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Developmental Biology 274 (2004) 478–489 www.elsevier.com/locate/ydbio Genomes & Developmental Control Identification of downstream of the ascidian muscle determinant Ci-macho1

Kasumi Yagi*, Nori Satoh, Yutaka Satou

Department of Zoology, Graduate School of Science, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan Received for publication 22 May 2004, revised 9 July 2004, accepted 14 July 2004 Available online 25 August 2004

Abstract

Autonomous differentiation of primary muscle cells in ascidian embryos is triggered by a maternal determinant recently identified as the macho-1 gene. macho-1 encodes a of the Zic family with five C2H2 zinc-finger motifs. In the present study, we firstly performed a screen, using a quantitative PCR method, of genes encoding transcription factors and components in major signaling pathways to identify those regulated downstream of Ci-macho1 in early embryos of Ciona intestinalis. The amount of transcripts for a total of 64 genes was altered at the 32-cell stage depending on the Ci-macho1 activity level. Whole-mount in situ hybridization assays revealed that the alteration of expression for at least 13 of them was adequately visualized to confirm the results of quantitative PCR. Second, we determined a possible binding sequence of Ciona macho1. macho1 recombinant of both C. intestinalis and Ciona savignyi recognized a sequence, 5V-GCCCCCCGCTG-3V, that resembles the mammalian Zic binding site. In addition, most of the genes identified as potential Ci-macho1 downstream genes, in particular Ci-Tbx6b and Ci-snail, possessed plausible Ci-macho1-binding sequences in their 5V upstream region, suggesting their direct activation by Ci-macho1. Furthermore, some of the genes including three Wnt genes noted in the quantitative analyses implied that Ci-macho1 is involved in the differentiation of endoderm and mesenchyme via intracellular communications. D 2004 Elsevier Inc. All rights reserved.

Keywords: Ciona intestinalis; Autonomous muscle differentiation; Determinant; macho1; Binding sequence; Downstream genes\

Introduction species), which lie on both sides of the notochord in the tail, and 28 cells in the anterior and middle parts originate from the Maternal information stored in the egg cytoplasm plays a B4.1 blastomeres of a bilaterally symmetrical 8-cell embryo. pivotal role in the establishment of embryonic axes or Differentiation of these B-line or primary muscle cells occurs specification of embryonic cells. Ascidians have provided a autonomously depending on a maternally provided muscle classical as well as a modern experimental system for determinant(s). Experimental approaches toward character- identifying morphogenetic determinants and for elucidating izing the molecular nature of the determinant suggested that it their function in subsequent differentiation (for recent is provided as maternal mRNA, localized to the so-called reviews, see Corbo et al., 2001; Jeffery, 2001; Nishida, myoplasm after fertilization, and segregated into blastomeres 2002a,b; Satoh, 2001, 2003). Muscle formation in ascidians of the B-line muscle lineage, and that the product is the best-studied system of autonomous differentiation. A from the mRNA triggers the genetic cascade responsible for tadpole-type larva of Ciona intestinalis has 36 unicellular and muscle differentiation (Deno et al., 1984; Marikawa et al., striated muscle cells (the number varies a little depending on 1994; Nishida, 1990; Whittaker, 1973, 1980). By a subtractive hybridization screen using Halocynthia roretzi embryos, Nishida and Sawada (2001) isolated * Corresponding author. Department of Zoology, Graduate School of Science, Kyoto University, Kitashirakawa Oiwake-cho, Sakyo-ku, Kyoto macho-1, which encodes a Zic-like zinc-finger protein with 606-8502, Japan. Fax: +81 75 705 1113. transcription factor activity. They showed that maternally E-mail address: [email protected] (K. Yagi). provided macho-1 mRNA is segregated to the myoplasm,

0012-1606/$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.ydbio.2004.07.013 K. Yagi et al. / Developmental Biology 274 (2004) 478–489 479 that overexpression and ectopic expression of the gene several genes including Ci-Tbx6b and Ci-snail are regulated promote ectopic differentiation of muscle cells, and that by Ci-macho1 and they possess plausible Ci-macho1- downregulation of the gene function results in failure of the binding sequences in their 5V upstream region, suggesting differentiation of B-line muscle cells. In a previous study, that they are direct target genes of Ci-macho1. we isolated orthologous genes of macho-1 from C. intestinalis (Ci-macho1) and Ciona savignyi (Cs-macho1), and showed that they are also responsible for initiation of Materials and methods muscle differentiation (Satou et al., 2002a). The molecular function of the macho1 protein and the Ascidian eggs and embryos genetic cascade triggered by macho1 remain to be clarified. So far, some transcription factor genes, including Tbx6 C. intestinalis individuals were maintained under con- (Mitani et al., 1999), snail (Fujiwara et al., 1998), and ZicL, stant light to induce oocyte maturation. Eggs and sperm which is another Zic family gene (Imai et al., 2002), have were obtained surgically from the gonoduct. After insemi- been shown to be involved in the differentiation of ascidian nation, eggs were reared at about 188C in Millipore-filtered muscle cells. The expression of Ci-Tbx6 genes starts at the seawater (MFSW) containing 50 Ag/ml streptomycin 16-cell stage (Takatori et al., 2004), Ci-snail expression is sulfate. detected from the 32-cell stage onwards (Fujiwara et al., 1998; Erives et al., 1998), and the expression of ZicL is Injection of morpholino antisense oligonucleotides and detected from the 32-cell stage (Imai et al., 2002; Yagi et al., synthetic capped mRNA 2004). In Ciona embryos, the expression of muscle protein genes becomes evident after gastrulation (for example, Cs- A 25-mer morpholino antisense oligonucleotide for Ci- MA1, Chiba et al., 1998). The actual function of these macho1 (hereafter simply called Ci-macho1 MO), 5V- transcription factor genes in the activation of muscle protein ATCCCATCGTACCAGTAAAGGCCAT-3V, was order- genes and their relationships with macho1 remain to be made (Gene Tools, LLC). The target sequence contained clarified. the first methionine codon (underlined). MOs act efficiently Recent decoding of the C. intestinalis draft genome to knock down Ciona developmental genes (Satou et al., allowed us to investigate this issue on a genome-wide scale. 2001a). Control embryos injected with a universal control The Ciona genome sequence (about 160 Mb) contains fewer MO (5V-CCTCTTACCTCAGTTACAATTTATA-3V,Gene paralogues than are usually seen in vertebrates, and many Tools, LLC) did not cause any irrelevant . genes represent their original features before the duplication In vitro synthesized capped mRNA of Ci-macho1 was that is presumed to have occurred twice in vertebrate prepared from cDNA cloned into pBluescript RN3 vector evolution (Dehal et al., 2002; Satoh et al., 2003; Satou and using a Megascript T3 kit (Ambion). Microinjections were Satoh, 2003). Taking advantage of these traits, as a scaffold performed as previously described (Imai et al., 2000), and for a future thorough understanding of development, we each injection contained about 30 pl of solution with 0.5 compiled a comprehensive list of C. intestinalis genes, mM MO or 1.2 mM synthesized mRNA. encoding transcription factors and components of major signaling transduction pathways (hereafter, signaling genes), Quantitative RT-PCR (real-time PCR) and determined their spatiotemporal expression profiles by whole-mount in situ hybridization and quantitative PCR Total RNA extraction from embryos and cDNA synthesis (Imai et al., 2004). The list includes, for transcription factor with oligo(dT) primers were performed as described genes, those encoding a factor with bHLH, , Fox, previously (Satou et al., 2001b). Each quantitative PCR ETS, bZIP, HMG, or T-box domains and specific categories was carried out using 0.4 embryo equivalent quantity of of zinc-fingers (Satou et al., 2003a; Wada et al., 2003; Yagi et cDNA as template in 10 Al of reaction mixture consisting of al., 2003; Yamada et al., 2003), and for signaling genes those 1Â SYBR Green Master Mix and 300 nM of each primer. encoding a major component involved in signaling pathways The relative amounts of mRNA for each of comprehen- of receptor tyrosine kinases, MAPK, Notch, TGFh, Wnt, and sively annotated genes encoding transcription factors and JAK/STAT (Hino et al., 2003; Satou et al., 2002b, 2003b). major components involved in signaling pathways (Imai et In the present study, we conducted a genome-wide screen al., 2004) were examined (see Supplementary Table 1). for these transcription factor genes and signaling genes Reactions were performed using the ABI Prism 7000 regulated by Ci-macho1 in vivo. Quantitative analysis and Sequence Detection System (Applied Biosystem) according whole-mount in situ hybridization analysis identified genes to manufacturer’s specifications with universal amplifica- whose zygotic expression is under the control of Ci-macho1 tion conditions. Dissociation curves were used to confirm from the early cleavage stage. In addition, we determined a that single PCR products were amplified. Control samples binding sequence of Ci-macho1 in vitro, which enabled us lacking reverse transcription failed to give specific products. to identify potential binding sites in 5Vupstream regions of Relative expression values were calculated by comparing the candidate target genes. We found that expressions of expression levels between injected embryos and noninjected 480 K. Yagi et al. / Developmental Biology 274 (2004) 478–489 control embryos. An alteration in the amount of transcripts in the C. intestinalis genome (Supplementary Table 1; for of a particular gene was confirmed by at least three detailed information, see Imai et al., 2004). Genes of which independent series of experiments using the constant level the relative amount of transcripts was reduced to one-half or of endogenous Ci-macho1 mRNA as a control. less by microinjection of Ci-macho1 MO or genes of which relative amount of transcripts was increased twofold or more Whole-mount in situ hybridization by microinjection of Ci-macho1 mRNA compared with Ci- macho1 MO-injected embryos were selected as possible Whole-mount in situ hybridization was performed using downstream genes. As a result, 45 transcription factor genes digoxigenin-labeled antisense probes as described previ- and 19 signaling genes were found transcriptionally affected ously (Satou et al., 2001b). Control embryos hybridized by alteration of Ci-macho1 function (Fig. 1 and Supple- with a sense probe did not show any signals above mentary Table 1). In particular, the relative amount of background. transcripts of Atonal, Tbx6a, Tbx6b, snail, FoxH-a, FoxI-a, Nkx-A, and Orphan Wnt-e was reduced in Ci-macho1- Preparation of GST fusion proteins suppressed embryos to less than 10% compared with control embryos (1%, 6%, 1%, 1%, 1%, 1%, 8%, and 9%, GST fusion proteins containing the zinc-finger motifs of respectively). In contrast, transcriptional levels were macho1 of C. intestinalis and C. savignyi, GST/Ci- increased in Ci-macho1-overexpressing embryos (499%, macho1(ZF), and GST/Cs-macho1(ZF), respectively, were 255%, 279%, 217%, 285%, 378%, 201%, and 173%, prepared basically as described by Yagi et al. (2004). The respectively) at the late 32-cell stage. We found no genes following primers and cDNAs were used as templates to that apparently showed the opposite response where the construct the recombinant plasmids: Ci-macho1,5V- expression was activated in Ci-macho1-suppressed embryos CGCGGATCCCGCGTCGACCTCACCTG-3V and 5V- or downregulated in Ci-macho1-overexpressing embryos. CCGCTCGAGATCCTGTTCGCATATGATG-3V, BAB84543; Cs-macho1,5V-CGCGGATCCCGCGTC- Expression pattern of possible Ci-macho1 target genes GAACTTGCCTG-3V and 5V-CCGCTCGAGGGTTGGC- TCGCAAATGAC-3V, BAB68349. The alteration of the transcription level determined by the quantitative analysis described above may be visualized by Binding site selection assays and mutational analyses whole-mount in situ hybridization analysis, and therefore we performed this analysis using manipulated embryos. DNA sequences optimal for binding with GST/ Spatial expression patterns of almost all the transcription macho1(ZF) fusion proteins were determined as described factor genes and signaling genes in Ciona embryos have previously (Yagi et al., 2004) by PCR-assisted binding site been described by Imai et al. (2004). It is expected that Ci- selection assays and mutational analyses together with macho1 protein generated from maternal Ci-macho1 tran- electrophoretic gel mobility shift assays (EMSAs). script promotes transcription of its target genes by the 32- cell stage. Of the 64 genes listed in Fig. 1, 14 genes (indicated by asterisks) start their zygotic expression to be Results detectable by in situ hybridization by the 32-cell stage, and thus they were subjected to further analyses. The following Comprehensive identification of genes downstream of is a description of their expression patterns in normal control Ci-macho1 embryos and in Ci-macho1-overexpressing or -suppressed embryos. To identify Ci-macho1 downstream genes, we prepared manipulated embryos in which Ci-macho1 function was E(spl)/hairy-b downregulated by its specific MO and those in which Ci- In a control embryo, the hybridization signal of this gene macho1 function was upregulated by its synthetic mRNA. was weakly detected in nuclei of vegetal blastomeres at the MO or synthetic mRNA was microinjected into fertilized 32-cell stage (Fig. 2A). Although some signal was detected eggs and total RNA was extracted from these manipulated in a similar manner in Ci-macho1-overexpressing embryos embryos and their controls at the 32-cell stage. The present (Fig. 2AV), Ci-macho1-suppressed embryos did not show study aimed to identify direct and indirect-but-close target any signal (Fig. 2AW). genes of Ci-macho1, and such genes are expected to begin their zygotic expression by the 32-cell stage. Then we Tbx6 determined the relative amount of mRNA for each of the Formerly, Halocynthia Tbx6 gene Hr-Tbx6 was shown to transcription factor genes and signaling genes by quantita- play a role in muscle development (Mitani et al., 1999). An tive PCR. intensive survey of the C. intestinalis genome yielded at In the present study, we analyzed a total of 366 least three Tbx6 class genes, namely Ci-Tbx6a, Ci-Tbx6b, transcription factor genes and 120 signaling genes encoded and Ci-Tbx6c, and their expression was confirmed by in situ K. Yagi et al. / Developmental Biology 274 (2004) 478–489 481

Fig. 1. Quantification of the amounts of transcripts of 45 transcription factor genes and 19 signaling genes in control embryos (green), and in experimental embryos developed from eggs injected with Ci-macho1 synthetic mRNA (red) or Ci-macho1 morpholino antisense oligonucleotide (blue). Transcripts in developing embryos at the late 32-cell stage were examined by quantitative RT-PCR. The relative amount of transcripts compared with that in control embryos is shown. The expression patterns of 14 genes indicated by asterisks were examined by whole-mount in situ hybridization (Fig. 2). hybridization (Takatori et al., 2004). Their zygotic expres- al., 1998)(Fig. 2F). Ci-snail was suggested to act as a sion was first detected in the B5.1 pair at the 16-cell stage, transcriptional repressor of Ci-Bra in non-notochord cells, and then in B6.1, B6.2, and B6.4 blastomeres at the 32-cell and maternal muscle determinant was inferred to be involved stage (Figs. 2B, C, D, and E). Their expression was in Ci-snail expression (Fujiwara et al., 1998). The expression maintained in cells of the primary muscle lineage at later of Ci-snail was observed ectopically in all vegetal blasto- stages. The spatiotemporal expression patterns of these three meres in Ci-macho1-overexpressing embryos (Fig. 2FV) and genes were similar except that the nuclear signal for Ci- lost in Ci-macho1-suppressed embryos (Fig. 2FW). Tbx6b and Ci-Tbx6c in B6.1 and B6.2 at the 32-cell stage was scarcely visible (Figs. 2B, C, D, and E) and the signal ZicL for Ci-Tbx6c at later stages was stronger in posterior cells. Ci-ZicL is expressed in B6.2, B6.4, A6.2, and A6.4 In Ci-macho1-overexpressing embryos, transcripts of all blastomeres at the 32-cell stage, and plays crucial roles in these Tbx6 genes were ectopically detected (Figs. 2BV,CV, the development of three types of cells originating from DV, and EV). However, the region of ectopic expression these blastomeres (muscle, nerve cord, and notochord) (Imai differed among them. Ci-Tbx6b and Ci-Tbx6c transcripts et al., 2002; Yagi et al., 2004)(Fig. 2G). ZicL expression in were detected in all vegetal blastomeres (Figs. 2DVand EV), A-line nerve cord–notochord cells was suggested to be while Ci-Tbx6a transcript was detected in animal blasto- under the control of FoxD in C. savignyi embryos (Imai et meres as well (Figs. 2BVand CV). In contrast, the expression al., 2002), but the regulation of its expression in B-line of all these Tbx6 genes disappeared in Ci-macho1-sup- muscle cells is unknown. Quantitative PCR demonstrated a pressed embryos (Figs. 2BW,CW,DW, and EW). 73% increase or 66% decrease in the amount of Ci-ZicL mRNA in Ci-macho1-overexpressing or -suppressed snail embryos, respectively, compared with control embryos The transcript of Ci-snail was detected from the 32-cell (Fig. 1). However, in situ hybridization failed to detect stage onwards in B6.1, B6.2, and B6.4 blastomeres (Erives et apparent changes in the Ci-ZicL expression pattern (Figs. 482 K. Yagi et al. / Developmental Biology 274 (2004) 478–489

Fig. 2. Effects of overexpression or functional suppression of Ci-macho1 revealed by whole-mount in situ hybridization. For each gene, the expression patterns at the late 32-cell stage in an intact embryo (left), that in a Ci-macho1-overexpressing embryo (middle), and that in a Ci-macho1-suppressed embryo (right) are shown. Expression of Ci-Tbx6a, Ci-ADMP, Ci-BMP3, Ci-Nodal, Ci-Wnt3, and Ci-Wnt5 is shown in both animal and vegetal views, and that of Ci-E(spl)/ hairy-b, Ci-Tbx6b, Ci-Tbx6c, Ci-snail, Ci-ZicL, Ci-Otx, Ci-lefty/antivin, and Ci-Orphan Wnt-e in a vegetal view. Arrows in R, RV, and RW indicate localized maternal transcripts. The scale bar represents 100 Am.

2GVand GW), although the intensity of signal showed some lefty/antivin changes in experimental embryos. The Ci-lefty/antivin transcript was first detected in B5.1 blastomeres and was weakly detected in A5.1 and Otx A5.2 blastomeres at the 16-cell stage, and in B6.4 at the Ci-Otx expression appears at the 32-cell stage in B6.1, 32-cell stage (Fig. 2M) (Satou et al., 2001b). Over- B6.2, and B6.4 blastomeres (Hudson and Lemaire, 2001; expression of Ci-macho1 resulted in ectopic expression Satou et al., 2001b)(Fig. 2H). Ci-macho1 overexpression of Ci-lefty/antivin in some blastomeres that varied caused ectopic activation of Ci-Otx in various vegetal depending on the embryo (Fig. 2MV), while Ci-macho1 blastomeres (Fig. 2HV), while the expression became suppression resulted in the loss of the hybridization undetectable as a result of Ci-macho1 suppression signal (Fig. 2MW). (Fig. 2HW). Nodal ADMP The amount of Ci-Nodal transcript was decreased to The hybridization signal for Ci-ADMP was detected in 28% in Ci-macho1-suppressed embryos compared with B5.1 cells at the 16-cell stage and in B6.1, B6.2, and B6.4 control embryos (Fig. 1). Interestingly, transcripts of this cells at the 32-cell stage (Figs. 2I and J). Ci-macho1- gene in Ci-macho1-overexpressing embryos were also overexpressing embryos displayed intense signal ectopically decreased to a similar extent. At the 32-cell stage, control in most blastomeres (Figs. 2IV and JV), while Ci-macho1- embryos showed Ci-Nodal expression in A6.1, A6.3, and suppressed embryos still showed weak signal in vegetal B6.1 cells of the vegetal hemisphere and also in b6.5 blastomeres (Fig. 2JW). cells of the animal hemisphere (Figs. 2N and O). In Ci- macho1-overexpressing embryos, hybridization signal in BMP3 vegetal blastomeres became less intense, while the signal The Ci-BMP3 transcript was detected in all vegetal in b6.5 blastomeres was lost (Figs. 2NV and OV). blastomeres in control embryos at the 32-cell stage, although Conversely, in Ci-macho1-suppressed embryos, the signal the in situ signal was weak (Fig. 2L). Ci-macho1 over- in b6.5 blastomeres was detected while that in vegetal expression caused weak ectopic signal in animal blastomeres blastomeres was lost (Figs. 2NW and OW). These observa- (Fig. 2KV), and its suppression caused a decrease of the signal tions can account for the result by quantitative PCR intensity in vegetal blastomeres (Fig. 2LW). method (Fig. 1). K. Yagi et al. / Developmental Biology 274 (2004) 478–489 483

Wnt3 consensus among the 26-bp dsDNAs was weaker than that In control embryos, Ci-Wnt3 expression was detected among the 16-bp dsDNAs (data not shown). Fig. 3B shows from the 32-cell stage onwards in primary muscle-lineage raw numbers of nucleotides obtained after selection with 16- cells (Figs. 2P and Q). The in situ hybridization analysis bp random dsDNAs. Eight and ten rounds of selection revealed that Ci-macho1 overexpression activated Ci-Wnt3 yielded a similar consensus sequence. This analysis transcription ectopically in most blastomeres (Figs. 2PVand demonstrated that most of the sequences selected had 5V- QV) and Ci-macho1 suppression inactivated gene expression GCCCCCCGCTGCG-3Vin dsDNAs. (Figs. 2PW and QW). The specific binding of the fusion protein to this sequence was confirmed by EMSA with a double-stranded Wnt5 oligonucleotide (5V-ACTAGTGCCCCCCGCTGCGCGG- Ci-Wnt5 is expressed maternally. Its maternal transcript 3V; Fig. 3C, top line) named macho1-b (Fig. 3D, lane 2). was concentrated and segregated into the posterior region of To examine the significance of each nucleotide for binding, early embryos (Imai et al., 2004), like that of Ci-macho1. we further performed gel shift assays with various oligonu- The zygotic expression of Ci-Wnt5 became evident in B6.1, cleotides that had a nucleotide substitution (A X CorTX G B6.2, and B6.4 blastomeres (Figs. 2R and S), and muscle- transversion) in the selected sequences. The results are and mesenchyme-lineage cells showed intense signal in shown in Fig. 3D. Neither a mutation Aa(TtoG)inthe cleaving embryos. Ci-Wnt5 expression was activated in region neighboring the consensus sequence, Am(CtoA), most blastomeres of Ci-macho1-overexpressing embryos nor An (G to T) in the 3V end region altered the binding (Figs. 2RV and SV), and was inactivated in Ci-macho1- affinity to GST/Ci-macho1(ZF). Mutations Ac, Ad, Ae, Ai, suppressed embryos (Figs. 2RW and SW) in a similar way to Aj, and Ak, in which a nucleotide near one of the ends of the Ci-Wnt3 expression. However, the localization of maternal consensus sequence was substituted, reduced the binding transcripts was not disturbed in manipulated embryos affinity. Mutations Ab (G to T) and Al (G to T), located at the (arrows in Figs. 2R–RW). ends of the sequence, dramatically reduced the binding affinity, and mutations Af (C to A), Ag (C to A), and Ah(Cto Orphan Wnt-e A), located in the center, resulted in a complete loss of Ten Wnt genes have been identified in the C. intestinalis binding affinity. genome, and this gene is one of five Wnt genes whose Altogether, these findings indicated that the most evolutionary relationships or orthologies have not been plausible binding sequence of GST/macho1(ZF) is 5V- confidently assigned (Hino et al., 2003). The expression GCCCCCCGCTG-3V, and that its core CCC and its 5V pattern of this gene in cleaving embryos resembled that of terminal G and 3V terminal G are critical for binding in Ci-Wnt3 described above (Fig. 2T), except that hybrid- vitro. ization signal was also detected in B5.1 blastomeres at the 16-cell stage. Transcripts of this gene were detected in all Probable Ci-macho1 downstream genes possess plausible vegetal blastomeres in Ci-macho1-overexpressing embryos Ci-macho1-binding elements in their 5Vupstream region (Fig. 2TV), but not in Ci-macho1-suppressed embryos (Fig. 2TW). To address whether the transcription of the candidate target genes of Ci-macho1 shown in Fig. 2 is activated Determination of macho1-binding sequences directly by Ci-macho1, we searched for potential Ci- macho1-binding elements in their 5V upstream regions. Next, the molecular characteristic of Ci-macho1 was Approximately 480,000 ESTs and the draft genome assessed to identify its possible direct target genes. We sequence of C. intestinalis provided us information of a 3- determined its recognition or binding sequence by PCR- kbp upstream region for each of these genes. Exceptions assisted binding site selection assays and then carried out were Ci-Tbx6c and Ci-ZicL, as the genomic sequences electrophoretic mobility shift assays (EMSAs) using around these genes are not completely determined. For Ci- mutated sequences to evaluate the binding efficiency as Wnt3, the longest EST lacked the entire ORF, and therefore described previously (Yagi et al., 2004 in the study of Ci- we performed RACE to predict the 5V-terminus of the ZicL-binding sequence). transcript. Recombinant proteins, GST/Ci-macho1(ZF) and GST/ It was thereby found that most of these genes Cs-macho1(ZF) (verified by SDS-PAGE: Fig. 3A), were possessed elements resembling the in vitro determined produced by fusion of zinc-finger domains of Ci-macho1 macho1-binding sequence 5V-GCCCCCCGCTG-3V in their and Cs-macho1 with the C-terminus of GST. Both proteins prospective upstream region. Moreover, 10 of these 12 gave identical results, therefore we describe here only those genes had potential Ci-macho1-binding elements in which for GST/Ci-macho1(ZF). dsDNAs consisting of 16-bp or more than 8 out of the 11 nucleotides were identical to the 26-bp random sequences were examined after 5, 8, or 10 GST/macho1(ZF)-binding consensus sequence (Fig. 4, rounds of selection. The same selection results were shown with the consensus sequence and their relative obtained for 16-bp and 26-bp dsDNAs, although the position in the 5Vflanking region). Ci-lefty/antivin and Ci- 484 K. Yagi et al. / Developmental Biology 274 (2004) 478–489

Fig. 3. Characterization of nucleotide sequences recognized by GST/macho1(ZF) fusion proteins. (A) SDS-PAGE of purified GST/Ci-macho1(ZF) fusion protein, GST/Cs-macho1 fusion protein, and GST protein. The molecular mass relative to Marker (lane 3) shows the successful production of the fusion proteins. (B) DNA sequences bound to the fusion proteins were selected and determined as described in "Materials and methods". Aligned are cloned random sequences after 8 or 10 rounds of selection for GST/Ci-macho1(ZF). The bottom sequence in each table represents the compiled most favored sequence at each nucleotide position. (C and D) A mutational analysis of the binding sequence of GST/Ci-macho1(ZF). Binding affinities for 16 types of oligonucleotides (C) were examined by a gel-shift assay (D). The boxed uppercase letters of the macho1-b oligonucleotides indicate the consensus macho1-binding sequence. The shaded lowercase letters in the Aa–Ao oligonucleotides indicate substituted nucleotides in each mutant oligonucleotide. The binding seemed specific because the binding was not detected under conditions of zinc-removed incubation (lane 1), preincubation with Â100 molar excess of unlabeled competitor DNA, or replacement with GST protein (data not shown).

Wnt3 are separated by an interval of about 0.7 and 1.3 kb downstream genes, suggesting that Ci-macho1 acts as a from a neighboring gene, respectively, and some of the direct activator of these genes. potential Ci-macho1-binding elements indicated by aster- As controls, we randomly selected four transcription isks were found located in the neighboring gene. How- factor genes (Ci-GATA-a, Ci-ATF2/7, Ci-HMG1/2, and Ci- ever, the others were located exclusively in the intergenic ) and three signaling genes (Ci-Eph1, Ci-GSK, and regions. Ci-JAK), which have not been identified as potential Ci- In particular, as shown in Fig. 4, the 5Vupstream region macho1 target genes (Supplementary Table 1), and exam- of Ci-Tbx6b contained two potential Ci-macho1-binding ined whether their 5Vupstream regions contain Ci-macho1- elements at À1095 and À1547, both retaining the central binding sequence. As a result, we did not find any (CCC) and the 5V and 3V terminal Gs. Ci-snail also promising Ci-macho1-binding sequence in the 5Vupstream contained three elements with the five nucleotides of high region of these genes (data not shown). binding affinity at À448, À525, and À2028. Zygotic expression of Ci-Tbx6b is activated as early as at the 16- cell stage and that of Ci-snail at the 32-cell stage. Discussion Therefore, it is highly likely that Ci-macho1 directly regulates the transcription of both Ci-Tbx6b and Ci-snail. macho-1 mRNA is a localized maternal determinant for The core sequence of the Ci-macho1-binding element was muscle cell differentiation in ascidian embryos, and there- also evident in the 5V upstream region of other possible fore macho-1 appears to trigger a genetic cascade leading to K. Yagi et al. / Developmental Biology 274 (2004) 478–489 485

Fig. 4. Potential Ci-macho1-binding sequences in the upstream region of possible downstream target genes of Ci-macho1. DNA sequences aligned with the determined Ci-macho1-binding consensus sequence 5V-GCCCCCCGCTG-3Vand their location relative to the first nucleotide of the longest EST are shown. The shaded letters indicate nucleotides identical to those in the consensus sequence, and the arrowheads indicate sequences in which nucleotides critical for binding in vitro are retained. muscle formation (Nishida and Sawada, 2001; Satou et al., engrailed and wingless in Drosophila embryos (Benedyk et 2001a). For the comprehensive elucidation of molecular al., 1994). In C. intestinalis and C. savignyi, two Zic-like mechanisms involved in ascidian muscle differentiation, the genes have been characterized; one is Ci-macho1 and Cs- present study identified transcription factor genes and macho1 (Satou et al., 2002a), and the other is Ci-ZicL (Yagi signaling genes whose zygotic expression was regulated et al., 2004) and Cs-ZicL (Imai et al., 2002). Also in H. downstream of Ci-macho1 in vivo. After confirmation of roretzi,twoZic genes have been identified, macho-1 the results by in situ hybridization, the 5Vupstream regions (Nishida and Sawada, 2001) and ZicN (Wada and Saiga, of candidate genes were screened for the presence of 2002). A molecular phylogenetic analysis suggests that macho1-binding sequences determined in vitro. these two are highly diverged Zic members (Yamada et al., 2003), and the ascidian genome is therefore likely to encode DNA sequence recognized by macho1 two members of the Zic family, macho1 and ZicL. The Ci-macho1-binding sequence (5V-G˙ CCCC˙ C˙ C˙ G- Ci-macho1 belongs to the Zic family of zinc-finger CTG˙ -3V) determined in the present study is partially over- proteins. The Zic (zinc-finger protein of the cerebellum; lapped by the Ci-ZicL-binding sequence (5V-CCCG˙ C˙ T˙ G˙ TG- Aruga et al., 1996) family proteins contain five C2H2 zinc- 3V)(Yagi et al., 2004). However, mutational analyses of the finger motifs and are known to have important roles in binding sequence in vitro indicated that Ci-macho1 and Ci- animal development, especially in neurogenesis (Aruga, ZicL possess different DNA binding affinities conferred by 2004; Mayor and Aybar, 2001). Mammals have five Zic distinct elements that account for their transcriptional genes and flies have only one gene, odd-paired. odd-paired function in vivo (cf. Figs. 3C and D, and Yagi et al., is one of the pair-rule genes and regulates the activation of 2004). The Ci-macho1-binding core is GdddCCCdddG 486 K. Yagi et al. / Developmental Biology 274 (2004) 478–489 while that of Ci-ZicL is dddGCTGdd. These two Ciona Zic Screening of macho1 downstream genes in C. intestinalis genes regulate distinct target genes, as shown in the present embryos and in the previous studies (Yagi et al., 2004). Namely, in A-line blastomeres of early Ciona embryos, Ci-ZicL Quantitative analyses based on the comprehensive gene directly activates the expression of Ci-Bra via two 5V- list (Imai et al., 2004; Supplementary Table 1) identified 45 CCAGCTGTG-3V sequences located in the first 400 bp transcription factor genes and 19 signaling genes regulated upstream of Ci-Bra, which share eight out of nine downstream of Ci-macho1 (Fig. 1). Of them, 14 genes nucleotides identical with the consensus sequence. On the showed expression as early as at the 32-cell stage by whole- other hand, in B-line blastomeres, Ci-ZicL appears to be mount in situ hybridization analysis, and most of them involved in muscle differentiation (Imai et al., 2002), but its indeed lost or decreased their expression under Ci-macho1 detailed molecular mechanism remains to be elucidated. functional suppression (Fig. 2). These genes are potentially The binding sequences of mammalian Zic proteins have direct targets of Ci-macho1 and play roles in specifying recently been investigated in mice. Salero et al. (2001) muscle fate. The presence of plausible macho1-binding reported that mouse Zic1 and Zic2 bind to the 5Vsequence in sequences in their upstream regions supports this notion. the proximal region of the apolipoprotein E promoter to Fig. 1 also contains genes whose expression was not activate this gene. They suggested three binding sequences, detected in embryos by in situ hybridization, probably due 5V-CCCTGGGGGAGGG-3V,5V-TCTGCCCTGCTTG-3V, to a low amount of transcript, but was downregulated in Ci- and 5V-CTGTGGGGGGTGG-3V. Ebert et al. (2003) dem- macho1-suppressed embryos. These genes could be directly onstrated that mouse Zic1 interacts with the enhancer of regulated by Ci-macho1 as well. Math1, a bHLH transcription factor gene expressed in In contrast, genes whose transcriptional level appeared neuronal progenitor cells, to repress the transcription of to be upregulated in Ci-macho1-overexpressing embryos, this gene. They showed that the binding sequence is 5V- but not significantly downregulated in Ci-macho1-sup- AGCTCCCCGGGGAGCG-3V. Ciona macho1 favored the pressed embryos, may not be direct targets of Ci-macho1. sequence 5V-GCCCCCCGCTG-3V, and this sequence is The increased level of expression of these genes could be shared with the mice Zic1 binding sequence. This due to indirect effects or direct inappropriate activation indicates that the Ciona macho1 has an affinity to due to the excessive amount of Ci-macho1 transcripts. In mammalian Zic from the point of view of their recognition the latter case, excess Ci-macho1 could drive artificial sequence. activation via favorable, even if low-affinity, binding. As shown above, the optimal DNA sequences preferred by Ciona Zic genes and their evolution two Zic family proteins encoded in the C. intestinalis genome, 5V-GCCCCCCGCTG-3V for Ci-macho1 and 5V- In addition to the maternal transcript, Ci-macho1 is also CCCGCTGTG-3Vfor Ci-ZicL (Yagi et al., 2004), partially expressed zygotically in the nervous system of the embryo overlap. (Satou et al., 2002a), similar to mammalian Zic genes. Ci- ZicL is expressed zygotically in early embryos and this gene Genes downstream of Ci-macho1 potentially mediate is involved in specification of the notochord, nerve cord, muscle differentiation and/or mesenchyme differentiation and muscle in Ciona embryos (Imai et al., 2002; Yagi et al., 2004). Later, Ci-ZicL is also expressed in the nervous Among genes transcriptionally regulated downstream of system of early tailbud embryos (Imai et al., 2002). Ci-macho1 (Fig. 1), Ci-E(spl)/hairy-b, Ci-Tbx6a, Ci-Tbx6b, Therefore, it is highly likely that after duplication of an Ci-Tbx6c, Ci-snail, Ci-Otx, Ci-ADMP, Ci-lefty/antivin, Ci- ancestral Zic gene, both macho1 and ZicL acquired novel Nodal, Ci-Wnt3, Ci-Wnt5, and Ci-Orphan Wnt-e showed unique roles in early ascidian development. macho1 might zygotic expression in blastomeres producing primary have obtained a new molecular mechanism for maternal muscle cells, and their expression was clearly downregu- expression, in addition to its original expression in the lated at the 32-cell stage in Ci-macho1-suppressed embryos embryonic nervous system. Maternally provided macho1 in (Fig. 2). These are highly likely direct or close indirect turn functions as a determinant to specify embryonic muscle targets of Ci-macho1 mediating the genetic network of cells, as one of essential factors conducting bmosaicQ autonomous differentiation of muscle cells. Besides these development of ascidian embryos. On the other hand, ZicL genes, Ci-MyoD(Ci-MDF), Ci-FoxB, Ci-Hox3, Ci-Irx-B, Ci- probably gained another role in the specification of Mnx, Ci-Otp, Ci-chordin, Ci-noggin, and Ci-DUSP1/2/4/5 notochord and muscle cells. showed detectable zygotic expression in muscle-lineage cells An alternative explanation is that a role of Zic in muscle after the 32-cell stage in intact embryos (Meedel et al., 1997 development may have been ancestral for chordates, and its for Ci-MDF; Imai et al., 2004; and data not shown). These expression and function in muscle cells were lost in the genes are possibly regulated indirectly by Ci-macho1 and lineage leading to vertebrates. This hypothesis is unlikely as also responsible for muscle fate determination. As for other Zic function in muscle differentiation is found only in genes that were not discussed in the present study, our ascidian embryos. spatiotemporal expression profiles of Ciona transcription K. Yagi et al. / Developmental Biology 274 (2004) 478–489 487 factor genes and signaling genes have revealed that some maternally provided h-catenin is responsible for develop- genes, including Ci-ROR, Ci-E(spl)/hairy-a, and Ci-Y-box ment of vegetal blastomeres, especially for endoderm protein 1/2/3, are expressed in primary muscle-lineage cells, (Imai et al., 2000; Satou et al., 2001b). Immunostaining especially at later stages (Imai et al., 2004). These genes may shows nuclear accumulation of h-catenin by the 32-cell also be involved in muscle differentiation, although it is not stage, although the mechanism regulating the accumulation known how many steps are involved in carrying out muscle is unknown. Our results raised the possibility that Ci- differentiation from the maternal determinant through macho1 also regulates endoderm differentiation via acti- expression of specific structural genes. vating Wnt genes. It is therefore intriguing that Ci-macho1 In addition, in control embryos, mesenchyme-lineage can affect the expression of Ci-lefty/antivin, Ci-ZicL, and cells show zygotic expression of Ci-Twist-like 2, Ci-FoxI-a, Ci-Otx, which all have been shown to be regulated by h- Ci-ets/pointed 1, Ci-Fli/ERG1, Ci-Orphan bZip-4, Ci- catenin (Imai, 2003; Satou et al., 2001b). Endoderm Hox4, Ci-SoxF, Ci-DUSP8/16, and Ci-Eph4 at later stages differentiation in ascidian embryos has also been consid- (Imai et al., 2004; and data not shown). Previously, Imai et ered to occur cell-autonomously under the control of al. (2003) showed that Twist-like genes of C. savignyi are localized cytoplasmic determinant(s), based on the results expressed in presumptive mesenchyme cells from later obtained from experiments involving endoderm-specific cleavage stages onwards and play critical roles in mesen- expression of alkaline phosphatase (AP) in cleavage- chyme formation. In Halocynthia, macho-1 is also proven arrested embryos (Whittaker, 1977), partial and continu- to be essential for mesenchyme specification (Kim and ously dissociated embryos (Nishida, 1992a,b; Whittaker, Nishida, 1999; Kim et al., 2000; Kobayashi et al., 2003). 1990), and cytoplasm ablation or transplantation (Nishida, Thus, if this role of macho1 is applicable to Ciona, our 1993; Yamada and Nishida, 1996). However, Kondoh et results are consistent with the model of mesenchyme al. (2003) showed that the intrinsic determinant(s) alone is differentiation they proposed. not sufficient for proper formation of posterior endoderm in Halocynthia embryos. Thus, together with the observa- Possible involvement of Ci-macho1 in endoderm tion that AP expression was seen in Ci-macho1-suppressed specification embryos at the later stages (data not shown), it is implied that redundant pathways for endodermal fate regulated by Differentiation of primary muscle cells takes place Ci-macho1 exist or that Ci-macho1 just ensures that, completely autonomously and does not require inductive although Ci-macho1 itself is not essential for endoderm intercellular communication, but in this study, we show that differentiation. Ci-macho1 overexpression can activate ectopic expression of genes encoding signaling molecules such as TGFh and Ci-macho1 as a transcriptional activator Wnt. In addition, expression of E(spl)/hairy-b, BMP3, and Nodal in anterior-vegetal blastomeres was downregulated In this study, we identified genes whose zygotic after functional suppression of Ci-macho1, even though expression was activated under the control of Ci-macho1 macho1 protein is supposed to be absent from anterior in early embryos (Fig. 1). And for a fraction of these genes, blastomeres. These data suggest that Ci-macho1 governs the whose transcription level in intact embryos was above the differentiation of not only muscle cells but also of other limit of detection, the alterations of the relative amounts of fates by yet unknown mechanisms involving signaling transcripts especially in Ci-macho1-suppressed embryos pathways. were confirmed by in situ hybridization (Fig. 2). The In particular, three Wnt genes (Ci-Wnt3, Ci-Wnt5, and presence of plausible recognition sequences by Ci-macho1 Ci-Orphan Wnt-e) appeared to be Ci-macho1 downstream in their presumptive upstream regions (Fig. 4), together with genes, and their expression was detected in primary the fact that our screen did not find any genes that appeared muscle-lineage cells of developing embryos (Fig. 2P–T to be directly repressed by Ci-macho1, suggests that Ci- and data not shown). Wnt3 and Wnt5 genes were shown to macho1 functions as a transcriptional activator to directly stimulate the canoninal dWnt/h-catenin pathwayT and the activate these genes. Previously, it was demonstrated in a dWnt/Ca2+ pathwayT, respectively, although the biological simple deletion experiment that the 465-bp upstream function of the latter pathway is unclear (Miller, 2001). sequence of Ci-snail can drive the expression of a reporter However, the relationship between specific Wnts and the gene in B4.1-lineage muscle cells in tailbud embryos, while intracellular pathway they use is not fixed, even if the a 321-bp sequence cannot (corresponding to 478- and 321- classification of Wnts in an evolutionary context provides bp sequences in the present study, respectively) (Erives et a useful tool for predicting their function. Overexpression al., 1998). Erives and Levine (2000) also demonstrated that of XWnt5a in combination with human Fz5 in Xenopus a 262-bp sequence (À356 to À618, corresponding to À358 embryos results in activation of the Wnt/h-catenin signal- to À615) of Ci-snail is sufficient to mediate reporter ing pathway, suggesting that the activity of Wnts in vivo expression within derivatives of the B4.1 blastomeres. In will be determined by the repertoire of Fz receptors (He et addition, Bertrand et al. (2003) reported that Ci-Otx al., 1997). In ascidians, the nuclear accumulation of expression in B-line cells requires upstream region located 488 K. Yagi et al. / Developmental Biology 274 (2004) 478–489 between À706 and À271 (À704 and À269 in the present Bertrand, V., Hudson, C., Caillol, D., Popovici, C., Lemaire, P., 2003. study). Here, plausible Ci-macho1-binding elements were Neural tissue in ascidian embryos is induced by FGF9/16/20, acting via a combination of maternal GATA and Ets transcription factors. Cell 115, found at À448 of Ci-snail and at À622 of Ci-Otx, 615–627. suggesting activation by Ci-macho1 via direct binding, Chiba, S., Satou, Y., Nishikata, T., Satoh, N., 1998. Isolation and although other elements outside this region have also been characterization of cDNA clones for epidermis-specific and shown to be important for Ci-snail activation (Erives et al., muscle-specific genes in Ciona savignyi embryos. Zool. Sci. 15, 1998). 239–246. Corbo, J.C., Di Gregorio, A., Levine, M., 2001. The ascidian as a macho1 is probably a nuclear protein as other Zic family model organism in developmental and evolutionary biology. 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Biol. 104, 322–328. their direct regulatory activities via direct DNA binding Ebert, P.J., Timmer, J.R., Nakada, Y., Helms, A.W., Parab, P.B., Liu, Y., were also shown (Ebert et al., 2003; Salero et al., 2001). Hunsaker, T.L., Johnson, J.E., 2003. Zic1 represses Math1 expression via interactions with the Math1 enhancer and modulation of Math1 Moreover, ZicL, another Zic family protein encoded in the autoregulation. Development 130, 1949–1959. Ciona genome, was shown to be a direct activator of Erives, A., Levine, M., 2000. Characterization of a maternal T-box gene in transcription (Yagi et al., 2004). So far, there is Ciona intestinalis. Dev. Biol. 225, 169–178. no direct evidence that Ci-macho1 functions as a transcrip- Erives, A., Corbo, J.C., Levine, M., 1998. Lineage-specific regulation of tional activator, and this issue should be elucidated via cis the Ciona snail gene in the embryonic mesoderm and neuroectoderm. Dev. 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