The zebrafish zic2a zic5 gene pair acts downstream of ... · The midbrain, one of the fastest...

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INTRODUCTIONIn the developing embryo, cells acquire distinct positional identities,or pattern, while simultaneously undergoing rapid division. Precisecoordination of pattern formation and proliferation is clearlyessential for normal embryogenesis, yet the molecular mechanismsresponsible for this coordination are not well understood for anyembryonic tissue. The midbrain, one of the fastest growingembryonic regions, is an attractive model system for dissecting thesemechanisms. In adults, the midbrain is located within the brainstemand is divided into the tectum dorsally and tegmentum ventrally. Thetectum controls visual reflexes and coordinated muscle movementsand relays auditory information, whereas the tegmentum containsnerve tracts that send information between the spinal cord andforebrain. These essential functions require patterns to be correctlyformed during development, while the midbrain tissue is rapidlygrowing.

Canonical Wnt signaling plays a major role during midbrainformation, as illustrated by midbrain deletion in Wnt1 mousemutants (McMahon and Bradley, 1990; Thomas and Capecchi,1990). Both patterning and proliferation-promoting roles have beenascribed to Wnt signaling during neural development. Wnt signalingplays a mitogenic role in the spinal cord (Chesnutt et al., 2004;Dickinson et al., 1994; Megason and McMahon, 2002), midbrainand hindbrain of higher vertebrates (Ikeya et al., 1997; Panhuysen etal., 2004), and in zebrafish midbrain (Lekven et al., 2003). WhetherWnts are patterning molecules, trophic factors or both (Buckles etal., 2004) in the forming midbrain is still unclear. However,canonical Wnt signaling is likely to have a central role in the geneticnetwork that links midbrain patterning and proliferation.Discovering genes regulated downstream of Wnt signaling is anecessary step toward elucidating this network.

Zic genes (Aruga, 2004; Aruga et al., 1994) are candidate targetsof 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 commonlyarranged in closely linked gene pairs. In humans, partial loss-of-function of ZIC2 is associated with holoprosencephaly and neuraltube closure defects (Grinberg and Millen, 2005). Mutant analysisof mouse Zic2 and Zic5, a linked gene pair, showed that these genesare 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 crestformation (Elms et al., 2003; Inoue et al., 2004). Several zic genesregulate neural precursor proliferation (Aruga et al., 2002b; Brewsteret al., 1998; Ebert et al., 2003), but no brain-specific regulators andfew transcriptional targets of zic genes have been identified (Ebertet al., 2003).

In this study, we use genetic and transgenic methods, combinedwith chromatin-binding assays, to show that Tcf/Lef factors canactivate zic2a and zic5 transcription in the tectum, and that thisactivation is likely to be direct. Using morpholino oligo (MO)knockdown assays, we demonstrate a requirement for zic2a and zic5in midbrain proliferation. Collectively, these data place zebrafishzic2a and zic5 either directly or very proximally downstream of Wntsignaling, and document an important role for these genes inregulating midbrain growth.

MATERIALS AND METHODSZebrafish strains and embryo cultureAdult zebrafish were maintained according to established methods(Westerfield, 1995). Embryos were obtained from natural matings andstaged according to Kimmel et al. (Kimmel et al., 1995). For transientanalysis, wild-type embryos were injected with 25-50 pg of plasmid DNAor PCR product at the one-cell stage. Deletion constructs were PCRamplified from zic2aD5:gfp (green fluorescent protein) plasmid.Zic2aD5:gfp stable transgenics were produced using established methods(Stuart et al., 1990).

The zebrafish zic2a-zic5 gene pair acts downstream ofcanonical Wnt signaling to control cell proliferation in thedeveloping tectumMolly 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 braindevelopment. In particular, canonical Wnt signaling is crucial for normal development of the dorsal midbrain, the future optictectum. 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 identifiedtwo new Wnt targets, the closely linked zic2a and zic5 genes. Using a combination of in vivo assays, we show that zic2a and zic5transcription is activated by Tcf/Lef transcription factors in the dorsal midbrain. Zic2a and Zic5, in turn, have essential, cooperativeroles in promoting cell proliferation in the tectum, but lack obvious patterning functions. Collectively these findings suggest thatWnts 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

Development 134, 735-746 (2007) doi:10.1242/dev.02756

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

*Author for correspondence (e-mail: [email protected])

Accepted 23 November 2006

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Sequence analysis and plasmid constructionA zic2a-zic5-containing BAC clone was identified by high-stringencyhybridization to the CHORI-211 library (BACAP Resources Center,Oakland, CA), and sequenced. Sequence was also obtained from the Daniorerio Sequencing Group at the Sanger Institute. Conserved regions wereidentified using Pipmaker (Schwartz et al., 2000). To generate thezic2aD5:gfp transgene, a portion of the zic2a-zic5 BAC clone was subclonedwith gfp into pBluescript KS (Stratagene).

In situ hybridization (ISH) and histologyAntisense RNA probes were transcribed from the following plasmidtemplates: zic2a (Grinblat and Sive, 2001), zic5 (Toyama et al., 2004), dlx3(dlx3b – Zebrafish Information Network) (Akimenko et al., 1994), pax7 (Seoet al., 1998), wnt1 (Molven et al., 1991), wnt3a (wnt3l – ZebrafishInformation 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 12medium (Ted Pella), and sections (5 �m) were cut with a steel blade on anAmerican Optical Company microtome. Nuclei were counterstained withMethyl Red.

Heat-shock induction of Tg(hs:GFP�tcf)Heterozygous Tg(hs:Gfp�tcf) embryos (Lewis et al., 2004) were heatshocked at 37°C for 30 or 45 minutes, then incubated at 29°C and fixed forISH. Embryos expressing Gfp�Tcf were identified using a Leica MZFLIIIstereoscope.

Ectopic activation of Wnt signalingTo 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 atthe 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-�-catplasmid 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. Fortreatments, injected embryos were incubated in 10 mM dexamethasone(DEX, Sigma) and/or 20 mM cycloheximide (CHX, Sigma) in E3 embryomedium for 2 hours, starting at 70% epiboly, until fixation at 90-100%epiboly. Control embryos were treated with equivalent concentrations ofvehicle (ethanol or DMSO) in E3.

Chromatin immunoprecipitation (ChIP) assaysChIP was performed as described previously (Lee, 2006; Weinmann et al.,2001) using an anti-Gfp antibody (Molecular Probes). The following PCRprimers were used:ngn1, 5�-GGGCTCATTGGAGCAAGTTTGATT-3� and 5�-CGC GGTAG -CCTACATTACTGCACA-3�;ngn1 negative, 5�-GTGACAGTTTTGTTGACCACGACG-3� and 5�-CTG GAGATTCGGCGTGGGGTTGGG-3�;nacre, 5�-GCAATTACCAAAGGCCCATCAGAC-3� and 5�-ACT GGC -TT ACGGCTAACTAACGTT-3�;zic2a-zic5 intergenic, 5�-TTGAACGGAGAAAATCACAACTAA-3� and5�-TATTACGAATCGATGTAACGGAGA-3�;zic5 3� enhancer, 5�-CTGCTTTGATGATTGACCATTTAG-3� and 5�-AC -TTTATGAAAAGCGGAATAGCAC-3�;zic5 intron, 5�-GGTGATGTGCATATTTAACTGGAA-3� and 5�-ACG -TTGTACAAATGCTATGTTGCT-3�.

PCR products were visualized on ethidium bromide-stained agarosegels. Each ChIP was repeated two to six times, except for hs:Gfp controlswhich were performed once.

MO knockdown assaysThe following antisense MOs were purchased from GeneTools:zic2a translation-blocking 2MOA, 5�-CGATGAAGTTCAATCCCCGCT-CACA-3� and 2MOB, 5�-CTCTTTCAAGCAGTCTATTCACGGC-3�;zic2a intron 1/exon 2 splice-blocking 2MOC, 5�-CTCACCTGAGAAG-GAAAACATCATA-3�;

zic5 intron 1/exon 2 splice-blocking 5MOC, 5�-GGCTTCTCACCTGT-CAAATGTAAAA-3�;tcf3a translation-inhibiting MO, 5�-TTTTTTGCTTACTTCGGAGTCT-GATG-3�;tcf3b splice-blocking MO, 5�-CGCCTCCGTTAAGCGGCATGTT-3�;tcf7 splice-blocking MO, 5�-AGCTGCGGCATGATCCAAACTTTCT-3�;lef1 exon 7/intron 7 splice-blocking MO, 5�-ACTGCCTGG AT -GAAACACTTACATG-3�; andstandard control conMO, 5�-CCTCTTACCTCAGTTACAATTTATA-3�.

MOs were diluted in 1� Danieau buffer (Nasevicius and Ekker, 2000) to2 ng/nl (2MOC, 5MOC, lef1MO, or 2MOC and 5MOC combined), 6.6ng/nl (2MOA and 2MOB combined), or 7-8 ng/nl (conMO). 1 nl of MO perembryo was injected at the one- to two-cell stage.

ImmunohistochemistryEmbryos were fixed in 4% formaldehyde in PBS and stained using thefollowing 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), Alexa488-conjugated goat anti-rabbit secondary (Molecular Probes, 1:500) andAlexa564-conjugated goat anti-rabbit secondary (Molecular Probes, 1:500).For double immunostaining, fixed embryos were treated with 1% hydrogenperoxide.

Cell proliferation analysisPH3-stained embryos were incubated in 0.1 �M ToPro3 (Molecular Probes),rinsed and mounted in DABCO antifade reagent (Sigma). A confocal z-serieswas generated at 40� using an Axiovert 100M (Carl Zeiss MicroImaging,Inc.) with Lasersharp Confocal Package (model 1024, Bio-Rad). Midbrainwas located relative to the retinae. Mitotic index (MI) was calculated bydividing PH3+ cell number by total cell number (ToPro3+). This was repeatedat nine dorsoventral (DV) positions for each embryo. Mean MIs wereconverted using the arc sin square root calculation to homogenize variancesbetween morphants and controls (Snedecor and Cochran, 1980). T2, ageneralization of the standard t-test for data with multivariate responses, wasused to compare combined MIs (all DV positions). MIs at individual DVpoints were compared using a standard independent-sample t-test. ForPH3+Pax7-stained embryos, confocal z-sections were processed using ImageJand Adobe Photoshop to generate three z-stacks: the dorsal half of the Pax7domain included MI points 1 and 2; the ventral half of the Pax7 domainincluded dorsal point 3 and hinge point; and the Pax7-negative domainincluded all ventral points. TUNEL staining was performed using the In SituCell Death Detection Kit, POD (Cole and Ross, 2001).

RESULTSZebrafish zic2a and zic5 are linked and co-expressedA linked, divergently transcribed genomic arrangement ischaracteristic of zic family members (Aruga, 2004; Grinberg andMillen, 2005). Zebrafish zic2a and zic5 are paired (Fig. 1A) and theirtranscription start sites (Grinblat and Sive, 2001; Toyama et al.,2004) are separated by 4.6 kb of intergenic sequence, suggesting thatzic2a and zic5 may share regulatory elements. The expressionpatterns of zic2a and zic5 have been described previously (Grinblatand Sive, 2001; Toyama et al., 2004), but not compared side-by-side.At midgastrula stages, zic2a was expressed throughout theneurectoderm (Fig. 1B), but zic5 was not expressed. By late gastrulastages, zic2a and zic5 were co-expressed at the neural–non-neuralborder, the presumptive dorsal neural tube (Fig. 1C and data notshown). During somitogenesis, zic2a and zic5 were co-expressed inthe dorsal brain, including the forming tectum (Fig. 1D,E,arrowheads). By 24 hours post-fertilization (hpf), tectal expressionwas restricted to the dorsal midline (Fig. 1F,G). Their close genomicproximity and similar expression patterns suggest that zic2a and zic5are likely to share regulatory elements and to have overlappingfunctions during development.

RESEARCH ARTICLE Development 134 (4)

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The zic2a-zic5 locus contains midbraintranscriptional enhancersTo identify potential regulatory regions, we compared the zic2a-zic5locus to the orthologous region in Fugu rubripes. Four conservedregions were identified (thick lines in Fig. 2A): the proximalpromoters of zic2a (P2) and zic5 (P5), part of the intergenic region(IG), and a region 3� to zic5 (D5) (see Fig. S1 in the supplementarymaterial). We designed several reporter constructs to test theenhancer activities of the conserved regions in vivo. The first two,zic5D5:gfp and zic2aD5:gfp, contained the IG and D5 regions, butdiffered in placement of gfp (Fig. 2B). In transient assays, Gfp wasexpressed strongly from both constructs, and Gfp-positive cells wereconcentrated 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 wasexpressed strongly and distributed similarly to the full-lengthconstruct (Fig. 2F). Zic2a:gfp was expressed in a few randomlydistributed cells (Fig. 2G). The latter pattern is characteristic ofreporter constructs that contain only proximal promoter sequencesand no enhancers. Zic2aIG�D5:gfp was not expressed in themidbrain and hindbrain, but was expressed strongly in thetelencephalon (data not shown). Based on these data, we concludethat D5 contains transcriptional enhancers that, independently oftheir genomic orientation, are sufficient to activate midbrain andhindbrain transcription.

Restricted expression of zic2aD5:gfp in the midbrain andhindbrain was confirmed in a stable transgenic line, Tg(zic2aD5:gfp)(Fig. 3). Gfp RNA in Tg(zic2aD5:gfp) embryos was first detected atthe 10-somite stage (14 hpf), and Gfp fluorescence was detected at

the 12- to 13-somite stage (15.5 hpf). Reporter expressionrecapitulated endogenous zic gene expression in the midbrain andposterior hindbrain at midsomitogenesis (Fig. 3, compare A,B,C)and late somitogenesis (Fig. 3D,E), whereas endogenous zic2a isexpressed throughout the dorsal brain (Fig. 3F). At 24 hpf, reporterexpression (Fig. 3G,H) was identical to endogenous expression (Fig.3I) in the midbrain and posterior hindbrain. Collectively, thisanalysis demonstrated that enhancers located in the D5 region directdorsal midbrain and hindbrain expression, and that additionalenhancers must control zic gene expression in other tissues.

Tcf/Lefs regulate zic2a and zic5 transcription anddirectly bind zic2a-zic5 genomic DNAThe zic2a-zic5 locus contains several consensus binding sites forTcf/Lefs, effectors of canonical Wnt signaling (Dorsky et al., 2000;Eastman and Grosschedl, 1999; Nusse, 1999; Ryu et al., 2001). Sixconsensus sites were found in the region (D5) that containsfunctional midbrain and hindbrain enhancers (see Fig. S1 in thesupplementary material). In addition, expression from zic2aD5:gfpoverlaps expression from Tg(TOP:GFP), a Wnt-signaling reporter(Dorsky, 2002) (see Fig. S2 in the supplementary material). Theseobservations led us to ask if Wnt signaling regulates zic2a and zic5.

We used hs:gfp�tcf transgenics to inhibit Tcf/Lef-mediatedtranscriptional activation (Lewis et al., 2004). Upon heat shock,Tg(hs:gfp�tcf) embryos produce a Gfp�Tcf fusion protein that isunable to bind �-catenin and acts as a dominant repressor of Wnttarget genes. Tg(hs:gfp�tcf) embryos were heat shocked duringsomitogenesis, allowed to recover and accumulate Gfp�Tcf protein,and assayed for zic expression (Fig. 4A). The result was a strongreduction in zic2a RNA only 20 minutes after Gfp�Tcf protein

737RESEARCH ARTICLEzic2a and zic5 regulate tectal proliferation

Fig. 1. Zic2a and zic5 are linked and co-expressed in presumptive dorsal brain. (A) Genomic arrangement of zic2a and zic5. Coding regionsare 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-neuralectoderm. (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 co-expressed in the optic stalk, telencephalon and dorsal diencephalon, and weakly in the tectum. All views are lateral, anterior to the left, except inB,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.

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began to accumulate (Fig. 4B-D, and see Table S1 in thesupplementary material). zic2a continued to be inhibited for at leasttwo hours post-heat-shock (Fig. 4E,F), as was zic5 (Fig. 4G,H).

We also tested the effect of Gfp�Tcf on zic2aD5:gfp transgeneexpression. zic2aD5:gfp contains enhanced gfp with severalmodified codons, whereas hs:gfp�tcf encodes unmodified Gfp. Thetwo gfp variants could therefore be distinguished at the RNA level.Embryos carrying both hs:gfp�tcf and zic2aD5:gfp were heatshocked and assayed for expression of zic2aD5:gfp RNA. The resultwas complete loss of zic2aD5:gfp expression (Fig. 4I,J, and seeTable S1 in the supplementary material). An overlapping dorsal

neural tube marker, wnt3a, was expressed normally in the presenceof Gfp�Tcf (Fig. 4K,L, and see Table S1 in the supplementarymaterial), indicating that Gfp�Tcf specifically repressed zic genetranscription.

Next we asked whether the �Tcf protein bound directly to theconserved IG and D5 regions using ChIP (Weinmann et al., 2001).Tg(hs:gfp�tcf) embryos were heat shocked and subjected to ChIPwith an anti-Gfp antibody to isolate DNA bound to Gfp�Tcf.Precipitated DNA was tested by PCR for the presence of known directtargets of canonical Wnt signaling – nacre (mitfa – ZebrafishInformation Network) (Dorsky et al., 2000) and ngn1 (neurog1 –Zebrafish Information Network) (Hirabayashi et al., 2004). For bothgenes, promoter DNA was amplified after ChIP with anti-Gfpantibody, but not after control ChIP (Fig. 4M, compare lane 1 withlanes 2-4). An upstream ngn1 region, which contains onenonconserved putative Tcf/Lef-binding site, failed to amplify (Fig.4M, lane 5). These controls verified the assay could efficiently andspecifically identify direct targets of canonical Wnt signaling inzebrafish embryos.

When the products of Tg(hs:gfp�tcf)–anti-Gfp ChIP weresubsequently assayed for IG and D5, both were amplified (Fig. 4N).The zic5 intron contained no conserved consensus Tcf/Lef sites andwas not amplified (Fig. 4N). To confirm that IG and D5 were boundto the Tcf portion of the fusion protein, ChIP was applied toTg(hs:gfp) embryos (Halloran et al., 2000), which expressunmodified Gfp protein after heat shock. Neither IG nor D5 wasdetected in this experiment (Fig. 4N, lane 5). The results from ChIPanalysis showed that transgenic Tcf protein was bound directly to D5and IG portions of the zic2a-zic5 DNA and demonstrated thepresence of Tcf/Lef-binding sites in these regions, thereby suggestingthat zic2a and zic5 are direct targets of the canonical Wnt pathway.

�-catenin and Tcf activate ectopic zic2atranscriptionHaving demonstrated a role for Tcf/Lefs in regulating zic genes, weasked if the zic genes could be activated by �-catenin, a keycomponent of the Wnt signal transduction pathway that interactswith 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 ofgastrulation, when zic2a expression is tightly restricted at the neuralborder (Fig. 5A-C), �-catenin failed to induce zic2a in the medialneural plate (Fig. 5A). However, it induced robust ectopic zic2aexpression in the lateral ectoderm (Fig. 5B,C). Ectopic zic2a(purple) overlapped �-catenin-gfp expression (orange), consistent


Fig. 2. Transient transgenic analysis of zic2a-zic5 genomicregions. (A) Conserved DNA regions (thick lines) at the zic2a-zic5locus. P2 and P5, each approximately 300 nt, are 70% identical withFugu. 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 IGand six in D5 (asterisks). (B) Transgenic constructs tested in transientassays. (C,D) Dorsal midbrain and hindbrain expression of Gfp inrepresentative zic5D5:gfp and zic2aD5:gfp transient transgenics. Insetsare dorsal views of the embryos shown. Asterisks mark mid-hindbrainorganizer. (E-G) gfp RNA in representative transient transgenics. Inembryos injected with 25 pg of zic2aD5:gfp (E) or zic2aD5�IG:gfp (F),expression was concentrated in dorsal midbrain and hindbrain. Embryosinjected with 25, 50 or 70pg of zic2a:gfp (G) did not show enhancedexpression. All views are lateral, except for insets.

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with �-catenin acting cell-autonomously to activate zic genetranscription. In control embryos, Gfp protein produced by transientexpression of hs:gfp failed to induce zic2a (Fig. 5D).

We next asked if the �-catenin-Tcf complex could activate zictranscription directly (i.e. without synthesis of an intermediatetranscriptional activator), using a hormone-inducible �-catenin-Leffusion protein, GR-Lef�N-�-cat (GR-Lef) (Domingos et al., 2001).GR-Lef contains the Tcf DNA-binding domain, the �-catenintransactivation domain, and the glucocorticoid-receptor hormone-binding domain (GR), which allows it to become fully active whenbound to dexamethasone. This construct has been demonstrated toactivate Wnt target genes in zebrafish (Ramel and Lekven, 2004).By activating GR-Lef in the presence of cycloheximide, an inhibitorof protein synthesis, it is possible to determine whether GR-Leffunctions independently of translation.

To test whether GR-Lef could induce zic gene expression, GR-Lef-injected zic2aD5:gfp embryos were assayed for gfpexpression (Fig. 5E-I). This analysis was performed at lategastrula stage, prior to the normal onset of zic2aD5:gfpexpression (Fig. 3); therefore, any gfp expression at this stage isectopic. GR-Lef alone induced some ectopic transgene expression(Fig. 5E). However, the ability of GR-Lef to activate gfpexpression was enhanced by addition of DEX (Fig. 5F), evenwhen translation was inhibited with CHX (Fig. 5G, and see Fig.S3 in the supplementary material). Expression of zic2aD5:gfp wasnever observed in uninjected, vehicle-treated controls (Fig. 5H).Endogenous zic2a, normally expressed in late gastrulaneurectoderm, was also ectopically induced by GR-Lefindependently of translation (not shown). Collectively, these datademonstrate that zic2a can be activated directly downstream ofWnt signaling in the zebrafish neurectoderm and suggest that thezic2aD5:gfp transgene contains a Wnt-responsive enhancer.

Multiple endogenous Tcf/Lefs regulate zic genetranscriptionThe data reported thus far provide strong evidence that the canonicalWnt pathway directly regulates zic2a and zic5. We next usedmorpholino analysis to ascertain which endogenous Tcf/Lefsmediate this regulation. Two zebrafish tcf3 homologs, tcf3 (hdl) andtcf3b (tcf7l1a and tcf7l1b, respectively – Zebrafish InformationNetwork) are ubiquitously expressed and function redundantly(Dorsky et al., 2003). Endogenous zic2a was reduced in stronglyaffected tcf3+tcf3b morphants (Fig. 6D, and see Fig. S4 in thesupplementary material), but still expressed in two discrete domainsin the midbrain and hindbrain (asterisks in Fig. 6D). Zic2aD5:gfp,which is normally transcribed in these domains, was also expressedin Tcf3-depleted embryos (Fig. 6C, and see Fig. S4 in thesupplementary material). Although midbrain size changes due tocaudalization in Tcf3 morphants and mutants (Dorsky et al., 2003;Kim et al., 2000) (see Fig. S4 in the supplementary material), wnt3awas strongly expressed in tcf3 morphants, suggesting that the dorsalmidbrain was not substantially reduced (see Fig. S4 in thesupplementary material, compare C with O). Altogether, these datasuggest that Tcf3 and Tcf3b are required to activate zic2atranscription outside the midbrain and hindbrain, and are partiallyresponsible 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 lef1morphants, zic2aD5:gfp expression in the tectum was stronglyreduced compared with controls (Fig. 6E, and see Table S2 in thesupplementary material). By contrast, endogenous zic2a expressionwas largely unaffected. Zic2aD5:gfp expression was mildly affectedin tcf7 morphants (Fig. 6G), and endogenous expression was notaffected (Fig. 6H). In double lef1+tcf7 morphants, zic2aD5:gfpexpression was strongly reduced or eliminated (Fig. 6I). Expression


Fig. 3. Stable zic2aD5:gfp expressionrecapitulates endogenous midbrain andposterior hindbrain expression. Gfpexpression in Tg(zic2aD5:gfp) embryosdetected by ISH (A,D,G,H), or by fluorescence(B,E). (C,F,I) Endogenous zic2a expressiondetected by ISH. (A-C) gfp RNA at the 11- to12-somite stage (A) and Gfp protein at 14-somites (B) are restricted to dorsal midbrainand posterior hindbrain. Endogenous zic2a istranscribed in the dorsal diencephalon,midbrain and hindbrain at 11-somites (C). (F-H) Transgenic (D,E) and endogenous (F)expression overlap in the midbrain andposterior hindbrain at the 20-somite stage.At 24 hpf, transgenic expression (G,H)overlaps endogenous expression (I) in thedorsal midbrain. All views are lateral, anteriorto the left, except H,I which are dorsal views.Arrowheads mark anterior and posteriorlimits of tectum. m, midbrain; h, hindbrain.

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of zic2a was reduced, but only mildly (Fig. 6J). Taken together, thesedata indicate that several endogenous Tcf/Lefs regulate zic2aexpression: Lef1 and Tcf7 in the midbrain, acting through themidbrain enhancer in zic2aD5:gfp; and Tcf3 and Tcf3b throughoutthe endogenous zic2a expression domain.

Antisense MOs effectively knockdown zic2a andzic5 and disrupt formation of the dorsal brainAs Wnt signaling targets, zic2a and zic5 are likely to mediate someof its functions. To analyze the functions of zebrafish zic2a and zic5,MOs were used to knockdown zic2a and zic5 separately or in


Fig. 4. Tcf/Lefs are required for zic gene expression. (A) The method used to disrupt Tcf/Lef signaling at controlled times. (B) Temporalcorrelation between Gfp�Tcf induction (by fluorescence) and zic2a RNA (by ISH). (C-L) Representative embryos after ISH. Stage of heat-shock andrecovery time are indicated at upper right. Zic2a expression is normal in �Tcf-negative heat-shocked controls (C,E), and reduced or absent in �Tcf-positive siblings (D,F). Zic5 expression was normal in �Tcf-negative (G) embryos, and absent in �Tcf-positive embryos (H). Zic2aD5:gfp expressionwas 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 ofheat-shocked Tg(hs:gfp�tcf) and Tg(hs:gfp) embryos. Genotype of embryos and PCR templates are shown above, as follows: GFP Ab, chromatinafter anti-Gfp IP; input, total chromatin before IP; no Ab, no-antibody ChIP; mock, no chromatin. Regions amplified are labeled next to thecorresponding 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.

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combination. Three non-overlapping MOs were designed for zic2a(Fig. 7A); two to block translation (2MOA and 2MOB), and one toinhibit splicing (2MOC). In addition, a splice-blocking MO wasdesigned against zic5 (5MOC).

We observed two distinct defects in zic2a morphants: (1)abnormal morphology of the dorsal neural tube, includingincomplete 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 thesame or a higher amount of conMO formed well-definedsymmetrical ventricles, and were indistinguishable from uninjectedcontrols. 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 increasedpenetrance of the ventricle phenotype (100%, n=40), supporting apartially redundant function for these genes.

The morphological abnormalities in zic morphants werevisualized by expression of wnt1 and wnt3a, markers of the dorsalmidbrain and hindbrain (Fig. 7B-J). Control morphants displayedwild-type wnt gene expression at 24 hpf (Fig. 7B-D). By contrast,the tectal wnt expression domain was disorganized and shortened inthe AP direction (see Fig. S5 in the supplementary material) in zic2amorphants, zic5 morphants, and zic2a+zic5 morphants (Fig. 7B).For all MO combinations, a range of phenotypic severity wasobserved, 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 appearednormal in zic morphants (see Fig. S6 in the supplementary material).Interestingly, co-injection of 2MOC and 5MOC at half the dose usedin single knockdowns resulted in a dramatic increase in theproportion of severely affected embryos, from 46.5% in zic2amorphants to 80.5% in zic2a+zic5 morphants (Fig. 7B). Thisobservation again suggests functional redundancy. Together, thesedata validate the MOs as effective and specific tools for functionalanalysis of zic2a and zic5.

DV pattern forms correctly in the midbrains ofzic2a and zic5 morphantsThe abnormal wnt gene expression in zic morphants suggested thatzic genes pattern the dorsal neural tube. To test this, we analyzed theDV pattern in zic2a+zic5 morphant midbrains at two stages ofdevelopment. At the 19-somite stage, just as brain ventricles begin toinflate, wnt3a was expressed correctly in morphants as comparedwith controls (Fig. 8A,B). pax7, an alar plate marker immediatelyventral 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-positivecells in zic2a+zic5 morphant midbrains (an average 15 cells persection, n=3; Fig. 6G) and control embryos (an average 16.5 cells persection, n=3; Fig. 8J). However, morphant wnt3a-positive cells weredisorganized. The pax7 domain was misshapen in zic2a+zic5morphants, but positioned correctly in whole mounts (Fig. 8K,L,N,O)and in midbrain sections (Fig. 8M,P). hlx1, expressed near the


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 ectopicallyexpressed (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 embryosexpressing gfp for each treatment condition. The number of embryos scored for each condition is listed along the x-axis.

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alar/basal plate junction, was also positioned correctly in morphantmidbrains (Fig. 8Q,R), as was shh, a marker of the ventral midline(Fig. 8S,T). Although all examined midbrain markers were expressedin the proper DV positions in zic morphants, the morphant pax7domain was smaller (Fig. 8D,N). Indeed, nuclear counts in midbraincross-sections at 24 hpf revealed significantly fewer cells inmorphants (an average 95 cells, n=4) compared with controls (anaverage 195.5 cells, n=4, P=3.4�10–6). This delayed growth affectedthe ventral midbrain, where zic genes are not expressed, in additionto the dorsal midbrain, suggesting that signals from the tectumpromote proliferation in the tegmentum. Together, these data failedto identify DV patterning defects, but demonstrated abnormalmorphology and a reduced cell number in morphant midbrains.

Cell proliferation in the dorsal midbrain isreduced in zic morphantsReduction in the pax7 domain and overall cell number in 24-hpfzic morphants suggested that the zic genes function before 24 hpfto promote midbrain growth. To test this, we asked whetherzic2a+zic5 morphants show increased cell death or decreased cell

proliferation in the dorsal midbrain. TUNEL assays performed atthe 16- and 19-somite stages and at 24 hpf showed normalapoptosis in morphants (n=69). Proliferating (M-phase) cells weredetected by anti-PH3 immunostaining and mitotic indices atseveral defined DV levels were calculated (see Materials andmethods). At the 19-somite stage, five representative zic2a+zic5morphants and five conMO-injected embryos were analyzed. Zicmorphants 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 comparisonsbetween individual DV positions revealed significantly lowermitotic indices in the three dorsal-most points, i.e. dorsal midlineand 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 andcontrols had the same total cell number and mitotic indices earlierin development at 13 somites (n=8, data not shown), suggestingthat proliferation is reduced in morphants after the 13-somitestage.

To better pinpoint the most-affected DV region in morphants, 19-somite-stage embryos were immunostained simultaneously for Pax7and PH3. In zic morphants, PH3-positive cells were stronglyreduced within the dorsal half of the Pax7 domain (Fig. 9, compareH with K), and only mildly reduced in the ventral Pax7 domain (Fig.9, compare I with L). There was no reduction in PH3-stained cellsin the basal plate (Fig. 9J,M). In summary, the proliferation defectin zic2a+zic5 morphants originated within the alar plate, wherezic2a and zic5 are co-expressed, between the 13- and 19-somitestages (15.5 and 18.5 hpf, respectively). The cumulative result of acontinued decrease in cell proliferation can explain the smallermidbrains and morphological defects observed at 24 hpf. Thus, theearliest demonstrable zic2a and zic5 function in the midbrain is tostimulate proliferation.

DISCUSSIONZic genes encode a family of zinc-finger TFs with essentialfunctions during neural development. We present evidence that inzebrafish, canonical Wnt signaling activates transcription of thezic2a-zic5 gene pair throughout the developing dorsal brain,including the tectum, and that this activation is very likely to bedirect. We also show that the primary function of zic2a and zic5 isto promote cell proliferation in the forming tectum. Together, thesefindings identify a novel component of the mechanism that regulatesbrain development downstream of Wnt signaling, and areparticularly relevant to understanding the crucial mitogenic roleWnts play in the midbrain.

Canonical Wnt signaling activates transcription ofzic genesExpression of zic2a and zic5 is restricted spatially and temporally,and this restriction is crucial for their correct function, yet the geneticmechanisms of their regulation are virtually unknown (Aruga,2004). We have identified Tcf/Lefs, effectors of the canonical Wntsignaling pathway, as essential activators of the zic2a-zic5 gene pair.We demonstrate that dominant-negative Tcf3, �Tcf, represses zicgene transcription, whereas a constitutively active Tcf fusionprotein, GR-Lef-�-cat, is sufficient to activate it. We further showthat activation by GR-Lef-�-cat occurs independently of translation,and that repression by �Tcf is very rapid, suggesting that the zicgenes are direct or very proximal targets of Tcf/Lefs. zic2a was alsoinduced by �-catenin, confirming that it is regulated by canonicalWnt signaling.


Fig. 6. Multiple Tcf/Lefs are required for zic gene expression.Embryos injected with the MOs shown at the lower left of each paneland 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 andreduced zic2aD5:gfp (asterisks) and zic2a domains. (E-J) lef1 morphantsshowed a strong reduction in zic2aD5:gfp (E) and a mild reduction inzic2a (F). tcf7 morphants showed a mild reduction in zic2aD5:gfp (G),but normal zic2a (H). lef1+tcf7 morphants showed a strong reductionin zic2aD5:gfp (I) and mild reduction in zic2a (J). All views are lateral,anterior to the left. Arrowheads indicate the midbrain.

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We have identified a region of the zic2a-zic5 locus, D5, whichcontains enhancers of midbrain and posterior hindbraintranscription. D5 associates with �Tcf in vivo, as demonstrated inChIP assays, and contains six consensus Tcf/Lef-binding sites.Together, these data conclusively show that zic2a and zic5 are aproximal target of Wnt signaling, and argue that this regulation isprobably direct. Formal proof of direct interaction will, however,require identification of functional Tcf/Lef-binding sites and theendogenous Tcf/Lefs that bind to them.

Eliminating zic2a and zic5 expression using �Tcf, a dominantinhibitor of Tcf/Lef targets, showed that Tcf/Lefs are required forzic gene expression (Lewis et al., 2004). Several Tcf/Lefs have beenidentified in zebrafish, and we tested four of these with respect to zicregulation. Neither tcf3 nor lef1+tcf7 MOs completely inhibitedendogenous zic gene expression. A possible explanation for thisresult is that canonical Wnt signaling activates zic expressionthrough the D5 enhancer, but that other Wnt-independent enhancersare sufficient for partial activation of the zic genes in the absence ofTcf/Lef function. Alternatively, all four Tcf/Lefs may cooperate toregulate the zic genes. Quadruple morphant analysis aimed at testingthis hypothesis proved uninformative, as these morphants exhibitednon-specific MO toxicity.

Other regulators of zic gene transcriptionWnt signaling through Tcf/Lefs almost certainly cooperates withadditional regulatory inputs to shape the complex spatial andtemporal pattern of zic2a and zic5 expression. Previous studiesplaced zic1 downstream of BMP signaling (Rohr et al., 1999; Aruga

et al., 2002b). BMP signaling also regulates Wnt signaling (Chesnuttet al., 2004), and may therefore activate zic gene expressionindirectly, through the Wnt pathway. Hedgehog signaling represseszic gene expression in the ventral neural tube, but the mechanism ofthis repression has not been addressed (Aruga et al., 2002b; Brownet al., 2003; Rohr et al., 1999). It is probable that the critical regionD5 defined in our analysis contains binding sites for TFs thatmeditate these regulatory inputs. Detailed dissection of D5 and otherzic enhancers will help identify direct regulators and additionalsignaling pathways that regulate zic2a and zic5.

Zic genes and the cell cycleOur data document an important role for zic2a and zic5 in cell-cycleregulation in the zebrafish midbrain. This is the first analysis of zicfunction in zebrafish, and the first demonstration of a role for zicgenes in the developing vertebrate midbrain. zic1 has previouslybeen shown to promote proliferation, but a similar role for zic2 hasnot been demonstrated conclusively (Aruga et al., 2002b). zic2 hasbeen 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 havenormal proliferation rates (Elms et al., 2003). Our data definitivelydemonstrate a role for zic2 in regulating proliferation. In humans,ZIC proteins are expressed in the vastly proliferative cerebellar EGLand in its tumor derivative, medulloblastoma (Miyata et al., 1998;Yokota et al., 1996), making it very important to understand theirrole in proliferation. Our studies establish zebrafish as a promisingnew model for dissecting zic gene function in cell-cycle control.


Fig. 7. MO knockdown of zic2a and zic5 disrupts dorsal midbrain formation. (A) MO-binding sites in the zic2a-zic5 locus. Coding regions areshown 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) Embryoinjected with conMO (8 ng) showing normal wnt1 expression. (E,F) Severely affected embryo injected with 2MOC (2 ng) showing disorganizedwnt1 expression in dorsal midbrain and hindbrain. (G,H) Mildly affected embryo injected with 2MOA+2MOB (6.6 ng). (I,J) Embryos co-injected with2MOC 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 shownto the left; embryos are positioned with anterior to the left. Arrowheads indicate the midbrain.

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Patterning versus proliferation in the midbrainOur examination of zic2a+zic5 morphant midbrains identifiedproliferation defects, but no clear DV patterning defects. Althoughthe dorsal domains of wnt1 and wnt3a expression have theappearance of a patterning defect, this phenotype is best interpretedas a morphological defect, as morphant wnt gene-expressing cells

are disorganized, but no more numerous than controls.Overexpression studies in Xenopus (Aruga, 2004) reported neuraland neural-crest inducing roles for zic genes, suggesting that zicgenes pattern the future brain early during development, well beforethe stages examined in the current study. However, cell proliferationwas not examined in these studies.


Fig. 8. Knockdown of zic2a and zic5 disrupts midbrain morphology, but not DV pattern. Embryos were injected with conMO (8 ng), or2MOC and 5MOC combined (1 ng each), and stained for DV marker expression by ISH. (A-D) Marker expression in 19-somite embryos. wnt3amarks 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-Tare 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 inimages to left. Arrowheads indicate the midbrain.

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Although our results support a primary role for zic2a and zic5 inmidbrain proliferation, we cannot exclude a more subtle role in DVpatterning. Wnts, as well as other candidate regulators of zic genessuch as Shh, control both patterning and proliferation (Buckles et al.,

2004; Ishibashi and McMahon, 2002; Muroyama et al., 2002). Ourfindings support a hypothesis that zic2a and zic5 are patterned byWnt signaling, and subsequently function in cell proliferation.Identification of transcriptional targets of zic2a and zic5 will help usto better understand how these two genes regulate midbrain growthand pattern.

A role for zic genes in regulating tectalproliferationTaken together, our findings place zic2a and zic5 in the context ofan important signaling pathway that controls growth in thedeveloping midbrain (Fig. 10). Wnt signaling, a proven dorsalmidbrain mitogen (Ikeya et al., 1997; Panhuysen et al., 2004) actsthrough several Tcf/Lef proteins to activate zic2a and zic5transcription in the tectum. Zics in turn stimulate the mitotic cellcycle in the tectum, perhaps through regulating cyclin D1 (Aruga etal., 2002a) or other genes required for cell-cycle exit and neuronaldifferentiation (Ebert et al., 2003). Wnts may also regulateproliferation through a parallel pathway by directly activating thecell-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). Futuredetailed analysis of zic gene regulation and identification of directzic targets will test this model and contribute to a mechanisticunderstanding of how patterning and proliferation are coordinatedduring midbrain development.

We thank Rick Nordheim and Peter Crump for help with statistical analysis;Nancy Wall for optimizing the histology protocol; Melisa Shiroda, MattGillhouse, Suzie Sagues and Andrea Gallagher for technical assistance; ArneLekven, Bill Dobens and Vicky Prince for sharing reagents; and Vicky Prince,Sergei Sokol, Nick Sanek, Aaron Taylor and Ji Eun Lee for valuable discussionsduring manuscript preparation. This work was funded by NIH RO1GM076244-01 to Y.G.

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

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