Regulation of Dharma/Bozozok by the Wnt Pathway

Developmental Biology 231, 397–409 (2001) doi:10.1006/dbio.2000.0150, available online at http://www.idealibrary.com on Regulation of dharma/bozozok by the Wnt Pathway

Seung-Lim Ryu, Ritsuko Fujii, Yojiro Yamanaka, Takashi Shimizu, Taijiro Yabe, Tsutomu Hirata, Masahiko Hibi, and Toshio Hirano1 Division of Molecular Oncology, Biomedical Research Center, Osaka University Graduate School of Medicine, 2-2 Yamada-oka, Suita, Osaka 565-0871, Japan

The zebraﬁsh homeobox gene dharma/bozozok (boz) is required for the formation and/or function of the Nieuwkoop center and the subsequent induction of the Spemann organizer. dharma is expressed soon after the midblastula transition in the dorsal blastomeres and the dorsal yolk syncytial layer (YSL). We found that the expression of dharma was upregulated or ectopically induced by misexpression of a Wnt protein and cytoplasmic components of the Wnt signaling pathway and downregulated by the expression of dominant-negative Tcf3. A 1.4-kbp fragment of the dharma promoter region contains consensus sequences for Tcf/Lef binding sites. This promoter region recapitulated the Wnt-dependent and dorsal dharma expression pattern when it was fused to luciferase or GFP. Deletion and point mutant analyses revealed that the Tcf/Lef binding sites were required to drive this expression pattern. These data established that dharma/boz functions between the dorsal determinants-mediated Wnt signals and the formation of the Nieuwkoop center. © 2001 Academic Press Key Words: dharma; bozozok; Wnt signal; Nieuwkoop center; ␤-catenin; Tcf; Lef.

INTRODUCTION positively transmit the Frizzled signals downstream. In the absence of Wnt ligands, GSK3 and ␤-catenin bind and The program that regulates the formation of the dorso- form a complex with the scaffolding proteins Axin/Axil/ ventral (DV) axis is initiated soon after fertilization in Conductin and the tumor suppressor gene product, APC. Xenopus laevis as well as in zebrafish. Several lines of GSK3 phosphorylates the amino-terminal region of ␤-cate- evidence show that the dorsal determinants (DDs), which nin in the complex, resulting in the ubiquitin-dependent are localized in the vegetal pole, are transported to the degradation of ␤-catenin. Upon stimulation by Wnt, Dsh/ dorsal side of the embryo through a subcortical microtubule Dvl suppresses the phosphorylation of ␤-catenin by GSK3 array at the zygote or early cleavage stage (Jesuthasan and and releases the ␤-catenin from the complex. ␤-Catenin is Stahle, 1997; Mizuno et al., 1999b; Rowning et al., 1997). transported to the nucleus and forms a protein complex The DDs are thought to activate components of the Wnt with the DNA-binding protein Tcf or Lef–high mobility signaling cascades in the dorsal side and induce the genes group (HMG) proteins that specifically bind the element required for the formation of the Nieuwkoop center, which 5Ј-G/CTTTGA/TA/T-3Ј (Dorsky et al., 2000; van de Weter- subsequently induces the Spemann organizer and specifies ing et al., 1997) to regulate gene expression. In addition to the dorsoventral patterning (reviewed in Moon et al., 1997). these components, GSK3-binding protein (GBP) and casein Genetic and biochemical studies have revealed signal- kinase I (CKI) are involved in the positive regulation of the transduction cascades of Wnt family members (reviewed in Wnt signal, and nemolike kinase (NLK) is involved in its Dierick and Bejsovec, 1999; Peifer and Polakis, 2000; Sokol, negative regulation (Ishitani et al., 1999; Peters et al., 1999; 1999). Wnt proteins bind to the Frizzled family of seven- Sakanaka et al., 1999; Sumoy et al., 1999; Yost et al., 1998). pass transmembrane proteins and activate cytoplasmic sig- Activation of the Wnt signals by the overexpression of nals. Dishevelled (Dsh) and its homologues (the Dvls) Wnts (Sokol et al., 1991), Dsh (Sokol et al., 1995), dominant-negative (DN) GSK3 (He et al., 1995; Pierce and

1 Kimelman, 1995), or ␤-catenin (Funayama et al., 1995; To whom correspondence should be addressed at Division of Molecular Oncology (C7), Biomedical Research Center, Osaka Guger and Gumbiner, 1995) in the ventral marginal zone of University Graduate School of Medicine, 2-2 Yamada-oka, Suita, Xenopus embryos leads to secondary axis formation. LiCl Osaka 565-0871, Japan. Fax: ϩ81-6-6879-3889. E-mail: hirano@ treatment before the midblastula transition (MBT), which molonc.med.osaka-u.ac.jp. inhibits GSK3 activity (Hedgepeth et al., 1997; Klein and

FIG. 1. Regulation of dharma by Wnt signals. Synthetic RNAs for Wnt8b (200 pg), Dvl3 (520 pg), DN GSK3␤ (480 pg), an activated form of ␤-catenin (⌬N␤-catenin, 100 pg), GBP (480 pg), mNLK (100 pg), or DN Tcf3 (100 pg) were injected into one-cell-stage embryos. For yolk depletion, the vegetal yolk mass was removed at the one-cell stage. The embryos were ﬁxed at the sphere stage (4 hpf) and the dharma transcripts were detected by whole-mount in situ hybridization. (A) In situ data representing each condition. (B) The proportions of embryos displaying reduced, normal, and expanded expression of dharma are shown as percentages.

Melton, 1996), leads to the ectopic induction of dorsal depletion of ␤-catenin (Heasman et al., 1994) leads to mesoderm and the organizer in Xenopus and zebraﬁsh defects in dorsal axis formation. Consistent with the role of embryos (Backstrom, 1954; Kao and Elinson, 1988; Stachel Wnt signals in dorsal axis formation, nuclear accumulation et al., 1993). Inhibition of the Wnt signals by overexpression of ␤-catenin is observed in the dorsal side of Xenopus and of GSK3 (He et al., 1995; Pierce and Kimelman, 1995), Axin zebraﬁsh embryos. Several genes are regulated by the Wnt (Itoh et al., 1998; Zeng et al., 1997), Axil (Yamamoto et al., signals. Among them, the Xenopus homeobox gene siamois 1998), or dominant-negative Tcf3 (Molenaar et al., 1996) or and its related gene twin, and the Xenopus nodal-related

FIG. 2. dharma promoter sequence. Tcf/Lef consensus sequences are boxed (5Ј-C/GTTTGA/TA/T-3Ј and 5Ј-C/GTTTCA/TA/T-3Ј). Putative homeodomain-binding sites and TATA boxlike sequences are underlined. Transcriptional initiation sites were determined by primer extension and are indicated by arrows (black arrow, major initiation site; gray arrow, likely minor sites). The nucleotides that were mutated in Fig. 6 are indicated.

FIG. 3. The Ϫ1.4-kbp promoter can recapitulate dharma expression. (A) Schematic representation of the Ϫ1.4-kbp promoter region of dharma and the GFP reporter construct (pDha-1420). (B, C) GFP expression. 50 pg of pDha-1420 GFP (B) or 25 pg of pCS2ϩGFP (C, CMV-GFP) plasmid DNA was injected into one-cell-stage embryos, and expression of GFP proteins was detected by ﬂuorescence microscopy and in situ hybridization with antisense GFP and dharma probes (B). Embryos shown are sphere stage (upper row) and shield stage (lower row).

gene Xnr3, which are involved in the formation and/or 1998). The zebrafish recessive mutation bozozok (boz) leads functions of the Spemann organizer (Laurent et al., 1997; to defects in the development of axial structures and of the Lemaire et al., 1995; Smith et al., 1995), contain Tcf/Lef- anterior and ventral neuroectoderm (Fekany et al., 1999; binding sites and their expression in the dorsal blastoderm Solnica-Krezel et al., 1996) and the boz locus was found to is dependent on these sites (Brannon et al., 1997; Fan et al., encode the dharma gene (Fekany et al., 1999). dharma is 1998; Laurent et al., 1997; McKendry et al., 1997). However, first expressed in a few dorsal blastomeres shortly after the other targets for the Wnt signals for the dorsal axis forma- MBT and soon thereafter its transcripts are confined to the tion remain elusive. dorsal extraembryonic yolk syncytial layer (YSL), which dharma, also identified as nieuwkoid, encodes a paired- underlies the embryonic shield, which is equivalent to the type homeodomain protein that has dorsal organizer- Spemann organizer in teleosts (Yamanaka et al., 1998). The inducing activity (Koos and Ho, 1998; Yamanaka et al., formation of the embryonic shield and the expression of

FIG. 4. The dharma promoter can be regulated by Wnt but not Nodal signal. (A) pDha-1420 GFP plasmid DNA was coinjected with synthetic RNAs for wnt8b (200 pg), dvl3 (520 pg), DN gsk3␤ (480 pg), GBP (480 pg), ⌬N␤-catenin (100 pg), ⌬N␤-catenin (100 pg), and DN Tcf3 (100 pg) as indicated. Expression of GFP was detected at the 30% epiboly stage except for DN GSK3␤ RNA-injected embryos (sphere stage). (B) pDha-1420 GFP plasmid DNA was coinjected with synthetic RNAs for squint (1 pg) and antivin (5 pg) as indicated. Expression of GFP was detected at the shield stage. The squint RNA- and antivin RNA-injected embryos displayed a severely dorsalized phenotype and a cyclops;squint-like phenotype at pharyngula period, respectively (data not shown). Lateral view and dorsal is to the right.

organizer-specific genes are strongly perturbed in boz mu- normalized using ␤-galactosidase activity in embryos that were tant embryos (Shimizu et al., 2000; Solnica-Krezel et al., coinjected with pCS2 ϩ ␤-galactosidase (Turner and Weintraub, 1996). These reports indicate that dharma is required for 1994). For the GFP reporter assay, 50–100 pg of NotI-digested the formation and/or function of the zebrafish Nieuwkoop reporter DNA was injected in to one-cell-stage embryos or one center and subsequent induction and formation of the blastomere of eight-cell-stage embryos. For the constitutively active promoter, 25 pg of circular pCS2ϩMT EGFP was injected. dorsal organizer and anterior neuroectoderm. The expres- wnt8b was synthesized from pCS2ϩWnt8b, as described previ- sion of dharma ectopically expands to the ventral side in ously (Yamanaka et al., 1998; Hashimoto et al., 2000). To construct LiCl-treated embryos (Yamanaka et al., 1998). The expres- an active form of ␤-catenin, the EcoRI–HindIII fragment containing sion domain of dharma overlaps with the region in which the entire coding region of ␤-catenin from pT7Ts ␤-catenin (a gift of ␤-catenin accumulates in the nucleus (Koos and Ho, 1998; R. Moon) was subcloned into pcDNA3 (Invitrogen), and the ClaI– Schneider et al., 1996). These data suggest a role for the XbaI fragment was inserted into pCS2ϩ (Turner and Weintraub, maternally derived Wnt signal in the regulation of dharma 1994) (in pCS2ϩ⌬N␤-catenin, amino acids 1–80 have been de- gene expression. leted). ⌬N␤-catenin RNA was transcribed from the XbaI-digested ϩ⌬ ␤ Here we show that dharma is regulated by the Wnt pCS2 N -catenin using SP6 RNA polymerase. To make DN signals. The promoter region contains Tcf/Lef binding sites, Tcf3 RNA, the HindIII–XbaI fragment of DN XTcf3 was excised from pT7Ts XTcf3 (a gift of R. Moon) and subcloned into pcDNA3 which are required for the Wnt-dependent and dorsal ex- (pcDNA3-dnXTcf3), and the RNA was transcribed from the XbaI- pression of dharma. These results establish the role of digested pcDNA3-dnXTcf3 using T7 RNA polymerase. The DN dharma/boz in linking the DDs-mediated Wnt signal and GSK3␤ was constructed from pCS2ϩGSK3␤ (Shimizu et al., 2000) the formation of the Nieuwkooop center. by the mutation of lysine 85 to arginine (pCS2ϩDN GSK3␤). The DN GSK3␤ RNA was synthesized from the NotI-digested pCS2ϩDN GSK3␤ using SP6 RNA polymerase. To make zebrafish MATERIALS AND METHODS GBP RNA, the GBP cDNA was isolated from the maternal cDNA library by PCR and subcloned into the EcoRI and XbaI sites of Genomic DNA and Plasmid Construction pCS2ϩ, and the RNA was transcribed from the NotI-digested vector using SP6 polymerase. Zebrafish Dvl3 cDNA was isolated A genomic fragment of dharma was isolated from a lambda FixII by hybridization screening of an early gastrula cDNA library zebrafish genomic library (Clonetech) by a standard hybridization (Shimizu et al., 2000) using a fragment of the zebrafish EST clone screening with the dharma cDNA as a probe (Yamanaka et al., AI544486 (amplified by PCR) as the probe. The nucleotide se- 1998). A SalI fragment (1676 bp) containing the promoter and the quence of zebrafish Dvl3 has been deposited in a database under coding region of dharma was subcloned into pBluescript SKϩ Accession No. AB041734. The Dvl3 cDNA was inserted into the (Stratagene; pBLSK-Dha). To construct the GFP reporter plasmid, NcoI and XbaI sites of pCS2ϩMT, and the RNA was transcribed an NcoI site was generated at the translation initiation codon in the from the Acc65I-digested vector using SP6 polymerase. To make dharma promoter by PCR, and EGFP cDNA from pEGFP-C1 was NLK RNA, the mouse NLK cDNA (Brott et al., 1998) was isolated inserted in the NcoI and EcoRI sites of pCS2ϩMT (pCS2ϩMT by PCR and subcloned into pCS2ϩMT, and the RNA was tran- EGFP). The SalI–NcoI fragment from the dharma promoter and the scribed from the NotI-digested vector using SP6 polymerase. NcoI–NotI fragment containing EGFP cDNA and an additional poly(A) signal were inserted into pBluescript SKϩ, resulting in pDha-1420 GFP. pDha-1420 Luc was constructed by ligating the Transcripts Detection SalI-NcoI fragment and the XhoI-SalI fragment of the luciferase translation unit from pGV-B (Toyo inki). The deletion mutants Whole-mount in situ hybridization was performed principally as for dharma were constructed by PCR from pDha-1420 GFP or described previously (Jowett and Yan, 1996). BM purple AP pDha-1420Luc. Site-directed mutagenesis of Tcf/Lef-binding and (Boehringer-Manheim) was used as an alkaline phosphatase sub- homeodomain-binding sites was carried out by a combination of strate. The expression of dharma was detected as described previ- overlap extension and megaprimer PCR (Seraphin and Kandels- ously (Yamanaka et al. (1998)). To make an antisense GFP probe, Lewis, 1996). To make mutations of Tcf/Lef-binding sites, two base the BamHI–HpaI fragment of pEGFP-1 was subcloned to the pairs (three base pair for T3) were changed from the consensus as BamHI and HincII sites of pBluescript SKt (pBLSK-EGFP). pBLSK- previously reported (Fan et al., 1998). The same type of mutation EGFP was digested with BamHI and transcribed with T7 RNA abolishes the Tcf-mediated transcription of siamois. To make polymerase. Photographs were taken using an AxioPlan-2 micro- mutations of homeodomain-binding sites, the first two nucleotides scope (Carl Zeiss) and HC-2500 3CCD camera (Fuji Film). Figures A and T were changed to C and G, respectively. The precise were assembled using Adobe Photoshop, version 5. construction procedures and primers for PCR are available upon request. Primer Extension ␮ RNA and DNA Injection To find transcription initiation sites, 30 g of total RNA from the LiCl-treated early gastrula embryos was annealed with a For the luciferase assay, 50 pg of the reporter construct DNA and 32P-end-labeled dharma-specific primer GGGGATTCTGCTGTT- the synthetic RNA (described in the figure legend) were injected TGCCTGCTGGTGTCTCC, and the cDNA fragments were ex- into one-cell-stage embryos. The injected embryos were harvested tended using MMLV reverse transcriptase RNaseHϪ (ReverTra at the shield stage (6 hpf) and the luciferase activity in 10 injected Ace, Toyobo) at 42oC for 60 min. The reaction was stopped by embryos for each condition was determined using the PicaGene adding EDTA (10 mM). After the RNase A treatment (10 ␮g/ml, luciferase assay system (Toyo inki). The luciferase activity was 37oC for 30 min), the DNA fragment was extracted with phenol and

TABLE 1 GFP Expression by dharma Promoter and Constitutively Active Promoter

CMV-GFP injection pDha-1420GFP injection

One-cell stage Eight-cell stage One-cell stage Eight-cell stage Expression of GFP (%) (n ϭ 76) (n ϭ 67) (n ϭ 188) (n ϭ 191)

Ubiquitous 94.7 0 0 0 Dorsal (including dorsal YSL) 0 34.3 64.9 35.1 Ventral 0 26.9 0 0 Lateral or animal 0 3.0 0 0 Entire YSL 0 0 1.1 0 Not detected 5.3 35.8 34.0 64.9

Note. 50 pg of CMV-GFP (pCS2 ϩ MT EGFP) and pDha-1420GFP plasmid DNA was injected to one-cell-stage embryos or one blastomere (located on the lateral end) of eight-cell-stage embryos. Expression of GFP was detected at the shield stage. Expression domain was determined by comparison with the location of embryonic shield. YSL, yolk syncytial layer.

chloroform and precipitated with ethanol. The DNA was analyzed which are localized in the vegetal pole and transported to on a 6% polyacrylamide sequencing gel. the ventral blastomeres during the zygote and early cleavage stages (Jesuthasan and Stahle, 1997; Mizuno et al., Yolk Depletion 1999b). Depletion of the yolk material in the vegetal pole at the early zygote stage elicits complete ventralization of the Yolk depletion was performed as described previously (Mizuno embryos (Mizuno et al., 1999b). Depletion of the vegetal et al., 1999a,b). yolk abrogated the expression of dharma, suggesting that it depends on the DDs. These results implicate the DDs- mediated Wnt signal in the regulation of dharma. RESULTS dharma Expression Is Regulated by Wnt Signals The dharma Promoter Contains Tcf/Lef-Binding Sites We previously showed that dharma expression is enhanced by LiCl treatment before the MBT (Yamanaka et al., We isolated an approximately 1.4-kbp genomic DNA 1998), suggesting a role for Wnt in regulating dharma fragment upstream of the dharma coding region. This expression. However, LiCl does not specifically inhibit the fragment contained seven consensus sequences for Tcf/Lef activity of GSK3; it also influences other signals, such as binding, 5Ј-C/GTTTGA/TA/T-3Ј (van de Wetering et al., inositol phosphatase (Berridge et al., 1989; Hallcher and 1997; Fig. 2, T1, T2, T4–T6, T8, and T9). Recently, it was Sherman, 1980). To reveal whether dharma expression is shown that 5Ј-C/GTTTCA/TA/T-3Ј also binds Tcf/Lef in regulated by the Wnt signal, we examined the dharma the promoter of the Wnt-responsive gene nacre (Dorsky et expression in embryos expressing activators or inhibitors of al., 2000). There are two regions that match this sequence the Wnt signal. dharma expression expanded or was ectopi- in the 1.4-kbp promoter fragment (designated as T3 and T7 cally induced in embryos injected with RNAs for zebrafish in Fig. 2). In addition to the Tcf/Lef-binding sites, there are Wnt8b, Dvl3 (a Dishevelled homologue), DN GSK3␤, GBP, three consensus sequences for homeodomain binding (a and ⌬N␤-catenin (in which the amino-terminal phosphoac- palindromic repeat of 5Ј-ATTA-3Ј, Figs. 2, 2H1–3; Gehring ceptor sites for GSK3 were deleted), which all activate the et al., 1994). Primer extension analysis revealed that most Wnt signal at the sphere stage (Figs. 1A and 1B). In contrast, dharma transcripts mainly started 99 nucleotides upstream dharma expression was reduced or absent in embryos of the translation initiation site, a point that is 20–30 overexpressing DN Xenopus Tcf3 or mouse NLK, which nucleotides downstream of the TATA-like sequences (Fig. 2). phosphorylates Tcf/Lef and inhibits the formation of the To reveal whether the 1.4-kbp fragment could recapitu- ␤-catenin–Tcf/Lef complex (Ishitani et al., 1999; Figs. 1A late the native expression of dharma, we constructed a and 1B). These data were consistent with our previous reporter plasmid by connecting the 1.4-kbp promoter frag- observation that inhibiting the Wnt signals by misexpress- ment with either a GFP or a luciferase transcription unit ing Axin or Axil abrogated the dorsal expression of dharma (Figs. 3 and 4, pDha-1420-GFP, pDha-1420-Luc). When the (Shimizu et al., 2000). The effects of these activators or GFP reporter plasmid was injected into one-cell-stage em- inhibitors on dharma expression were essentially the same bryos, GFP expression was detected in dorsal side of the at the shield stage as at the sphere stage (data not shown). YSL at the sphere stage (4 hpf) by fluorescence microscopy The Wnt signal is thought to be activated by the DDs, (Fig. 3). In some injected embryos (5%), the fluorescence

FIG. 5. Wnt enhances the expression of dharma promoter-driven luciferase. (A) Schematic representation of the luciferase reporter construct, pDha-1420 Luc. (B) Luciferase activity. pDha-1420 Luc plasmid DNA was coinjected with the indicated synthetic RNAs into one-cell-stage embryos. The embryos were harvested at the shield stage (6 hpf) and the luciferase activities were determined from 10 embryos. The activities are indicated as fold induction in the experimental versus the reporter alone injected embryos. The averages and standard deviations from triplicate samples are shown.

was detected in a few dorsal blastomeres on the same side (Table 1). In the CMV-GFP-injected embryos, GFP was as the YSL fluorescence (data not shown). The fluorescence detected in blastomeres scatteringly located on dorsal, in the dorsal YSL continued until the midgastrula stage (the ventral, or lateral/animal pole sides at shield stage, where 60 to 70% epiboly stage; Fig. 3B, lower panel, the shield the reporter DNA was incorporated. In contrast, GFP was stage). The expression of GFP was confirmed by in situ detected only in the dorsal YSL in the pDha-1420 GFP- hybridization with an antisense GFP probe. The pattern of injected embryos at a lower percentage than the embryos the fluorescence was essentially the same as that of the injected at one-cell stage, suggesting that GFP was ex- dharma transcripts detected by whole-mount in situ hy- pressed only when the reporter DNA was incorporated on bridization (Yamanaka et al., 1998), but it was delayed the dorsal side. about 30 min by the transcripts, due to protein synthesis. These results indicate that the 1.4-kbp promoter region is Injection of the reporter DNA did not affect the endogenous sufficient to drive the dharma expression in the dorsal YSL dharma expression (Fig. 3B, right panels). When a constitu- from the late blastula to the midgastrula stage. tively active promoter (cytomegalovirus IE94 promoter) -driven GFP reporter plasmid (CMV-GFP) was injected into The dharma Promoter Is Regulated by Wnt Signal one-cell-stage embryos, GFP was expressed ubiquitously but Not by Nodal Signal throughout the blastoderm (Fig. 3C). It is known that DNA-mediated gene expression displays We examined whether the 1.4-kbp promoter is also re- mosaic patterns of the transcripts in zebrafish. This might sponsible for the Wnt signal-dependent regulation of affect specific expression driven by a given promoter. To dharma. The GFP construct (pDha-1420 GFP) was injected address this issue, CMV-GFP and pDha-1420GFP plasmid with RNAs for the activators or an inhibitor of the Wnt DNA was injected to one blastomere (on the lateral end) signal. Misexpression of Wnt8b, Dvl3, DNGSK3, GBP, or of eight-cell-stage embryos to produce severe mosaicism ⌬N␤-catenin expanded the GFP expression to the ventral

FIG. 6. Tcf/Lef-binding sites are required for ␤-catenin-dependent and dorsal expression of dharma. (A) Schematic representation of deletion and point mutants for the reporter constructs. Mutations in the consensus sequences are indicated in Fig. 2. (B) ␤-Catenin- dependent expression. The luciferase reporter plasmid was injected with (closed bars) or without (open bars) ⌬N␤-catenin RNA (300 pg) and the luciferase activities for various promoter mutants in the constructs were determined as described in Fig. 5. The averages and standard deviations from triplicate samples are shown. (C) Dorsal expression. The mutant GFP reporter plasmids were injected at the one-cell-stage and the expression of GFP was detected at the shield stage. Lateral view and dorsal is to the right. 406 Ryu et al. side, although the fluorescence remained stronger in the to the overexpression of ⌬N␤-catenin and conferred the dorsal side in these embryos. In contrast, expression of DN weak dorsal expression (data not shown), and this property Tcf3 completely abrogated the GFP expression, even in the was abolished by a point mutation of T1 and T2. These data presence of ⌬N␤-catenin (Fig. 4A). To quantify the respon- suggest that these Tcf/Lef-binding sites are sufficient for siveness of dharma expression to the Wnt signal, the the dharma expression at weak levels, but the other Tcf/Lef luciferase reporter gene driven by the 1.4-kbp promoter sites and the homeoprotein-binding sequences are required (pDha-1420 Luc) was injected with RNAs for the Wnt for the full activation. activators, and the luciferase activity was determined in shield-stage embryos (Fig. 5). Misexpression of Wnt8b or ⌬N␤-catenin induced the promoter-driven luciferase activ- DISCUSSION ity, and increasing the amount of these molecules further increased the luciferase activity. Expression of DN Tcf3 Regulation of dharma by the Maternally Derived inhibited the ␤-catenin-dependent induction of luciferase. Wnt Signal These results show that the 1.4-kbp promoter region can In this report, we demonstrated that dharma/boz expres- recapitulate the Wnt-dependent dharma expression. sion is directly regulated by the maternally derived Wnt We next examined whether the dharma promoter is signal. First, dharma expression expanded in embryos over- regulated by Nodal signal, which is required for expression expressing the Wnt signal activators Wnt8b, Dvl3, GBP, and of various dorsal-specific genes (Feldman et al., 1998; Grits- DN-GSK3␤ and ⌬N␤-catenin. Inhibition of the Wnt signal man et al., 1999). pDha-1420GFP was injected with RNAs by DN Tcf3 or NLK inhibited dharma expression. Deple- for the zebrafish Nodal-related molecule Squint and an tion of the vegetal yolk material, which elicits the complete Actvin/Nodal inhibitor Antivin (Gritsman et al., 1999; ventralization of embryos, abrogated dharma/boz expres- Meno et al., 1999; Thisse and Thisse, 1999). Misexpression sion (Fig. 1). Consistent disruption of microtubules by UV of squint and antivin did not significantly affect the irradiation at the zygote stage, which is thought to inhibit dharma promoter-driven GFP expression in the dorsal YSL the transportation of the DDs to the dorsal blastomeres (Fig. 4B). This is consistent with our previous data that the (Jesuthasan and Stahle, 1997), also diminished dharma/boz endogenous expression of dharma was not affected in the expression (data not shown). Second, the promoter region of squint RNA- and antivin RNA-injected embryos and dharma contains consensus sequences for Tcf/Lef binding squint-mutant embryos (Shimizu et al., 2000). (Fig. 2), and the promoter-driven expression of GFP or luciferase in the dorsal YSL in response to ␤-catenin was Tcf/Lef-Binding Sites Are Required induced by the Wnt activators and inhibited by DN Tcf3 for dharma Expression (Figs. 4 and 5). Mutation and/or deletion of these consensus sites abolished the ␤-catenin-dependent dharma expression To reveal which regions in the 1.4-kbp promoter are and its expression in the dorsal YSL in the reporter assays responsible for the responsiveness of dharma to Wnt signals (Fig. 6). These results indicate that the DDs-mediated Wnt and which regions are responsible for the dorsal expression signal is required for dharma expression in the dorsal YSL. of dharma, we performed deletion and point mutant anal- Overexpression of Wnt activators expanded both the yses on the promoter region using the luciferase (Fig. 6B) endogenous dharma expression (Fig. 1) and the dharma/boz and GFP reporter (Fig. 6C) constructs. Deletion of the region promoter-driven GFP expression (Fig. 4A). In contrast, ec- from Ϫ1420 to Ϫ619, which contains six Tcf/Lef-binding topic expression, such as in the ventral side and/or animal consensus sequences (T4–T9), diminished the ␤-catenin- pole, was rarely detected in these embryos. To express dependent expression, but only slightly reduced the dorsal dharma ectopically, higher amounts of Wnt8b or ⌬N␤- expression of GFP. A mutation in all the Tcf/Lef-binding catenin were required. These data suggest that ectopic consensus sequences in the context of pDha-619 strongly activation of the Wnt signal may not be sufficient to drive reduced the ␤-catenin-dependent expression [pDha-619 the ectopic expression of dharma or the dorsalizing signal. ⌬(T1, T2, T3)] and abolished the dorsal GFP expression. A It is also possible that other signaling pathways are involved mutation in the putative homeodomain-binding sequences in the normal activation of the dharma gene. (H1 and H2) in the context of pDha-619 reduced but did ␤ not abolish the -catenin-dependent expression and the ␤ dorsal expression. Deletion of a region containing the dharma Expression and TGF- Signaling homeodomain-binding sequences and a mutation of the The Xenopus homeobox genes siamois and twin, which Tcf/Lef-binding sequences [pDha-322 ⌬(T1, T2, T3)] com- are expressed in the dorsovegetal region of embryos, are pletely abolished the ␤-catenin-dependent transcription and regulated directly by the maternally derived Wnt signals the dorsal expression. These data indicate that the Tcf/Lef- (Brannon et al., 1997; Fan et al., 1998; Laurent et al., 1997; and homeodomain-binding sequences cooperatively regu- McKendry et al., 1997). These homeoproteins are capable of late the dorsal expression of dharma and the Wnt- inducing a secondary axis when they are expressed ven- dependent expression. The 220-bp region, which contains trally, and their expression rescues ventralized embryos the two Tcf/Lef-binding sequences (pDha-220), responded (Laurent et al., 1997; Lemaire et al., 1995). Misexpression of

Copyright © 2001 by Academic Press. All rights of reproduction in any form reserved. Regulation of dharma/bozozok by the Wnt Pathway 407 dharma is also capable of inducing a complete secondary Although it is not known which homeodomain proteins axis in zebrafish embryos (data not shown) (Yamanaka et bind these sequences, a point mutation of H1 and H2 sites al., 1998), and its expression is regulated by the Wnt signals, in the context of 619-bp promoter diminished the suggesting a similar role for Siamois and Twin and Dharma/ ␤-catenin-dependent expression and the dorsal expression Boz in the formation of the dorsal axis. However, there are (Figs. 6B and 6C), suggesting that these sites play a role in some differences between the regulation of siamois and dharma expression. Intriguingly, the 619-bp region lacking twin expression and that of dharma. The expression of all the Tcf/Lef sites but containing H1 and H2 sites [Fig. 6B; siamois is induced more strongly in animal caps expressing pDha-619 ⌬(T1, T2, T3)] still responded to the overex- ␤-catenin or Wnt8 together with Smad2 than in caps pressed ␤-catenin, suggesting that homeodomain protein(s) expressing ␤-catenin or Wnt8 alone (Crease et al., 1998). which binds H1 and H2 might also act downstream of the The overexpression of a DN activin type II receptor inhibits maternal Wnt signal. Previously we reported that dharma the expression of siamois, suggesting that Activin or its expression is not maintained after the late blastula stage in related molecules, such as the Nodals or Vg1, are involved boz mutant embryos (Fekany et al., 1999). These sites may in the marginal and vegetal expression of siamois (Crease et cooperate with Tcf/Lef-binding sites to maintain dharma al., 1998; Fan et al., 1998). These data suggest that Activin expression. or Nodal-related signals confer the dorsal vegetal and dorsal In summary, dharma/boz is a direct target of the mater- marginal expression on siamois. In contrast, we previously nally derived Wnt signals, and it links the DDs-mediated showed that misexpression of the zebrafish Nodal-related signals with the formation of the dorsal organizer. protein Squint does not affect the expression of dharma (Shimizu et al., 2000). dharma expression is not affected in squint mutant embryos (Shimizu et al., 2000), in maternal– ACKNOWLEDGMENTS zygotic one-eyed pinhead (MZ-oep) mutant embryos (Grits- man et al., 1999), or in embryos overexpressing Antivin, an We thank R. Moon for kindly providing the plasmid constructs. inhibitor for both Activin and the Nodals (Shimizu et al., We also thank R. Masuda and A. Kubota for secretarial assistance 2000), indicating that the Activin/Nodal signal is not in- and Y. Kuga for fish maintenance and technical assistance. 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