef-injected embryo showing weak ectopic gfp (arrow). (F,G) GR-Lef-injected embryos treated with DEX (F) or DEX and CHX (G).
(H) Uninjected zic2aD5:gfp control treated with DEX. (I) Summary of GR-Lef overexpression experiments, showing the proportion of embryos
expressing gfp for each treatment condition. The number of embryos scored for each condition is listed along the x-axis.
misshapen third ventricles (not shown). Wnt signaling appeared
normal in zic morphants (see Fig. S6 in the supplementary material).
Interestingly, co-injection of 2MOC and 5MOC at half the dose used
in single knockdowns resulted in a dramatic increase in the
proportion of severely affected embryos, from 46.5% in zic2a
morphants to 80.5% in zic2a+zic5 morphants (Fig. 7B). This
observation again suggests functional redundancy. Together, these
data validate the MOs as effective and specific tools for functional
analysis of zic2a and zic5.
DV pattern forms correctly in the midbrains of
zic2a and zic5 morphants
The abnormal wnt gene expression in zic morphants suggested that
zic genes pattern the dorsal neural tube. To test this, we analyzed the
DV pattern in zic2a+zic5 morphant midbrains at two stages of
development. At the 19-somite stage, just as brain ventricles begin to
inflate, wnt3a was expressed correctly in morphants as compared
with controls (Fig. 8A,B). pax7, an alar plate marker immediately
ventral to wnt3a, was also patterned correctly (Fig. 8C,D). By 24 hpf,
wnt3a expression appeared misshapen in zic2a+zic5 morphants (Fig.
8E,F,H,I). Cross-sections showed similar numbers of wnt3a-positive
cells in zic2a+zic5 morphant midbrains (an average 15 cells per
section, n=3; Fig. 6G) and control embryos (an average 16.5 cells per
section, n=3; Fig. 8J). However, morphant wnt3a-positive cells were
disorganized. The pax7 domain was misshapen in zic2a+zic5
morphants, but positioned correctly in whole mounts (Fig. 8K,L,N,O)
and in midbrain sections (Fig. 8M,P). hlx1, expressed near the
DEVELOPMENT
combination. Three non-overlapping MOs were designed for zic2a
(Fig. 7A); two to block translation (2MOA and 2MOB), and one to
inhibit splicing (2MOC). In addition, a splice-blocking MO was
designed against zic5 (5MOC).
We observed two distinct defects in zic2a morphants: (1)
abnormal morphology of the dorsal neural tube, including
incomplete inflation of midbrain and hindbrain ventricles (86%,
n=188); and (2) reduced neural retina with open choroid fissure
(coloboma, 65%, n=99). By contrast, embryos injected with the
same or a higher amount of conMO formed well-defined
symmetrical ventricles, and were indistinguishable from uninjected
controls. All three zic2a MOs produced the same defects,
confirming their specificity. zic5 morphants showed ventricle defects
(69%, n=116) similar to zic2a morphants, but did not have coloboma
(0%, n=54). Co-injection of 2MOC and 5MOC resulted in increased
penetrance of the ventricle phenotype (100%, n=40), supporting a
partially redundant function for these genes.
The morphological abnormalities in zic morphants were
visualized by expression of wnt1 and wnt3a, markers of the dorsal
midbrain and hindbrain (Fig. 7B-J). Control morphants displayed
wild-type wnt gene expression at 24 hpf (Fig. 7B-D). By contrast,
the tectal wnt expression domain was disorganized and shortened in
the AP direction (see Fig. S5 in the supplementary material) in zic2a
morphants, zic5 morphants, and zic2a+zic5 morphants (Fig. 7B).
For all MO combinations, a range of phenotypic severity was
observed, from mildly (Fig. 7G,H) to severely affected (Fig.
7E,F,I,J). Both mild and severe wnt defects were accompanied by
RESEARCH ARTICLE
Fig. 6. Multiple Tcf/Lefs are required for zic gene expression.
Embryos injected with the MOs shown at the lower left of each panel
and stained for zic2aD5:gfp (A,C,E,G,I) or zic2a (B,D,F,H,J) at 18 hpf.
(C-D) Strongly affected tcf3 morphants had anterior trunctions and
reduced zic2aD5:gfp (asterisks) and zic2a domains. (E-J) lef1 morphants
showed a strong reduction in zic2aD5:gfp (E) and a mild reduction in
zic2a (F). tcf7 morphants showed a mild reduction in zic2aD5:gfp (G),
but normal zic2a (H). lef1+tcf7 morphants showed a strong reduction
in zic2aD5:gfp (I) and mild reduction in zic2a (J). All views are lateral,
anterior to the left. Arrowheads indicate the midbrain.
alar/basal plate junction, was also positioned correctly in morphant
midbrains (Fig. 8Q,R), as was shh, a marker of the ventral midline
(Fig. 8S,T). Although all examined midbrain markers were expressed
in the proper DV positions in zic morphants, the morphant pax7
domain was smaller (Fig. 8D,N). Indeed, nuclear counts in midbrain
cross-sections at 24 hpf revealed significantly fewer cells in
morphants (an average 95 cells, n=4) compared with controls (an
average 195.5 cells, n=4, P=3.4⫻10–6). This delayed growth affected
the ventral midbrain, where zic genes are not expressed, in addition
to the dorsal midbrain, suggesting that signals from the tectum
promote proliferation in the tegmentum. Together, these data failed
to identify DV patterning defects, but demonstrated abnormal
morphology and a reduced cell number in morphant midbrains.
Cell proliferation in the dorsal midbrain is
reduced in zic morphants
Reduction in the pax7 domain and overall cell number in 24-hpf
zic morphants suggested that the zic genes function before 24 hpf
to promote midbrain growth. To test this, we asked whether
zic2a+zic5 morphants show increased cell death or decreased cell
Development 134 (4)
proliferation in the dorsal midbrain. TUNEL assays performed at
the 16- and 19-somite stages and at 24 hpf showed normal
apoptosis in morphants (n=69). Proliferating (M-phase) cells were
detected by anti-PH3 immunostaining and mitotic indices at
several defined DV levels were calculated (see Materials and
methods). At the 19-somite stage, five representative zic2a+zic5
morphants and five conMO-injected embryos were analyzed. Zic
morphants had lower mitotic indices (Fig. 7A; T2 test, P=0.002),
but the same total cell number (average morphant, 1110.6 cells;
average control, 1165.5 cells; P=0.5). Statistical comparisons
between individual DV positions revealed significantly lower
mitotic indices in the three dorsal-most points, i.e. dorsal midline
and alar plate regions where zic2a and zic5 are expressed
(asterisks in Fig. 9A; dorsal 1, P=0.04; dorsal 2, P=0.05; dorsal 3,
P=0.009; Fig. 9, compare B and E with C,D,F,G). Morphants and
controls had the same total cell number and mitotic indices earlier
in development at 13 somites (n=8, data not shown), suggesting
that proliferation is reduced in morphants after the 13-somite
stage.
To better pinpoint the most-affected DV region in morphants, 19somite-stage embryos were immunostained simultaneously for Pax7
and PH3. In zic morphants, PH3-positive cells were strongly
reduced within the dorsal half of the Pax7 domain (Fig. 9, compare
H with K), and only mildly reduced in the ventral Pax7 domain (Fig.
9, compare I with L). There was no reduction in PH3-stained cells
in the basal plate (Fig. 9J,M). In summary, the proliferation defect
in zic2a+zic5 morphants originated within the alar plate, where
zic2a and zic5 are co-expressed, between the 13- and 19-somite
stages (15.5 and 18.5 hpf, respectively). The cumulative result of a
continued decrease in cell proliferation can explain the smaller
midbrains and morphological defects observed at 24 hpf. Thus, the
earliest demonstrable zic2a and zic5 function in the midbrain is to
stimulate proliferation.
DISCUSSION
Zic genes encode a family of zinc-finger TFs with essential
functions during neural development. We present evidence that in
zebrafish, canonical Wnt signaling activates transcription of the
zic2a-zic5 gene pair throughout the developing dorsal brain,
including the tectum, and that this activation is very likely to be
direct. We also show that the primary function of zic2a and zic5 is
to promote cell proliferation in the forming tectum. Together, these
findings identify a novel component of the mechanism that regulates
brain development downstream of Wnt signaling, and are
particularly relevant to understanding the crucial mitogenic role
Wnts play in the midbrain.
Canonical Wnt signaling activates transcription of
zic genes
Expression of zic2a and zic5 is restricted spatially and temporally,
and this restriction is crucial for their correct function, yet the genetic
mechanisms of their regulation are virtually unknown (Aruga,
2004). We have identified Tcf/Lefs, effectors of the canonical Wnt
signaling pathway, as essential activators of the zic2a-zic5 gene pair.
We demonstrate that dominant-negative Tcf3, ⌬Tcf, represses zic
gene transcription, whereas a constitutively active Tcf fusion
protein, GR-Lef-␤-cat, is sufficient to activate it. We further show
that activation by GR-Lef-␤-cat occurs independently of translation,
and that repression by ⌬Tcf is very rapid, suggesting that the zic
genes are direct or very proximal targets of Tcf/Lefs. zic2a was also
induced by ␤-catenin, confirming that it is regulated by canonical
Wnt signaling.
DEVELOPMENT
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zic2a and zic5 regulate tectal proliferation
RESEARCH ARTICLE
743
Fig. 7. MO knockdown of zic2a and zic5 disrupts dorsal midbrain formation. (A) MO-binding sites in the zic2a-zic5 locus. Coding regions are
shown as gray boxes, non-coding transcribed regions as white boxes, and introns as lines. MO-binding sites are designated with red lines.
(B-J) Embryos injected with indicated MOs were assayed for expression of wnt1 or wnt3a at 24-25 hpf. Error bars in B show s.e.m. (C,D) Embryo
injected with conMO (8 ng) showing normal wnt1 expression. (E,F) Severely affected embryo injected with 2MOC (2 ng) showing disorganized
wnt1 expression in dorsal midbrain and hindbrain. (G,H) Mildly affected embryo injected with 2MOA+2MOB (6.6 ng). (I,J) Embryos co-injected with
2MOC and 5MOC (1 ng each) showing severe defects in wnt1 expression. C,E,G,I are lateral views; D,F,H,J are dorsal views of the embryos shown
to the left; embryos are positioned with anterior to the left. Arrowheads indicate the midbrain.
Other regulators of zic gene transcription
Wnt signaling through Tcf/Lefs almost certainly cooperates with
additional regulatory inputs to shape the complex spatial and
temporal pattern of zic2a and zic5 expression. Previous studies
placed zic1 downstream of BMP signaling (Rohr et al., 1999; Aruga
et al., 2002b). BMP signaling also regulates Wnt signaling (Chesnutt
et al., 2004), and may therefore activate zic gene expression
indirectly, through the Wnt pathway. Hedgehog signaling represses
zic gene expression in the ventral neural tube, but the mechanism of
this repression has not been addressed (Aruga et al., 2002b; Brown
et al., 2003; Rohr et al., 1999). It is probable that the critical region
D5 defined in our analysis contains binding sites for TFs that
meditate these regulatory inputs. Detailed dissection of D5 and other
zic enhancers will help identify direct regulators and additional
signaling pathways that regulate zic2a and zic5.
Zic genes and the cell cycle
Our data document an important role for zic2a and zic5 in cell-cycle
regulation in the zebrafish midbrain. This is the first analysis of zic
function in zebrafish, and the first demonstration of a role for zic
genes in the developing vertebrate midbrain. zic1 has previously
been shown to promote proliferation, but a similar role for zic2 has
not been demonstrated conclusively (Aruga et al., 2002b). zic2 has
been shown to inhibit cell-cycle exit, and thereby promote mitosis,
using assays that may not have been specific to zic2 (Brewster et al.,
1998). By contrast, mouse mutants lacking zic2 function have
normal proliferation rates (Elms et al., 2003). Our data definitively
demonstrate a role for zic2 in regulating proliferation. In humans,
ZIC proteins are expressed in the vastly proliferative cerebellar EGL
and in its tumor derivative, medulloblastoma (Miyata et al., 1998;
Yokota et al., 1996), making it very important to understand their
role in proliferation. Our studies establish zebrafish as a promising
new model for dissecting zic gene function in cell-cycle control.
DEVELOPMENT
We have identified a region of the zic2a-zic5 locus, D5, which
contains enhancers of midbrain and posterior hindbrain
transcription. D5 associates with ⌬Tcf in vivo, as demonstrated in
ChIP assays, and contains six consensus Tcf/Lef-binding sites.
Together, these data conclusively show that zic2a and zic5 are a
proximal target of Wnt signaling, and argue that this regulation is
probably direct. Formal proof of direct interaction will, however,
require identification of functional Tcf/Lef-binding sites and the
endogenous Tcf/Lefs that bind to them.
Eliminating zic2a and zic5 expression using ⌬Tcf, a dominant
inhibitor of Tcf/Lef targets, showed that Tcf/Lefs are required for
zic gene expression (Lewis et al., 2004). Several Tcf/Lefs have been
identified in zebrafish, and we tested four of these with respect to zic
regulation. Neither tcf3 nor lef1+tcf7 MOs completely inhibited
endogenous zic gene expression. A possible explanation for this
result is that canonical Wnt signaling activates zic expression
through the D5 enhancer, but that other Wnt-independent enhancers
are sufficient for partial activation of the zic genes in the absence of
Tcf/Lef function. Alternatively, all four Tcf/Lefs may cooperate to
regulate the zic genes. Quadruple morphant analysis aimed at testing
this hypothesis proved uninformative, as these morphants exhibited
non-specific MO toxicity.
RESEARCH ARTICLE
Development 134 (4)
Fig. 8. Knockdown of zic2a and zic5 disrupts midbrain morphology, but not DV pattern. Embryos were injected with conMO (8 ng), or
2MOC and 5MOC combined (1 ng each), and stained for DV marker expression by ISH. (A-D) Marker expression in 19-somite embryos. wnt3a
marks dorsal midline and adjacent cells in midbrains of conMO-injected embryos (A) and in zic morphants (B). pax7 marks alar midbrain in controls
(C) and morphants (D). (E-T) Marker expression in 24-hpf embryos. wnt3a is expressed in dorsal tectum of conMO-injected embryos (E-G). (H-J)
wnt3a domain in zic morphants. (K-M) pax7 in the alar tectum of controls. (N-P) pax7 in zic morphants is reduced, but correctly positioned. (Q,R)
hlx1 marks basal plate midbrain of controls (Q) and zic morphants (R). (S,T) shh in ventral midbrain of controls and zic morphants. A-D,E,H,K,N,Q-T
are lateral views, anterior to the left; F,I,L,O are dorsal views of embryos at left; G,J,M,P are midbrain cross-sections at positions indicated by lines in
images to left. Arrowheads indicate the midbrain.
Patterning versus proliferation in the midbrain
Our examination of zic2a+zic5 morphant midbrains identified
proliferation defects, but no clear DV patterning defects. Although
the dorsal domains of wnt1 and wnt3a expression have the
appearance of a patterning defect, this phenotype is best interpreted
as a morphological defect, as morphant wnt gene-expressing cells
are disorganized, but no more numerous than controls.
Overexpression studies in Xenopus (Aruga, 2004) reported neural
and neural-crest inducing roles for zic genes, suggesting that zic
genes pattern the future brain early during development, well before
the stages examined in the current study. However, cell proliferation
was not examined in these studies.
DEVELOPMENT
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zic2a and zic5 regulate tectal proliferation
RESEARCH ARTICLE
Wnt
745
N-myc and/or
Cyclin D
β-catenin
Tcf/Lefs
Zic2a
Zic5
Fig. 10. A proposed role for zic genes in the developing
midbrain. Expression of zic2a and zic5 is activated through canonical
Wnt signaling in the dorsal midbrain. Activation of zic tectal expression
is mediated through Tcf/Lefs, including Lef1 and Tcf7 acting through an
enhancer in the downstream zic5 (D5) region. Zics subsequently control
proliferation in the alar plate.
2004; Ishibashi and McMahon, 2002; Muroyama et al., 2002). Our
findings support a hypothesis that zic2a and zic5 are patterned by
Wnt signaling, and subsequently function in cell proliferation.
Identification of transcriptional targets of zic2a and zic5 will help us
to better understand how these two genes regulate midbrain growth
and pattern.
A role for zic genes in regulating tectal
proliferation
Taken together, our findings place zic2a and zic5 in the context of
an important signaling pathway that controls growth in the
developing midbrain (Fig. 10). Wnt signaling, a proven dorsal
midbrain mitogen (Ikeya et al., 1997; Panhuysen et al., 2004) acts
through several Tcf/Lef proteins to activate zic2a and zic5
transcription in the tectum. Zics in turn stimulate the mitotic cell
cycle in the tectum, perhaps through regulating cyclin D1 (Aruga et
al., 2002a) or other genes required for cell-cycle exit and neuronal
differentiation (Ebert et al., 2003). Wnts may also regulate
proliferation through a parallel pathway by directly activating the
cell-cycle genes n-myc (mycn – Zebrafish Information Network)
and cyclin D1, as they do in other model systems (Shtutman et al.,
1999; Shu et al., 2005; Tetsu and McCormick, 1999). Future
detailed analysis of zic gene regulation and identification of direct
zic targets will test this model and contribute to a mechanistic
understanding of how patterning and proliferation are coordinated
during midbrain development.
Although our results support a primary role for zic2a and zic5 in
midbrain proliferation, we cannot exclude a more subtle role in DV
patterning. Wnts, as well as other candidate regulators of zic genes
such as Shh, control both patterning and proliferation (Buckles et al.,
We thank Rick Nordheim and Peter Crump for help with statistical analysis;
Nancy Wall for optimizing the histology protocol; Melisa Shiroda, Matt
Gillhouse, Suzie Sagues and Andrea Gallagher for technical assistance; Arne
Lekven, Bill Dobens and Vicky Prince for sharing reagents; and Vicky Prince,
Sergei Sokol, Nick Sanek, Aaron Taylor and Ji Eun Lee for valuable discussions
during manuscript preparation. This work was funded by NIH RO1
GM076244-01 to Y.G.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/134/4/735/DC1
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