Overlapping expression of FoxA and Zic confers responsiveness to FGF signaling to specify notochord in ascidian embryos

Developmental Biology 30 (2006) 770 – 784 www.elsevier.com/locate/ydbio

Genomes & Developmental Control

Overlapping expression of FoxA and Zic confers responsiveness to FGF signaling to specify notochord in ascidian embryos Gaku Kumano ⁎, Satoshi Yamaguchi, Hiroki Nishida Department of Biological Sciences, Graduate School of Science, Osaka University, 1-1 Machikaneyama-cho, Toyonaka, Osaka 560-0043, Japan Received for publication 1 March 2006; revised 18 July 2006; accepted 26 July 2006 Available online 4 August 2006

Abstract Differences in cell responsiveness to an inductive signal contribute to the emergence of a variety of tissue types during animal development. In ascidian embryos, the Fibroblast Growth Factor (FGF) signal secreted from endoderm cells induces several different tissue types, such as notochord, mesenchyme and brain, at different positions in the embryo at the 32-cell stage. We show here in Halocynthia roretzi that FoxA and Zic are required for notochord formation in cells that receive the FGF signal. We also show that these transcription factors, only when both are supplied, are able to induce ectopic expression of the brachyury gene, a notochord-specific marker, in cells of all the three germ layers in an FGFdependent manner. These results suggest that FoxA and Zic confer notochord-specific responsiveness to FGF signaling. Further analyses including knockdown and over-expression experiments showed that combinatorial inputs from maternally supplied and zigotically activated factors lead to overlapping expression of FoxA and Zic in the presumptive notochord cells, which eventually activate the expression of the brachyury gene in cooperation with FGF signaling. Our data illustrate how a complex gene network specifies the notochord at its specific position within the embryo. © 2006 Elsevier Inc. All rights reserved. Keywords: Zic; FoxA; FGF; Competence; Induction; Notochord; Ascidian

Introduction During embryogenesis, a variety of tissue types arise in precisely defined spatial and temporal patterns. These patterns are established by combinatorial inputs from domains of activity that converge at specific positions within the embryo. A number of factors are known to be responsible for these combinatorial actions, such as maternally derived localized factors that are inherited into specific regions within the embryo and those zygotically activated by these maternal factors. These factors include transcription factors that act cell autonomously to regulate gene expression in specific regions and inductive signals that emanate from localized areas but influence relatively broad neighboring regions. In order to understand animal embryonic development, it is necessary to identify these factors and to determine the mechanism by which their activities are integrated at the developmental and molecular levels. The ascidian embryo is an ideal system with which to study this issue. It develops into a tadpole-type larva that possesses a ⁎ Corresponding author. Fax: +81 6 6850 5472. E-mail address: [email protected] (G. Kumano). 0012-1606/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.ydbio.2006.07.033

notochord and dorsal neural tube, features that are characteristic of all chordates. When gastrulation starts, the embryo has only 110 cells, whose developmental fates are already mostly restricted and determined by this stage (Nishida, 1987). These features make it possible to draw a comprehensive picture of how different tissues arise in an embryo at the single-cell level. Recent studies with several ascidian species have identified a number of molecules that are developmentally important, and have often shown that multiple factors are required independently in order for a tissue to form. For example, in Halocynthia roretzi embryos, fate determination for the mesenchyme is known to require both a maternally localized factor, Macho-1, and a FGF signal secreted from the endoderm cells (Kobayashi et al., 2003). We have been particularly interested in the intersection of activities from intrinsic factors within signalreceiving cells and extrinsic inductive signals in the context of cell fate determination. The state of a cell that is conferred by intrinsic factors, enabling it to respond to an inductive signal, is known as “competence”. Competence is one of the means by which embryos produce different kinds of outcomes using a relatively small number of inductive signals. In ascidian embryos, competence to the FGF signal that emanates from

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the vegetally located endoderm cells differs between cells in the anterior and posterior parts of the marginal zone and those in the animal region. As mentioned above, Macho-1, which is suggested to be present in the posterior part of the embryo (Kondoh et al., 2003), confers responsiveness to the signal such that cells give rise to mesenchyme (Kobayashi et al., 2003), while GATA is activated only in the animal cells and is required for the cells to become brain in response to FGF signaling (Bertrand et al., 2003). In the anterior part of the embryo, notochord is known to be induced by FGF signaling (Nakatani and Nishida, 1994; Nakatani et al., 1996; Shimauchi et al., 2001a; Imai et al., 2002a). Although zygotically activated Zic (ZicL in Ciona and ZicN in Halocynthia) is speculated to be a competence factor for notochord induction since it is necessary for notochord-specific brachyury and fibrinogen-like expression (Wada and Saiga, 2002; Imai et al., 2002b) and its expression is independent of FGF signaling (Wada and Saiga, 2002), it has not been shown whether the transcription factor cooperates with FGF signaling to specify notochord. The notochord is one of the features characteristic of the phylum Chordata. Therefore, it is important to understand the mechanism by which notochord is formed in ascidians, which are primitive chordates. Notochord induction by FGF signaling takes place at the 32- and early 44-cell stages (Nakatani and Nishida, 1994; Nakatani et al., 1996; Kim and Nishida, 2001) in ascidian embryos. The notochord fate in the A-line (the primary notochord) is restricted at the 44-cell stage when it divides off from the nerve cord lineage. The presumptive notochord/nerve cord cell at the 32-cell stage is exposed presumably on one side to FGF signaling from adjacent endoderm cells (Nakatani and Nishida, 1994; Nakatani et al., 1996; Imai et al., 2002a). At the 44-cell stage, one of its sister cells that is positioned closer to the endoderm cells continues to receive the signal and goes on to become notochord. On the other hand, the other sister cell that does not receive FGF signaling gives rise to nerve cord. This is thought to be a binary decision between these two fates with the nerve cord fate as a default (Minokawa et al., 2001): both the presumptive notochord and nerve cord cells assume their default nerve cord fate without FGF signaling, while they both become notochord when treated with FGF (Nakatani et al., 1996; Minokawa et al., 2001). A similar mechanism also regulates the fate decision between mesenchyme and muscle, with the muscle fate as a default in the posterior part of the embryo (Kim and Nishida, 1999; Kim et al., 2000). The molecular nature of notochord formation has also been studied extensively. Brachyury is the key transcription factor in notochord formation (Nishida, 2005), since expression of its gene starts specifically in the notochord lineage immediately after the notochord fate is restricted (Yasuo and Satoh, 1993, 1994). The expression depends on FGF signaling (Nakatani et al., 1996; Kim and Nishida, 2001; Imai et al., 2002a) and it regulates the expression of a number of downstream notochord-specific genes (Takahashi et al., 1999a; Hotta et al., 1999, 2000). In Halocynthia embryos, Hr-ZicN is shown to be located upstream of Halocynthia brachyury (Hr-Bra) (Wada and Saiga, 2002), as mentioned above. Hr-FGFR, a receptor for the FGF signal, and Hr-Ets, a transcription factor that is activated by FGF signaling,

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are involved in notochord formation (Shimauchi et al., 2001a; Miya and Nishida, 2003). Although a module between −289 and −250 bp of the 5′ flanking region of the Hr-Bra gene is known to be responsible for notochord-specific enhancer activity at the tailbud stage (Takahashi et al., 1999b), whether it is also essential for the early expression of the gene remains to be seen. Some other factors are known to play roles in notochord formation in Ciona embryos. Cs-β-catenin, Cs-FoxD, Ci-FoxDa/b, CiFoxAa, Cs- and Ci-ZicLs (orthologues of Hr-ZicN) are all known to be necessary for Ciona savignyi or Ciona intestinalis brachyury (Cs- or Ci-Bra) or notochord-specific Cs-Fibrn (fibrinogen-like) expression (Imai et al., 2000, 2002b,c, 2006), although Cs- and Ci-ZicLs are not required for the B-line expression (the secondary notochord, Imai et al., 2002b, 2006). Cs-β-catenin initiates the expression of the Cs-FoxD gene via TCF binding elements (Imai et al., 2002c). Cs-ZicL binds directly to the enhancer region of the Cs-Bra gene (Yagi et al., 2004a). Finally, Cs-FoxD and Ci-FoxDa/b are known to be located upstream of Cs- and Ci-ZicLs, respectively (Imai et al., 2002b, 2006). These results from Ciona embryos link all the factors into the following cascade for the formation of A-line notochord: βcatenin/TCF → FoxD → → ZicL → brachyury. These factors in Halocynthia and Ciona embryos described above are essential both for early notochord specification (with respect to notochordspecific gene expression at the 110-cell stage) and for late notochord differentiation (with respect to notochord-specific gene or antigen expression or morphology at the tailbud stage). Cs- and Ci-FGF9/16/20 are also known to be required for early notochord specification in Ciona embryos (Imai et al., 2002a, 2006), and is therefore proposed to be an endogenous inducer. In the present study, we isolated Halocynthia orthologues of FoxD and FGF9/16/20, and investigated how the notochord forms at the specific position of the Halocynthia embryo by using knockdown and over-expression analyses. Our model proposes that a more complicated gene network than previously acknowledged is necessary for notochord formation. We also clarified that Hr-FoxA and Hr-ZicN serve as competence factors for notochord induction in Halocynthia embryos. Either of these factors alone is not sufficient to activate Hr-Bra expression even in the presence of FGF signaling. In the anterior part of the embryo, from which the notochord arises, Hr-FoxA is expressed in the presumptive notochord and endoderm cells (Shimauchi et al., 1997), while Hr-ZicN is expressed in the notochord and nerve cord precursor cells (Wada and Saiga, 2002). Therefore, overlapping expression of these genes in the presumptive notochord cells confers responsiveness to FGF signaling during notochord induction, allowing brachyury expression. Materials and methods Animals and embryos Adults of H. roretzi were collected in the vicinity of the Asamushi Research Center for Marine Biology and the Otsuchi International Coastal Research Center, and kept alive in tanks without food during the spawning season. Eggs were spawned under temperature and light control, fertilized with a suspension of non-self sperm, and allowed to develop in Millipore-filtered seawater containing 50 μg/ml streptomycin and 50 μg/ml kanamycin at 11°C.

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Isolation of Hr-FGF9/16/20 and Hr-FoxD by RT-PCR and 5′ and 3′ RACE

described previously (Minokawa et al., 2001) with the exception that the treatment was initiated immediately after microinjection.

A 236-bp-long cDNA fragment of Halocynthia FGF9/16/20 was isolated by RT-PCR with the degenerate upstream oligonucleotide (5′-GGICTNTAYCTNGSNATG-3′) and the downstream oligonucleotide (5′-KKYCTVGGYAARAANTG-3′). Two types of 147-bp-long cDNA fragments of Halocynthia FoxD were isolated by another RT-PCR with degenerate oligonucleotides, 5′-TAYATHGCIYTNATHACNATG-3′ for the upstream, 5′AARTTYCCNGCNTGGCARAA-3′ for the nested upstream and 5′SWICCNSWYTCRAACATRTC-3′ for the downstream primers. Full-length cDNAs were obtained by 5′ and 3′ RACE using the SMART RACE cDNA Amplification Kit (Clontech). The Hr-FGF9/16/20 cDNA consisted of 1916 nucleotides and encoded a predicted protein of 330 amino acids. The Hr-FoxDa and Hr-FoxDb cDNAs were 1658 bp and 3347 bp long and encoded forkhead proteins of 456 and 863 amino acids, respectively. There were trans-splicing leader sequences represented by ATTGGAA(T)TATTTGAGTATTAAG at the 5′ ends of the Hr-FGF9/16/20 and Hr-FoxDb cDNAs.

Histochemistry, immunohistochemistry and in situ hybridization Histochemical staining for endodermal alkaline phosphatase was performed as described previously (Meedel and Whittaker, 1989). Immunohistochemical staining for notochord-specific Not1 (Nishikata and Satoh, 1990) and mesenchyme-specific Mch-3 (Kim and Nishida, 1998) antigens was carried out as described previously (Kobayashi et al., 2003). Detection of Hr-FoxDa, Hr-FoxDb, Hr-FGF9/16/20, Hr-FoxA (Shimauchi et al., 1997), Hr-ZicN (Wada and Saiga, 2002) and Hr-Bra (Yasuo and Satoh, 1993) expression by in situ hybridization was performed according to the standard protocol (Miya et al., 1997) with the exception that hybridization was done at 50°C.

Results Cloning of Hr-FGF9/16/20 and Hr-FoxD

Morpholino oligonucleotides and RNA injection The morpholino antisense oligonucleotides to knockdown the expression of Hr-β-catenin, Hr-FoxDa, Hr-FoxDb, Hr-FGF9/16/20, Hr-FoxA and Hr-ZicN were purchased from Gene Tools and their sequences were as follows: Hr-β-catenin MO, 5′-GGCTCATTAACATCTCGGCCATGAT-3′ Hr-FoxDa MO, 5′-TGTTTCTGCCGTACTGGAATGCAAT-3′ Hr-FoxDb MO, 5′-ATGTTCGCACGGAAACTTGACGCAT-3′ Hr-FGF9/16/20 MO, 5′-TACCATTTGTACTGAAGGCATTTTC-3′ Hr-FoxA MO, 5′-TTGACGGAGGCGAAGAAAGCATCAT-3′ Hr-ZicN MO, 5′GCTGTTGCGTATGCCATTTTTGCTT-3′ (Wada and Saiga, 2002). The specificity of the MOs is shown elsewhere for Hr-ZicN MO (Wada and Saiga, 2002), by our rescue experiments by co-injecting synthetic RNAs for Hrβ-catenin, Hr-FGF9/16/20 and Hr-FoxA MOs (data not shown), and by using the second MO (5′-TGTGATGAGATTCGTCGGTAGTCTT-3′) that recognize a different region in the 5′ UTR of the Hr-FoxDa gene (data not shown). The standard control (c-MO) provided by Gene Tools was used as a control. Plasmids for in vitro RNA synthesis were prepared using PCR-amplifying fragments that contained the entire or the most of the ORFs and by inserting them into home-made injection vectors, pBS-HTB for Hr-FoxDa and Hr-ZicN, and pBS-HTB(N) for Hr-β-catenin and Hr-FoxA. For the PCR, cDNA pools prepared using the SMART RACE cDNA Amplification Kit (Clontech) were used for HrFoxDa, Hr-FoxA and Hr-ZicN, and the Hr-β-catenin plasmid kindly provided by Dr. H. Saiga (Tokyo Metropolitan University) was used for Hr-β-catenin as a template. pBS-HTB is a pBluescript-based vector that has the 5′ and 3′ UTRs from Hr-Tbb2 tubulin cDNA (Miya and Satoh, 1997) and its multi-cloning sites flanked by them. A NotI site was inserted behind the 3′ UTR in pBS-HTB(N). To obtain a stable form of Hr-β-catenin, the region encoding amino acids 2–48 that includes four putative phosphorylation sites, Serines 30, 34 and 42 and Threonine 38, was deleted (Y. Iida and H. Nishida, unpublished). As a control for RNA injection, a Venus YFP construct was made by subcloning the BamHI/EcoRI fragment of the Venus plasmid (Nagai et al., 2002) kindly provided by Dr. A Miyawaki (Brain Science Institute, RIKEN) into the BamHI/EcoRI sites of the pBS-RN3 vector (Lemaire et al., 1995). Capped RNAs were transcribed with mMessage mMachine (Ambion) and subsequently PolyA was added to the RNAs with Poly(A) Tailing Kit (Ambion). Microinjection was carried out 45 min to 2 h after fertilization, as described previously (Miya et al., 1997).

Blastomere isolation, FGF and UO126 treatment and cell cleavage arrest Isolation of blastomeres, treatment with bFGF proteins (Sigma) and inhibition of cell cleavage were performed according to Kobayashi et al. (2003). Treatment with the MEK inhibitor, UO126 (Promega), was done as

The key factors for notochord specification proposed in Ciona embryos are β-catenin, FoxD and the genes downstream from them, ZicL and FGF9/16/20 (Imai et al., 2000, 2002a,b,c, 2006). As a first step to clarify the mechanism by which different sets of combinatorial inputs specify the notochord at its specific position in Halocynthia embryos, we isolated Halocynthia versions of FGF9/16/20 (Fig. S1A) and FoxD (Figs. S3A and B) by the classical RT-PCR method and 5′ and 3′ RACE. A Halocynthia version of β-catenin is found in MAGEST (Kawashima et al., 2000; Y. Iida and H. Nishida, unpublished) and that of Zic (Hr-ZicN) has already been published (Wada and Saiga, 2002). In situ hybridization for HrFGF9/16/20 (Figs. S1B-Q) shows that the expression pattern of this gene is identical to that of Cs-FGF9/16/20 with the exception that the expression is still detectable in notochord cells at the 64-cell stage in Halocynthia and in muscle cells at the 110-cell stage in Ciona (Imai et al., 2002a), consistent with its role in notochord induction: it is expressed in the endoderm and other cells at the 32- and 44-cell stages. Knockdown of this gene by MO injection resulted in almost complete loss of notochord-specific Hr-Bra expression at the 110-cell stage (100%, n = 7; Figs. S2A and B) and at the mid-tailbud stage (91%, n = 11; Figs. S2C and D). Therefore, Hr-FGF9/16/20 appears to be fully responsible for notochord induction, in contrast to the situation in Ciona embryos where brachyury expression is restored by the tailbud stage after FGF9/16/20 knockdown (Imai et al., 2002a). We found two types of FoxD in Halocynthia (designated as Hr-FoxDa and Hr-FoxDb, Figs. S3A and B). Hr-FoxDa is expressed only at the cleavage stages from the 16-cell through the 64-cell stages (Figs. S3C-M), while Hr-FoxDb is activated only after the neurula stage (Fig. S3N– V). C. savignyi has one FoxD, whose expression is observed both at the cleavage stages and after the neurula stage onwards with a spatial expression pattern very similar to that of HrFoxDa and Hr-FoxDb combined (Imai et al., 2002c), suggesting that the two Halocynthia FoxDs split their roles while the Ciona FoxD performs them all with these single genes. C. intestinalis, on the other hand, has two FoxDs, Ci-FoxDa/b (Yagi et al., 2003). Knockdown experiments using MO injection showed that Hr-FoxDa, but not Hr-FoxDb, is necessary for

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notochord formation (96.7% loss of Not-I staining for HrFoxDa MO (n = 32) and 12.5% reduction for Hr-FoxDb MO (n = 24) compared to 0% loss for c-MO (n = 7) in Fig. S4). From its expression pattern and the results of the knockdown experiments, we suggest that Hr-FoxDa plays a role in early notochord specification. Epistasis analysis of notochord genes In the beginning, we attempted to reveal the molecular cascades in Halocynthia embryos that lead to notochord-specific Hr-Bra expression at the 110-cell stage (Fig. 1). From studies with Ciona embryos, a cascade, β-catenin → FoxD → ZicL → brachyury, is proposed (see Introduction). We included HrFoxA (former Hr-HNF3-1, Shimauchi et al., 1997) in the gene pool to be analyzed, because it has been shown in another ascidian, Molgula oculata, that loss of function of the FoxArelated gene Mocu-FH1 resulted in abnormal axis formation (Olsen and Jeffery, 1997), and also because Ci-FoxAa is recently shown to regulate brachyury expression (Imai et al., 2006). A series of knockdown experiments by MO injection using the Halocynthia notochord genes (Hr-β-catenin, HrFoxDa, Hr-FoxA, Hr-ZicN and Hr-Bra) led us to conclude that Hr-β-catenin is upstream of Hr-FoxDa, Hr-FoxA and HrFGF9/16/20 (Figs. 2B, F and L compared to A, E and K, respectively), and that Hr-FoxDa and Hr-FoxA are dependent on each other (Figs. 2H and N compared to E and K, respectively), although the B-line expression of Hr-FoxDa does not depend on Hr-FoxA (Fig. 2H). These two genes are also necessary for Hr-ZicN expression with the exception that the B-line expression of Hr-ZicN does not require Hr-FoxDa (Figs. 2Q and R compared to O). In other experiments, HrFoxDa, Hr-FoxA and Hr-ZicN are all expressed in Hr-FGF9/ 16/20 knockdown embryos (Figs. 2G, J, M and P compared to E, I, K and O, respectively) with the exception that the expression of Hr-FoxA in the notochord lineage at the 64-cell stage (A7.3 and A7.7), but not at the 32-cell stage (A6.2 and A6.4), is largely decreased (arrowheads in Fig. 2M compared to K for 64-cell stage and those in Fig. 2J compared to I for 32-cell stage). This is consistent with previous results in which notochord precursor cells isolated at the 32-cell stage had the expression of Hr-FoxA up-regulated when cultured in the presence of bFGF proteins (Shimauchi et al., 2001b). These results suggest that the expression of Hr-FoxDa and Hr-ZicN is independent of Hr-FGF9/16/20 signaling, whereas that that of Hr-FoxA depends on the signal for its maintenance, but not for its initiation, in the notochord lineage. Thus, while these factors all could function as intrinsic competence factors in notochord induction, it is also possible, with regard to HrFoxA, that it functions only downstream of, but not together with, FGF signaling in specifying notochord, and that the role of FGF signaling in the induction is only to maintain its gene expression in the notochord lineage. Finally, Hr-FoxA, Hr-ZicN and Hr-FGF9/16/20 are required for the early Hr-Bra expression at the 64-cell stage (Figs. 2T–V compared to S), and these three genes and Hr-FoxDa at the 110-cell stage (Wada and Saiga, 2002; Figs. 2X–ZZ compared to W).

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In order to better understand the relationship between HrFoxDa and Hr-FoxA, we carried out additional experiments involving over-expression of RNA. Over-expression of a stable form of Hr-β-catenin RNA (Y. Iida and H. Nishida, unpublished) caused ectopic, although very weak, expression of Hr-FGF9/16/ 20 (Fig. 3B), Hr-FoxDa (Fig. 3D) and Hr-FoxA (Fig. 3G). The amount of the RNA injected is sufficient to convert the ectoderm cells to endoderm as revealed by alkaline phosphatase (AP) expression (Y. Iida and H. Nishida, unpublished), indicative of endoderm differentiation. Whereas loss of function analysis by MO injection revealed that expression of Hr-FoxDa and HrFoxA is inter-dependent (Figs. 2H and N), over-expression analysis showed that injection of RNA encoded by either of the two genes did not cause ectopic expression of the other gene (Figs. 3E and H), but rather inhibited their endogenous expression (data not shown). At present, it is unclear why the endogenous expression was eliminated, although the results of over-expression experiments are often difficult to interpret due to the fact that the injected RNAs are translated and become excessively functional at too early a stage. In any event, these results suggest that Hr-β-catenin is sufficient for Hr-FGF9/16/ 20, Hr-FoxDa and Hr-FoxA and that it can activate Hr-FoxDa and Hr-FoxA expression in a way that does not involve the regulatory loop between Hr-FoxA and Hr-FoxDa. This is consistent with results from Ciona embryos in which Cs-βcatenin directly controls the expression of Cs-FoxD via TCF binding elements (Imai et al., 2002c). In other experiments, HrFoxA, when over-expressed, was able to induce ectopic Hr-ZicN expression (Fig. 3J) without inducing Hr-FoxDa (Fig. 3E). Injection of Hr-FoxDa RNA also resulted in ectopic expression of Hr-ZicN (Fig. 3K) without activating Hr-FoxA (Fig. 3H). These results suggest that either Hr-FoxDa or Hr-FoxA is capable of inducing Hr-ZicN expression alone, independently from the other. Unfortunately, from these results, it is unclear which one of them or whether both is an endogenous activator of Hr-ZicN expression because of their dependency on each other for their expression (Figs. 2H and N). Finally, injection of HrZicN MO resulted in ectopic AP expression in the notochord cells (average number of positive cells is 14.5 (n = 13) in Fig. 3M, compared to c-MO-injected embryos with an average number of 9.6 (n = 8) in Fig. 3L), suggesting that another role of Hr-ZicN is suppression of endoderm formation in the notochord lineage. Hr-FoxA and Hr-ZicN are intrinsic competence factors in notochord induction In an effort to identify an intrinsic factor(s) that cooperates with Hr-FGF9/16/20 signaling to specify notochord, we adopted a candidate approach. From what we had found in the previous section and what others have shown (Wada and Saiga, 2002), we tested whether Hr-FoxDa, Hr-FoxA or Hr-ZicN behaves as the intrinsic factor(s). These are all transcription factors that are necessary for notochord formation with respect to Hr-Bra expression at the 64- and 110-cell stages (Wada and Saiga, 2002; Figs. 2S–ZZ) and notochord-specific Not-1 antigen expression at the tailbud stage (Figs. S4B, 100% loss of Not-I staining for Hr-FoxA (n = 8) and Hr-ZicN (n = 7) MOs in Figs. 4B and C,

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Fig. 1. Schematic representation of the notochord lineage. Shown in the diagrams are primary (dark orange) and secondary (light orange) notochord precursors at the 16-cell (A), 32-cell (B), 64-cell (C) and 110-cell (D) stages. Vegetal views with anterior to the top. (E) Lineage tree of the primary (dark orange) and the secondary (light orange) notochord cells. The blastomeres that develop into notochord are colored orange. Notochord fate is restricted in the A7.3, A7.7 and B8.6 blastomeres. (F) Expression pattern of the notochord genes. Vegetal views. Anterior is up. Shown in the diagrams are the expression patterns, at the 16-cell, 32-cell, 64-cell and 110-cell stages, of the four genes, Hr-FGF9/16/20 (red), Hr-FoxA (blue), Hr-FoxDa (yellow) and Hr-ZicN (green), which were shown either in this study or elsewhere (Shimauchi et al., 1997; Wada and Saiga, 2002) to be essential for notochord formation, and of the notochord-specific Hr-Bra gene (purple).

respectively). Whereas Hr-ZicN expression starts at the 32-cell stage only in notochord cells (Fig. 1, Wada and Saiga, 2002), HrFoxDa is expressed in cells that are fated to become both notochord and endoderm at the 16-cell stage and only in endoderm (inducing) cells after they divide off from the notochord lineage (Figs. S3 and 1). Hr-FoxA is also expressed in both the notochord and endoderm cells (Fig. 1, Shimauchi et al.,

1997) at the 32-cell stage when the induction occurs. Thus, initially, we attempted to clarify whether Hr-FoxDa and HrFoxA are required in the notochord (responding) blastomeres for notochord induction, and performed blastomere isolation experiments. In these experiments, we isolated notochord blastomeres from 24-cell stage embryos that had been injected with MO against Hr-FoxDa or Hr-FoxA and grew them in the

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Fig. 2. Epistasis analyses of the notochord genes 1. Expression of Hr-FGF9/16/20 (A–D at the 32-cell stage), Hr-FoxDa (E, F, G and H at the 32-cell stage), Hr-FoxA (I and J at the 32-cell stage, and K, L, M and N at the 64-cell stage), Hr-ZicN (O, P, Q and R at the 64-cell stage) and Hr-Bra (S–V at the 64-cell stage, and W, X, Y, Z and ZZ at the 110-cell stage) in the vegetal hemisphere of embryos that had been injected with different kinds of MOs was examined by in situ hybridization. The MOs injected are shown above the images. From the left-most column, either 300 pg or 1000 pg of control MO, 300 pg of Hr-β-catenin MO, 300 pg of Hr-FGF9/16/20 MO, 1000 pg of Hr-FoxDa MO, 300 pg of Hr-FoxA MO or 300 pg of Hr-ZicN MO were injected. Arrowheads represent Hr-FoxA expression in the A-line notochord lineage (A6.2 and A6.4 in I and J, and A7.3 and A7.7 in K) and the absence of Hr-FoxA expression in the notochord (M and N). Notochord precursors could not be identified in the β-catenin-injected embryo (L). Note that only A-line expression of Hr-FoxDa or Hr-ZicN was eliminated by injecting Hr-FoxA or Hr-FoxDa MO, respectively (H and Q). All the embryos are viewed vegetally with anterior to the top. Scale bar: 100 μm.

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Fig. 3. Epistasis analyses of the notochord genes 2. Expression of Hr-FGF9/16/20 (A and B at the 32-cell stage), Hr-FoxDa (C, D and E at the 32-cell stage), Hr-FoxA (F, G and H at the 64-cell stage), and Hr-ZicN (I, J and K at the 110-cell stage) in embryos that had been injected with 100 pg of different kinds of synthetic mRNAs. The RNAs injected are indicated above the images. The RNA-injected embryos are viewed animally so that ectopic expression can easily be recognized (B, D, E, G, H, J and K). Effects of RNA injection are often seen only in the lateral half of the embryo due to slow diffusion of the injected RNA before the first cell cleavage. Arrowheads in panels B, D, G, J and K indicate ectopic expression. (L and M) Histochemical staining for endoderm-specific alkaline phosphatase (AP) after injection of 300 pg of c-MO or Hr-ZicN MO into embryos in which cell division was arrested by cytochalasin B treatment from the 110-cell stage onwards. Arrowheads in M represent ectopic AP staining in the A-line notochord lineage. (A, C, F, I, L and M) Vegetal views with anterior up. Scale bars: 100 μm.

presence of bFGF proteins to the tailbud stage, when we stained them for Not-1 antigen expression (Fig. 4G). We found that injection of either Hr-FoxDa or Hr-FoxA MO resulted in almost complete loss of Not-1 staining (88% loss (n = 8) for Hr-FoxDa MO in Fig. 4I, 100% loss (n = 15) for Hr-FoxA MO in Fig. 4J, 0% loss (n = 26) for c-MO in Fig. 4H). These results indicate that the both factors are required in the responding cells for notochord induction. Since the expression domains of HrFoxDa and Hr-ZicN do not overlap during the cleavage stages (Fig. 1F), the above results suggest that Hr-FoxDa proteins translated at the 16-cell stage may be inherited into the notochord/nerve cord lineage at the division to the 32-cell stage and activate Hr-ZicN expression. We next examined whether they are capable of inducing ectopic Hr-Bra expression in an FGF-dependent manner. We

used an animal cap assay in which the four animal blastomeres are isolated from an 8-cell stage embryo that has been injected with RNA, and then cultured in the absence or presence of bFGF proteins until the equivalent of the 110-cell stage for Hr-Bra in situ hybridization (Fig. 5A). The ectopic expression of Hr-Bra in the animal cells was observed only when Hr-FoxA RNA was injected, regardless of whether or not FGF was used (Figs. 5D, H and Table 1). However, this ectopic Hr-Bra expression was reduced to the background level when the isolated animal cells were treated with the MEK inhibitor, UO126 (Fig. 5K, Table 1), suggesting that the induction of Hr-Bra by Hr-FoxA is dependent on FGF signaling. Since injection of Hr-FoxA RNA leads to weak ectopic expression of Hr-FGF9/16/20 in animal cells (data not shown), this might be the source of the FGF signal that caused Hr-FoxA without bFGF treatment to promote Hr-

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Fig. 4. Cell autonomous requirement of Hr-FoxDa and Hr-FoxA for notochord formation. (A–C, H–J) Notochord-specific Not1 immunostaining by the TSA fluorescein system. (D–F, K–M) Morphology of the MO-injected whole and partial embryos. (A–F) Tailbud-stage embryos injected with 300 pg of control (A and D), Hr-FoxA (B and E) or Hr-ZicN MO (C and F). See Supplementary Figure S4 for injection of Hr-FoxDa MO. (G) Schematic illustration of blastomere isolation experiments done for H-M. (H–M) Isolated notochord cells from embryos that have been injected with MOs. They were treated with bFGF protein immediately after isolation to the equivalent of the tailbud stage. The MOs injected were 1000 pg of control (H and K) and Hr-FoxDa MOs (I and L), and 300 pg of Hr-FoxA MO (J and M). (K–M) Note that the individual cells constituting the partial embryo in panel K are larger, and that the number of the cells is smaller, when compared to those in panels L and M. The notochord precursor cells at the stage when isolation was performed normally undergo only four more cell divisions and stop dividing to become fully differentiated notochord (Nishida, 1987), while the presumptive nerve cord cells continue to divide (Nishida, 1987). Scale bar: 100 μm.

Bra expression in the animal cap assay. Alternatively, the isolation itself might have caused an elevation of the FGF/ MAPK signaling pathway in the cells, as has been reported for dissected Xenopus animal cap cells (LeBonne and Whitman, 1997; Curran and Grainger, 2000; Kumano et al., 2001). In contrast to Hr-FoxA injection, no ectopic Hr-Bra expression was observed even in the presence of bFGF proteins when HrFoxDa, Hr-ZicN or control YFP RNA was injected (Figs. 5B, C, E–G, I, J and Table 1). These results suggest that Hr-FoxA functions as an intrinsic competence factor, cooperating with FGF signaling, despite the fact that its expression is dependent on FGF signaling within the signal-receiving cells. FGF signaling appears to have two distinct functions in notochord induction: it maintains the expression of Hr-FoxA in the notochord lineage (Figs. 2J and M) and activates Hr-Bra expression together with Hr-FoxA. The results above showed that Hr-FoxA activates Hr-Bra expression in an FGF-dependent manner. However, not only FGF

signaling but also Hr-ZicN was required for the induction of HrBra by Hr-FoxA (Fig. 6). Co-injection of Hr-ZicN MO with HrFoxA RNA resulted in reduction of the ectopic Hr-Bra expression in the animal region of the whole embryo (22% positive (n = 9) in Fig. 6B vs. c-MO-injected, 83% (n = 6) in Fig. 6A). Hr-Bra induction by Hr-FoxA was again dependent on FGF signaling in the whole embryo (0% positive (n = 9) with UO126 treatment in Fig. 6D vs. 100% (n = 9) with DMSO in Fig. 6C). However, this was not because Hr-FoxA is unable to induce Hr-ZicN expression without FGF signaling, since the treatment with UO126 did not affect the level of ectopic Hr-ZicN expression induced by HrFoxA (100% positive (n = 10) with UO126 in Fig. 6F vs. 100% (n = 10) with DMSO in Fig. 6E). Given that Hr-ZicN alone failed to induce ectopic Hr-Bra expression even in the presence of FGF signaling (Figs. 5E and I), the present results suggest that both Hr-ZicN-dependent and -independent pathways are necessary in order for Hr-FoxA to induce Hr-Bra expression, and that FGF signaling is an absolute requirement for this induction.

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Fig. 5. FGF-dependent induction of Hr-Bra expression by Hr-FoxA. (A) Schematic illustration of blastomere isolation experiments done for panels B–K. (B–K) Expression of Hr-Bra by in situ hybridization in partial embryos derived from the animal halves of 8-cell-stage embryos that have been injected with synthetic mRNAs. The isolated blastomeres were cultured to the equivalent of the 110-cell stage in the presence of either BSA/DMSO (B–E), bFGF protein (F–I) or the MEK inhibitor, UO126 (J and K). 100 pg of venus yfp (B, F and J), Hr-FoxDa (C and G), Hr-FoxA (D, H and K) or Hr-ZicN (E and I) RNA was injected. Scale bar: 100 μm.

Hr-FoxA/Hr-ZicN are capable of altering competence in other cells Although Hr-FoxA seems to be a competence factor for notochord induction, there are several other tissues besides notochord that are induced by FGF signaling. Therefore, our next objective was to clarify the extent to which Hr-FoxA is responsible for the difference in competence between induction of notochord and other tissues. We injected fertilized eggs with a lower amount of Hr-FoxA RNA (10 pg, one-tenth of the amount used above) so that the injected embryos underwent a sufficiently normal cleavage pattern to allow identification of the blastomeres that might show ectopic HrBra expression at the 110-cell stage. We found that Hr-Bra was ectopically expressed in the posterior mesenchyme cells as well as the posterior endoderm cells upon over-expression (75% (n = 16), Figs. 7A and B). The ectopic Hr-Bra expression in these tissues was dependent on both Hr-ZicN (0% positive (n = 7), Fig. 7C) and FGF signaling (0% positive (n = 6), Fig. 7D). These results indicate that the cells that normally receive the FGF signal in the developing embryo, with the exception of the anterior mesenchyme cells, can change their fate to Table 1 Expression of Hr-Bra in the isolated animal blastomeres

Discussion

Isolated blastomeres with Hr-Bra expression/blastomeres examined (%)

BSA/DMSO bFGF UO126

notochord when a single factor, Hr-FoxA, is over-expressed, and suggest that Hr-FoxA is a factor that makes the difference in competence to respond to FGF signaling. In contrast to the ectopic notochord formation in the posterior mesenchyme and endoderm lineages in response to Hr-FoxA over-expression, however, knockdown of Hr-FoxA by MO injection did not cause ectopic formation of either mesenchyme or endoderm in the notochord lineage (0% ectopic formation of endoderm (n = 6), Figs. 7E and F, 0% ectopic mesenchyme formation (n = 7), Figs. 7G–J). Therefore, it is likely that there are other factors in the mesenchyme and endoderm lineages that do not exist in the notochord, and that these positively regulate the formation of the two tissues in response to FGF signaling. One such factor is likely to be Macho-1, which functions in mesenchyme induction (Kobayashi et al., 2003). Although no ectopic AP expression in the notochord lineage upon Hr-FoxA MO injection (Fig. 7F) appears to be inconsistent with one of the earlier results showing that knockdown of Hr-ZicN, a downstream target of Hr-FoxA, results in the ectopic AP expression in that lineage (Fig. 3M), it could be possible that knockdown of Hr-FoxA causes loss of other downstream target genes that are required for the ectopic endoderm formation in the notochord lineage.

YFP

Hr-FoxDa

Hr-FoxA

Hr-ZicN

0/10 (0%) 0/9 (0%) 0/7 (0%)

0/13 (0%) 0/12 (0%)

23/31 (74%) 31/31 (100%) 0/10 (0%)

0/10 (0%) 0/9 (0%)

In this study, we have identified Hr-FoxA and Hr-ZicN as intrinsic factors that act with Hr-FGF9/16/20 signaling to activate notochord-specific Hr-Bra expression. This is consistent with a model proposed by Wada and Saiga (2002) in which Hr-ZicN may be required for the response to FGF

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Fig. 6. Dependency on Hr-ZicN for Hr-Bra induction by Hr-FoxA. Expression of Hr-Bra in whole embryos that have been injected with Hr-FoxA RNA together with either control (A) or Hr-ZicN MO (B). Expression of Hr-Bra (C and D) and Hr-ZicN (E and F) in whole embryos injected with Hr-FoxA RNA and then treated with either DMSO (C and E) or UO126 (D and F). All the embryos are at the equivalent of the 110-cell stage and viewed animally so that ectopic expression can easily be recognized. Scale bar: 100 μm.

signaling in notochord induction. Although Hr-FoxA expression at the 64-cell stage is dependent on Hr-FGF9/16/20 signaling (Fig. 2M) and Hr-FoxA might not be qualified as an intrinsic competence factor in this regard, we still do not know whether the FGF-independent Hr-FoxA expression at the 32cell stage (Fig. 2J), the FGF-dependent expression at the 64cell stage, or the both are responsible for the activation of the Hr-Bra gene at the 64-cell stage. Hr-FoxA/Hr-ZicN is the third group of factors that are responsible for cell-type-specific responses to FGF signaling and subsequent tissue-specific differentiation. The first of such factors to be identified was Macho-1 for mesenchyme induction (Kobayashi et al., 2003), and the second was GATA for brain induction (Bertrand et al., 2003). A group of such factors yet to be identified is an intrinsic factor(s) for induction of the posterior endoderm, which is also known to require FGF signaling to form in Halocynthia embryos (Kondoh et al., 2003). With the identification of intrinsic factors for notochord induction, we now understand more details about how the notochord is specified in the embryo, and this is discussed below. Molecular cascades that lead to A-line Hr-Bra expression We have isolated Halocynthia orthologues of FGF9/16/20 and FoxD. Hr-FGF9/16/20 is likely to be the endogenous

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inducer for notochord and mesenchyme for the following reasons. The first reason is that the expression pattern of HrFGF9/16/20 is in agreement with the predicted one both spatially and temporally (Nakatani and Nishida, 1994; Kim and Nishida, 1999). The second reason is that the results obtained from Hr-FGF9/16/20 knockdown and over-expression experiments are all consistent with the previous results from experiments using MEK and FGFR inhibitors and recombinant bFGF proteins (Nakatani et al., 1996; Kim et al., 2000; Kim and Nishida, 2001). Knockdown of Hr-FGF9/16/20 by MO injection resulted in loss of Hr-Bra and mesenchyme-specific Mch-3 antigen expression with concomitant ectopic expression of Hr-ETR-1, a nerve cord marker, and muscle actin in the notochord and mesenchyme lineages, respectively (this study; data not shown). On the other hand, over-expression of HrFGF9/16/20 caused loss of Hr-ETR-1 and muscle actin expression (data not shown) and ectopic Hr-Bra and Mch-3 expression (G.J. Kim, G. Kumano and H. Nishida, in preparation) in the nerve cord and muscle lineages, respectively. The results from our knockdown and over-expression analyses (Figs. 2 and 3) are consistent with what occurs in early notochord formation in the primary lineage in Ciona embryos. The cascade by which β-catenin activates FoxD which then activates ZicL to promote transcription of brachyury (Imai et al., 2000, 2002b,c, 2006; Yagi et al., 2004a) is conserved between Ciona and Halocynthia embryos. In this study, we have included FoxA in addition to the factors mentioned above and proposed a model for the molecular cascades that lead to A-line Hr-Bra expression (Fig. 8). Recent comprehensive in situ hybridization analysis and gene disruption assay with C. intestinalis embryos also suggest that Ci-FoxDa/b, Ci-FoxAa, Ci-ZicL and Ci-FGF9/16/20 signaling are the main zygotic components that regulate brachyury expression at the 64-cell stage (Imai et al., 2006).Although our model is likely to be far from complete and to involve more factors such as BMP2/4 (Darras and Nishida, 2001) and Wnt5 (Nakamura et al., 2006), it does explain, in its present form, how only the cells of notochord lineage respond to FGF signaling and express Hr-Bra. In the model, maternal βcatenin activates the expression of Hr-FoxA, Hr-FoxDa and Hr-FGF9/16/20 at the 16-cell stage (graded yellow and purple in Fig. 8). Then, Hr-FoxDa and Hr-FoxA seem to form a positive regulatory loop to maintain their mutual expression. In the notochord lineage, Hr-FoxDa and Hr-FoxA activate HrZicN expression at the 32-cell stage (graded purple in Fig. 8). About the same time, the Hr-FGF9/16/20 signal from endoderm (yellow) phosphorylates and activates Hr-Ets in the notochord precursors (graded purple). Finally, Hr-FoxA, Hr-ZicN and phosphorylated Hr-Ets activate the expression of Hr-Bra at the 64-cell stage (pink in Fig. 8). The expression of HrFoxA in the notochord lineage is maintained by FGF signaling (yellow and pink in Fig. 8). In the nerve cord lineage, on the other hand, Hr-ZicN activates Hr-ETR-1 expression at the 110-cell stage (purple in Fig. 8). Accordingly, Hr-Bra expression is activated only in the cells where the domains of Hr-FoxA (notochord and endoderm) and HrZicN (notochord and nerve cord) activities overlap (Fig. 1).

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Secondary notochord In addition to the A-line primary notochord cells, the posterior eight notochord cells of the tadpole tail are derived from the B-line (posterior-vegetal) blastomeres, and are known as the secondary notochord (Nishida, 1987). In situ hybridization for brachyury has shown that the secondary lineage is already evident at the 110-cell stage as two isolated spots of staining in the posterior half of the embryo (Yasuo and Satoh, 1993, 1994, Fig. 1R). The notochord fate in the B-line is restricted at the 110-cell stage when it divides off from the mesenchyme lineage. Formation of the secondary notochord also requires FGF signaling from adjacent endoderm cells at the 32-cell stage (Darras and Nishida, 2001). Our knockdown analyses and findings from another study showed that Hr-FGF9/ 16/20, Hr-FoxDa, Hr-FoxA and Hr-ZicN are all required for formation of the secondary notochord as well as the primary notochord in Halocynthia (Wada and Saiga, 2002, Figs. 2W– ZZ). This contradicts with results from analyses of Ciona embryos, where secondary notochord formation does not involve Cs-ZicL (Imai et al., 2002b), but rather Notch signaling (Imai et al., 2002c). In Halocynthia embryos, however, Notch signaling does not seem to be involved in notochord formation (Akanuma et al., 2002). It appears that relatively similar genetic cascades involving Hr-ZicN regulate both A-line and B-line notochord formation in Halocynthia embryos. On the other hand, the two lineages differ in that expression of Hr-FoxDa and Hr-ZicN in the B-line does not require Hr-FoxA and HrFoxDa, respectively (Figs. 2H and Q). Primary notochord, nerve cord and endoderm

Fig. 7. Notochord formation in the mesenchyme and endoderm lineages by over-expression of Hr-FoxA. Expression of Hr-Bra (A, C and D) at the 110cell stage in whole embryos injected with 10 pg of Hr-FoxA RNA either alone (A), together with 300 pg of Hr-ZicN MO (C) or with UO126 treatment after the injection of Hr-FoxA (D). (B) Diagram of the Hr-Bra expression shown in panel A. (E and F) Histochemical staining for AP in embryos that have been injected with 300 pg of c-MO (E) or Hr-FoxA MO (F). (G and H) Expression of mesenchyme-specific Mech-3 antigen in embryos that have been injected with 300 pg of c-MO (G) or Hr-FoxA MO (H). In these embryos (E–H), cell division was arrested from the 110-cell stage onwards. (I and J) Nomarsky images of the cleavage-arrested embryos shown in panels G and H. All the embryos are viewed vegetally. Scale bars: 100 μm.

Hr-FoxDa appears to contribute to Hr-Bra expression only through activation of Hr-ZicN and does not seem to be required for Hr-Bra expression at the 64-cell stage. This may be supported by its mRNA expression pattern, which does not persist in the notochord lineage at the 32- and 64-cell stages (Fig. 1).

Maternal β-catenin is known to be one of the factors positioned at the very top in a hierarchy of molecular cascades that lead to the formation of several tissues in the vegetal hemisphere, such as endoderm, notochord, mesenchyme and a subset of muscle (this study; Imai et al., 2000; Y. Iida and H. Nishida, unpublished). The β-catenin requirement for endoderm formation is accomplished through activation of one of the endodermal key transcription factors (Nishida, 2005), the LIMhomeobox gene Lhx3 (Satou et al., 2001), while that for notochord and mesenchyme formation occurs via activation of FGF9/16/20 (this study; Imai et al., 2002a). In the notochord, βcatenin also acts through FoxD and FoxA (this study; Imai et al., 2002c). Given the fact that Zic is downstream of FoxD and FoxA (this study; Imai et al., 2002b, 2006) and that Hr-ZicN is both necessary and sufficient for a nerve cord marker, Hr-ETR-1 (Wada and Saiga, 2002), the nerve cord would not form without β-catenin, either. Therefore, β-catenin seems to regulate the formation of three major tissues in the A-line: the notochord, nerve cord and endoderm. Although it remains unclear how these three tissues arise in the different regions of the anterior part of the early embryo, despite the fact that in all cases the process seems to begin with nuclear localization of β-catenin, some or all of this issue may be resolved by clarifying how the downstream genes such as FGF9/16/20, FoxD and FoxA are expressed in different cells from the 32-cell stage onwards after

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Fig. 8. A model for the molecular cascades that lead to notochord-specific Hr-Bra expression in the A-line. The En/Not/NC cell divides to produce the Not/NC and En (yellow) cells and the Not/NC cell divides to create the Not (pink) and NC (purple) cells. The position of each cell in the embryo is shown in the same color in the diagram of the embryo at the top. Solid arrows represent activation of the target genes that is shown to be both necessary and sufficient, while broken arrows represent activation that is supported by MO injection, but not by mRNA injection. The proteins are shown in the regular font, while the transcripts in the italic font. Since HrFoxDa mRNA is not detected in the Not/NC lineage at the 32-cell stage, we suggest that its protein is inherited by the cells of the lineage and activates Hr-ZicN expression (see text). The activation scheme drawn at each stage depends solely on when the target genes become first expressed. For example, activation of Hr-FGF9/ 16/20, Hr-FoxDa and Hr-FoxA by Hr-β-catenin is placed in the 16-cell stage embryo because the expression of these three genes starts at the 16-cell stage in the endoderm (En)/notochord (Not)/nerve cord (NC) cell (graded yellow and purple). Likewise, Hr-ZicN and Hr-Bra expression starts at the 32-cell stage in the Not/NC cell (graded purple) and at the 64-cell stage in the Not cell (pink), respectively. There are two exceptions that do not follow this rule. One is that in the developing embryo Hr-ETR-1 is activated at the 110-cell stage in the NC cell, not at the 64-cell stage as depicted in the diagram. The other exception is that we still do not know when Hr-Ets is activated (phosphorylated). The green and blue vertical bars on the right side represent expression of Hr-ZicN and Hr-FoxA at the 64-cell stage, respectively, showing the overlapping expression of these genes in the notochord cell. For more details, see text.

activation by β-catenin (Fig. 1F). In separate sections below, we will discuss this issue, focusing on what might explain differences between the notochord and nerve cord, and between the notochord and endoderm. Notochord and nerve cord The notochord and nerve cord lineages separate at the 64-cell stage. These tissues are similar in that they both express Zic at the 64- and 110-cell stages (Wada and Saiga, 2002; Imai et al., 2002b, Fig. 1). However, Zic in the notochord lineage activates notochord-specific brachyury and fibrinogen-like expression (Wada and Saiga, 2002; Imai et al., 2002b, 2006), while that in the nerve cord activates ETR-1 expression (Wada and Saiga, 2002, Fig. 8). How might Zic activate one gene in one lineage and another gene in the other? FGF signaling is known to mediate binary fate specification between notochord and nerve cord (Minokawa et al., 2001). Consistent with this notion, activated MAPK is observed in the nuclei of notochord lineage cells, but not in those of nerve cord cells at the 64-cell stage (Nishida, 2003). The present study also showed that FGF signaling is required for the maintenance of Hr-FoxA expression only in the notochord lineage at the 64-cell stage (Fig. 2M). The situation, however, may not be as simple as FGF signaling acting as a sole determinative factor between notochord and nerve cord when these tissues are specified. Results from previous studies and the expression pattern of Hr-FGF9/16/20 (Fig. S1) have suggested that the cells in the nerve cord lineage are also exposed to FGF signaling and yet somehow resistant to have the signaling activated with respect to the activation of MAPK in these cells (Nakatani and Nishida, 1994; Nishida,

2003). Therefore, FGF signaling is a key factor, but may not be a sole factor, that determines the fate between the notochord and nerve cord. Notochord and endoderm Compared to the relationship between the notochord and nerve cord, the notochord and endoderm appear to be more distant because these two lineages separate one cell cycle earlier than the notochord and nerve cord, i.e., upon division to the 32cell stage. Whereas the endoderm in the A-line (anterior endoderm) arises cell autonomously by inheritance of a yetunknown maternal cytoplasmic determinant(s) (Nishida, 1992), the notochord fate requires cell–cell communication (Nishida, 1992; Nakatani and Nishida, 1994). However, their relationship may be closer than it appears. For example, over-expression of Hr-Bra by injection of its RNA results in ectopic notochord formation in the endoderm as well as in the nerve cord, but not in other tissues (Yasuo and Satoh, 1998). Also, knockdown of Cs-FoxD in Ciona embryos causes notochord cells to express an endoderm marker, AP (Imai et al., 2002c). These fates can be inter-converted by loss or addition of a single factor. We showed in the present study that suppression of endoderm formation in the notochord lineage by FoxD is mediated by Zic (Fig. 3M). Therefore, Zic plays a role not only in promoting notochord and nerve cord differentiation but also in preventing the notochord cells from becoming endoderm. In addition, FoxD might suppress notochord formation in the endoderm lineage. Injection of a low amount of MO against Hr-FoxD, which is not sufficient to inhibit Hr-Bra expression, as observed in Fig. 2Y, caused ectopic Hr-Bra expression in the endoderm (data not

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shown). This result suggests a dose-dependent difference in the function of FoxD: a low concentration activates brachyury expression through Zic, whereas a high concentration inhibits it. FoxD is expressed in notochord/nerve cord/endoderm cells at the 16-cell stage and its transcripts are detectable only in endoderm cells after they divided off from the notochord/nerve cord lineage upon the division to the 32-cell stage (Fig. 1). Therefore, the endoderm is likely to have more FoxD protein. This speculation may also be supported by the observation that over-expression of Hr-FoxDa resulted in elimination of Hr-Bra expression (data not shown). Difference in competence to respond to FGF signaling We have identified Hr-FoxA/Hr-ZicN as competence factors for notochord induction. This is consistent with our promoter analysis of the Hr-Bra gene, where binding sites for Zic and Ets, and to lesser extent for FoxA, on the 5′ flanking region of the gene are necessary for enhancer activity at the 110-cell stage (J. Matsumoto, G. Kumano and H. Nishida, in preparation). Ets is one of the transcription factors that are ultimately phosphorylated and activated by FGF signaling, and in Halocynthia embryos Ets is known to be necessary for induction of tissues that are induced by FGF, such as notochord, mesenchyme and brain (Miya and Nishida, 2003). The existence of functional Ets binding sites on the 5′ region of the Hr-Bra gene supports the idea that FGF signaling activates Hr-Bra expression in a way that does not involve the maintenance of Hr-FoxA expression. Accordingly, activities of the two intrinsic factors, Hr-FoxA and Hr-ZicN, and Hr-FGF9/16/20 signaling could be integrated at the level of enhancer of the target gene Hr-Bra to specify notochord. Since Ets is known to bind to different sequences on enhancer regions and activate the expression of different genes, depending on the transcription factor (known as a partner protein) with which it forms a heterodimer (Verger and Duterque-Coquillaud, 2002), this might explain how the presumptive notochord, mesenchyme and brain cells respond differently to FGF signaling. It would be interesting to determine whether FoxA/ZicN, Macho-1 or GATA proteins bind to Ets and confer target site specificities. As shown in Fig. 7A, over-expression of Hr-FoxA/Hr-ZicN is sufficient for ectopic expression of Hr-Bra in the posterior mesenchyme (B7.7) and endoderm lineages, yet knockdown of Hr-FoxA/Hr-ZicN is not sufficient to specify ectopic mesenchyme in the notochord lineage (Fig. 7H). In contrast, overexpression of Macho-1 is sufficient to specify ectopic mesenchyme in the anterior part of the embryo, and knockdown of Macho-1 is also sufficient for ectopic notochord formation in both the anterior and posterior mesenchyme lineages (Kobayashi et al., 2003). In addition, Halocynthia and Ciona Snail (HrSna and Ci-sna), downstream targets of Macho-1 (Kobayashi et al., 2003; Yagi et al., 2004b), are known to inhibit brachyury expression when over-expressed (Fujiwara et al., 1998; Kobayashi et al., 2003). These results suggest that Macho-1 might suppress Hr-FoxA/Hr-ZicN expression through activation of Hr-Sna in the posterior mesenchyme to guarantee that it differentiates as mesenchyme, and not as notochord. The

situation in the anterior mesenchyme (B8.5), however, would not be so simple because both Hr-FoxA and Hr-ZicN are expressed in this blastomere at the 64-cell stage (Shimauchi et al., 1997; Wada and Saiga, 2002, Fig. 1), which coincides with nuclear localization of activated MAPK (Nishida, 2003), and yet they do not become notochord cells. Since these cells at the 64-cell stage divide to provide the mesenchyme and the secondary notochord cells at the next cleavage, there might be a mechanism other than repression of Hr-FoxA/Hr-ZicN expression by which notochord fate is actively inhibited in the B8.5, so that this asymmetric division takes place properly at the next division. Several lines of evidence suggest that development of the anterior and posterior mesenchyme cells is controlled differently (Imai et al., 2003; Tokuoka et al., 2004). In the anterior part of the embryo (notochord), responsiveness to FGF signaling is conferred by Hr-ZicN (and Hr-FoxA), while in the posterior part (mesenchyme), it is regulated by Macho-1. A genome-wide survey of Zic genes in ascidians has suggested that ZicL/ZicN and Macho-1 are the only Zic family groups (Yamada et al., 2003). Therefore, these two similar genes are responsible for conferring competence, one in the anterior half and the other in the posterior half of the embryo. An interesting area of future investigation would be to clarify how the anterior and posterior halves of the embryo are set up by interaction between these competence factors before induction by FGF signaling takes place. Acknowledgments We are grateful to the staff of the Asamushi Research Center for Marine Biology and the Otsuchi International Coastal Research Center for their help in collecting ascidian adults, and to the staff of the Misaki Marine Biological Station and the Seto Marine Biological Laboratory for their assistance in maintaining them. We also thank Dr. Takahito Nishikata (Konan University) for providing the Not1 monoclonal antibody, Dr. Hidetoshi Saiga (Tokyo Metropolitan University) for the β-catenin plasmid, Dr Nori Satoh (Kyoto University) for the Hr-Bra plasmid and Dr. Atsushi Miyawaki (Brain Science Institute, RIKEN) for the venus YFP plasmid. This work was supported by Grants-in-aid for Scientific Research from the JSPS, Japan (16107005) to H.N. and from the MEXT, Japan (13044003 and 16770161) to H.N. and G.K., and by a Toray Science and Technology Grant to H.N. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.ydbio.2006.07.033.

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