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First published online 10 January 2007
doi: 10.1242/dev.02756

Development 134, 735-746 (2007)
Published by The Company of Biologists 2007
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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,*

1 Departments of Zoology and Anatomy, University of Wisconsin, Madison, WI, 53706, USA.
2 Department of Neurobiology and Anatomy, University of Utah, Salt Lake City, UT 84132, USA.

* Author for correspondence (e-mail: ygrinblat{at}wisc.edu)

Accepted 23 November 2006


   SUMMARY
TOP
 SUMMARY
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
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


   INTRODUCTION
TOP
 SUMMARY
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
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, 1990Go; Thomas and Capecchi, 1990Go). 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., 2004Go; Dickinson et al., 1994Go; Megason and McMahon, 2002Go), midbrain and hindbrain of higher vertebrates (Ikeya et al., 1997Go; Panhuysen et al., 2004Go), and in zebrafish midbrain (Lekven et al., 2003Go). Whether Wnts are patterning molecules, trophic factors or both (Buckles et al., 2004Go) 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.

Zic genes (Aruga, 2004Go; Aruga et al., 1994Go) 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., 1994Go), encode zinc-finger transcription factors (TFs). Five mammalian (Aruga, 2004Go) and seven zebrafish zic genes (Grinblat et al., 1998Go; Grinblat and Sive, 2001Go; Parinov et al., 2004Go; Toyama et al., 2004Go) 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, 2005Go). 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., 2003Go; Inoue et al., 2004Go; Nagai et al., 2000Go), and for midbrain-derived neural crest formation (Elms et al., 2003Go; Inoue et al., 2004Go). Several zic genes regulate neural precursor proliferation (Aruga et al., 2002bGo; Brewster et al., 1998Go; Ebert et al., 2003Go), but no brain-specific regulators and few transcriptional targets of zic genes have been identified (Ebert et al., 2003Go).

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
TOP
 SUMMARY
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Zebrafish strains and embryo culture
Adult zebrafish were maintained according to established methods (Westerfield, 1995Go). Embryos were obtained from natural matings and staged according to Kimmel et al. (Kimmel et al., 1995Go). 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., 1990Go).

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., 2000Go). 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, 2001Go), zic5 (Toyama et al., 2004Go), dlx3 (dlx3b - Zebrafish Information Network) (Akimenko et al., 1994Go), pax7 (Seo et al., 1998Go), wnt1 (Molven et al., 1991Go), wnt3a (wnt3l - Zebrafish Information Network) (Buckles et al., 2004Go), shh (Krauss et al., 1993Go), hlx1 (Fjose et al., 1994Go), and krox-20 (egr2b - Zebrafish Information Network) (Oxtoby and Jowett, 1993Go). ISH was performed as previously described (Gillhouse et al., 2004Go). Stained embryos were embedded in Eponate 12 medium (Ted Pella), and sections (5 µm) 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{Delta}tcf)
Heterozygous Tg(hs:Gfp{Delta}tcf) embryos (Lewis et al., 2004Go) were heat shocked at 37°C for 30 or 45 minutes, then incubated at 29°C and fixed for ISH. Embryos expressing Gfp{Delta}Tcf were identified using a Leica MZFLIII stereoscope.

Ectopic activation of Wnt signaling
To ectopically activate Wnt signaling, 15-25 pg of hs:ß-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{Delta}N-ß-cat plasmid was used as previously described (Ramel and Lekven, 2004Go). Following in vitro transcription (mMessage Machine, Ambion), GR-lef{Delta}N- ß-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, 2006Go; Weinmann et al., 2001Go) 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';

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 1x Danieau buffer (Nasevicius and Ekker, 2000Go) 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 µl/ml, Developmental Studies Hybridoma Bank, University of Iowa, developed by Atushi Kawakami, NICHB), Alexa488-conjugated 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 µM ToPro3 (Molecular Probes), rinsed and mounted in DABCO antifade reagent (Sigma). A confocal z-series was generated at 40x 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, 1980Go). 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, 2001Go).


   RESULTS
TOP
 SUMMARY
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Zebrafish zic2a and zic5 are linked and co-expressed
A linked, divergently transcribed genomic arrangement is characteristic of zic family members (Aruga, 2004Go; Grinberg and Millen, 2005Go). Zebrafish zic2a and zic5 are paired (Fig. 1A) and their transcription start sites (Grinblat and Sive, 2001Go; Toyama et al., 2004Go) 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, 2001Go; Toyama et al., 2004Go), 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.

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).


Figure 1
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  		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.

 
We next tested three deletions of zic2aD5:gfp: zic2aD5{Delta}IG:gfp, which lacks IG; zic2aIG{Delta}D5, which lacks D5; and zic2a:gfp, which lacks both IG and D5 (Fig. 2B). Zic2aD5{Delta}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{Delta}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 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., 2000Go; Eastman and Grosschedl, 1999Go; Nusse, 1999Go; Ryu et al., 2001Go). 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, 2002Go) (see Fig. S2 in the supplementary material). These observations led us to ask if Wnt signaling regulates zic2a and zic5.

We used hs:gfp{Delta}tcf transgenics to inhibit Tcf/Lef-mediated transcriptional activation (Lewis et al., 2004Go). Upon heat shock, Tg(hs:gfp{Delta}tcf) embryos produce a Gfp{Delta}Tcf fusion protein that is unable to bind ß-catenin and acts as a dominant repressor of Wnt target genes. Tg(hs:gfp{Delta}tcf) embryos were heat shocked during somitogenesis, allowed to recover and accumulate Gfp{Delta}Tcf protein, and assayed for zic expression (Fig. 4A). The result was a strong reduction in zic2a RNA only 20 minutes after Gfp{Delta}Tcf protein 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).


Figure 2
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  		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{Delta}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.

 
We also tested the effect of Gfp{Delta}Tcf on zic2aD5:gfp transgene expression. zic2aD5:gfp contains enhanced gfp with several modified codons, whereas hs:gfp{Delta}tcf encodes unmodified Gfp. The two gfp variants could therefore be distinguished at the RNA level. Embryos carrying both hs:gfp{Delta}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 neural tube marker, wnt3a, was expressed normally in the presence of Gfp{Delta}Tcf (Fig. 4K,L, and see Table S1 in the supplementary material), indicating that Gfp{Delta}Tcf specifically repressed zic gene transcription.

Next we asked whether the {Delta}Tcf protein bound directly to the conserved IG and D5 regions using ChIP (Weinmann et al., 2001Go). Tg(hs:gfp{Delta}tcf) embryos were heat shocked and subjected to ChIP with an anti-Gfp antibody to isolate DNA bound to Gfp{Delta}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., 2000Go) and ngn1 (neurog1 - Zebrafish Information Network) (Hirabayashi et al., 2004Go). 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{Delta}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., 2000Go), 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.

ß-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 ß-catenin, a key component of the Wnt signal transduction pathway that interacts with Tcf/Lefs to activate target genes (reviewed by Stadeli et al., 2006Go). We used hs:ß-catenin-gfp to transiently overexpress a ß-catenin-Gfp fusion protein during gastrulation. At the end of gastrulation, when zic2a expression is tightly restricted at the neural border (Fig. 5A-C), ß-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 ß-catenin-gfp expression (orange), consistent with ß-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).


Figure 3
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  		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 14-somites (B) are restricted to dorsal midbrain and posterior hindbrain. Endogenous zic2a is transcribed in the dorsal diencephalon, midbrain and hindbrain at 11-somites (C). (F-H) 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.

 
We next asked if the ß-catenin-Tcf complex could activate zic transcription directly (i.e. without synthesis of an intermediate transcriptional activator), using a hormone-inducible ß-catenin-Lef fusion protein, GR-Lef{Delta}N-ß-cat (GR-Lef) (Domingos et al., 2001Go). GR-Lef contains the Tcf DNA-binding domain, the ß-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, 2004Go). 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, GR-Lef-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., 2003Go). 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., 2003Go; Kim et al., 2000Go) (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., 1999Go; Lee et al., 2006Go), 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 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.


Figure 4
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  		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{Delta}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 {Delta}Tcf-negative heat-shocked controls (C,E), and reduced or absent in {Delta}Tcf-positive siblings (D,F). Zic5 expression was normal in {Delta}Tcf-negative (G) embryos, and absent in {Delta}Tcf-positive embryos (H). Zic2aD5:gfp expression was normal in {Delta}Tcf-negative embryos (I) and absent in {Delta}Tcf-positive embryos (J). Wnt3a expression was normal in heat-shocked controls (K) and in {Delta}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{Delta}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.

 
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 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).


Figure 5
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  		Fig. 5. Ectopic activation of the canonical Wnt pathway induces zic gene transcription. (A-C) Embryos were injected with hs:ßCat-gfp DNA, heat shocked at shield-stage, and assayed for zic2a (purple) and gfp (orange) at late-gastrula. (A) ßCat-gfp is expressed in the medial neural plate; the zic2a pattern is normal (no ectopic expression; n=10, two experiments). (B,C) ßCat-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.

 
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 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 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=3.4x10-6). 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.


Figure 6
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  		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.

 
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 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, 19-somite-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
TOP
 SUMMARY
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
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, 2004Go). 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, {Delta}Tcf, represses zic gene transcription, whereas a constitutively active Tcf fusion protein, GR-Lef-ß-cat, is sufficient to activate it. We further show that activation by GR-Lef-ß-cat occurs independently of translation, and that repression by {Delta}Tcf is very rapid, suggesting that the zic genes are direct or very proximal targets of Tcf/Lefs. zic2a was also induced by ß-catenin, confirming that it is regulated by canonical Wnt signaling.

We have identified a region of the zic2a-zic5 locus, D5, which contains enhancers of midbrain and posterior hindbrain transcription. D5 associates with {Delta}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.


Figure 7
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  		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.

 
Eliminating zic2a and zic5 expression using {Delta}Tcf, a dominant inhibitor of Tcf/Lef targets, showed that Tcf/Lefs are required for zic gene expression (Lewis et al., 2004Go). 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.

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., 1999Go; Aruga et al., 2002bGo). BMP signaling also regulates Wnt signaling (Chesnutt et al., 2004Go), 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., 2002bGo; Brown et al., 2003Go; Rohr et al., 1999Go). 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., 2002bGo). 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., 1998Go). By contrast, mouse mutants lacking zic2 function have normal proliferation rates (Elms et al., 2003Go). 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., 1998Go; Yokota et al., 1996Go), 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.

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, 2004Go) 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.


Figure 8
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  		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.

 
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., 2004Go; Ishibashi and McMahon, 2002Go; Muroyama et al., 2002Go). 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.


Figure 9
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  		Fig. 9. Cell proliferation is reduced in the dorsal midbrain of zic morphants. (A) A comparison of average mitotic index in control (blue) and 2MOC+5MOC (pink) morphant midbrains. Mitotic index (y-axis) was calculated at nine DV positions (x-axis) at the 19-somite stage. Error bars show s.e.m. Asterisks mark statistically significant reductions in mitotic index. (B-D) PH3 (green) staining in representative confocal z-sections through midbrain dorsal position 3 in control (B) and zic MO-injected (C,D) embryos. (E-G) A merge of PH3 and nuclear ToPro3 (red) staining in embryos shown above. (H-M) Confocal z-stacks of 19-somite conMO (H-J) and 2MOC+5MOC-injected (K-M) embryos immunostained for Pax7 (red) and PH3 (green). H,K are stacks of dorsal-most Pax7 domain; I,L are stacks of ventral Pax7 domain; J,M are stacks of ventral midbrain.

 


Figure 10
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  		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.

 
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., 1997Go; Panhuysen et al., 2004Go) 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., 2002aGo) or other genes required for cell-cycle exit and neuronal differentiation (Ebert et al., 2003Go). 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., 1999Go; Shu et al., 2005Go; Tetsu and McCormick, 1999Go). 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.

Supplementary material
Supplementary material for this article is available at http://dev.biologists.org/cgi/content/full/134/4/735/DC1


   ACKNOWLEDGMENTS
 
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.


   REFERENCES
TOP
 SUMMARY
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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