A time delay gene circuit is required for palp formation in the ascidian embryo

Transcription

1 2013. Published by The Company of Biologists Ltd Development (2013) 140, doi: /dev RESEARCH REPORT A time delay gene circuit is required for palp formation in the ascidian embryo Tatsuro Ikeda, Terumi Matsuoka and Yutaka Satou* ABSTRACT The ascidian larval brain and palps (a putative rudimentary placode) are specified by two transcription factor genes, ZicL and FoxC, respectively. FGF9/16/20 induces ZicL expression soon after the bipotential ancestral cells divide into the brain and palp precursors at the early gastrula stage. FGF9/16/20 begins to be expressed at the 16-cell stage, and induces several target genes, including Otx, before the gastrula stage. Here, we show that ZicL expression in the brain lineage is transcriptionally repressed by Hes-a and two Blimp-1-like zinc finger proteins, BZ1 and BZ2, in the bi-potential ancestral cells. ZicL is precociously expressed in the bi-potential cells in embryos in which these repressors are knocked down. This precocious ZicL expression produces extra brain cells at the expense of palp cells. The expression of BZ1 and BZ2 is turned off by a negative autofeedback loop. This auto-repression acts as a delay circuit that prevents ZicL from being expressed precociously before the brain and palp fates split, thereby making room within the neural plate for the palps to be specified. Addition of the BZ1/2 delay timer circuit to the gene regulatory network responsible for brain formation might represent a key event in the acquisition of the primitive palps/placodes in an ancestral animal. KEY WORDS: Ciona intestinalis, Gene circuit, Palp, Brain, Placode INTRODUCTION The ascidian Ciona intestinalis is a simple chordate belonging to the tunicate group. The anterior animal cells (a-line) of the eight-cell Ciona embryo give rise to the trunk epidermis, brain and palps, adhesive organs containing sensory neurons (Nishida and Satoh, 1983) (Fig. 1A). FGF9/16/20, which is expressed in vegetal cells from the 16-cell to gastrula stage, then induces expression of the neural markers Otx (Hudson and Lemaire, 2001; Bertrand et al., 2003; Hudson et al., 2003) and DMRT1 (Tresser et al., 2010) in the future brain/palp (a7.9 and a7.10) and brain/epidermis (a7.13) cells at the 32- and 64-cell stages. At the gastrula stage, bi-potential brain/palp progenitor cells in the anterior part of the neural plate become further fate restricted, separating into distinct palp progenitors and brain progenitors (Nicol and Meinertzhagen, 1988) (Fig. 1B). After this fate restriction, only the brain progenitors express ZicL (Imai et al., 2004) in response to FGF9/16/20 signaling, because the brain progenitors, but not the palp progenitors, abut on FGF9/16/20-expressing vegetal cells (Wagner and Levine, 2012). Thus, even though FGF9/16/20 signals are present as early as the 16-cell stage, the brain progenitors respond to these signals to express ZicL only after the developmental fates Department of Zoology, Graduate School of Science, Kyoto University, Sakyo-ku, Kyoto, , Japan. *Author for correspondence (yutaka@ascidian.zool.kyoto-u.ac.jp) Received 20 June 2013; Accepted 4 September 2013 of the brain and palps diverge. The mechanism by which this timing is coordinated is still unknown. RESULTS AND DISCUSSION Identification of novel zinc finger genes expressed at the 16-cell stage During a microarray screen for zygotically activated genes in early Ciona embryos (Matsuoka et al., 2013), we identified a gene that is expressed in all anterior animal cells (a-line cells) at the 16-cell stage (Fig. 1C). Prior to the 64-cell stage, expression of this gene diminishes in all but one descendant, a7.13, and then is lost in a7.13 by the early gastrula stage. This gene is also transiently expressed in b6.5 at the 32-cell stage, and in a pair of posterior vegetal cells between the 32-cell and early gastrula stages. The gene we identified encodes a C2H2-type zinc-finger protein with sequence similarity to Blimp-1/PRDM1 in other animals. However, it lacks the PR/SET-domain common to Blimp-1 in other animals. Whereas Blimp-1 proteins in other animals have four or five zinc fingers, this has only three. A molecular phylogenetic analysis failed to give strong support (data not shown). Therefore, we named this gene Blimp-1-like Zinc finger gene 1 [BZ1; DNA Data Bank of Japan (DDBJ) accession number AB819270], although it is possible that this gene is a divergent Blimp-1. Another gene encoding a protein with 67% overall amino acid identity to BZ1 and an identical zinc finger domain was found 15 kb upstream of BZ1, and we named this BZ2 (supplementary material Fig. S1; DDBJ accession number AB819271). The expression pattern of BZ2 was almost identical to that of BZ1, except that BZ2 expression at the 16-cell stage was undetectable and that BZ1 and BZ2 were expressed in non-overlapping tissues of tailbud embryos (Fig. 1D). BZ1 and BZ2 are required for proper differentiation of the brain and palp lineages We knocked down BZ1 and BZ2 by injecting specific morpholino oligonucleotides (MOs) into fertilized eggs. All BZ1 morphant larvae show clear brain defects, with 6% also losing palps (n=51), whereas BZ2 morphant larvae appear normal (n=7) (supplementary material Fig. S2A,B). However, when both MOs are simultaneously injected, the brain and palps are severely disrupted compared with normal larvae (n=34, 100%; Fig. 2A,B), indicating functional redundancy between BZ1 and BZ2. Indeed, the palp markers islet and ZF220 are absent in BZ1 and BZ2 double morphants (BZ1/2 morphants) at the tailbud stage (Fig. 2C-F). EphrinA-d, one of the earliest markers for the palp lineage (Imai et al., 2004; Wagner and Levine, 2012), is lost at the late gastrula stage in BZ1/2 morphants, whereas FoxC, another earliest marker for the palp lineage, is not affected (Fig. 2G-J). Six3/6, which is normally expressed in the anterior row of the two rows of the brain progenitor cells at the late gastrula state (Imai et al., 2004; Moret et al., 2005), is ectopically expressed in the palp progenitor cells in BZ1/2 morphants, whereas 4703

2 RESEARCH REPORT Fig. 1. BZ1 and BZ2 are expressed in the progenitors of the neural plate. (A) Cell lineages of the anterior animal cells. Cells fated to give rise to the brain and palps are shown in blue and red, respectively. Blue and green arrows indicate the onset of Otx and DMRT1 expression. (B) Schematics of the brain and palp lineages of cells in the early and late gastrulae. The brain and palp progenitors are shown in blue and red, respectively. The names of individual cells, indicated at the left half of each illustration, are prefixed with the IDs shown next to the illustration. Vertical bars indicate sister relationships between blastomeres. (C,D) In situ hybridization for BZ1 (C) and BZ2 (D) from the 16- cell to early tailbud stages. All embryos except the tailbud embryos are shown in animal view. The tailbud embryos are in lateral view. MYTF, which is normally expressed in the posterior row, is not affected (Fig. 2K-N). ZicL, which is normally expressed in the brain lineage at the early gastrula stage, is ectopically expressed in the palp lineage in BZ1/2 morphants (Fig. 2O,P). Thus, BZ1 and BZ2 are necessary for proper specification of the brain and palp lineages. Previous studies revealed essential roles for ZicL and FoxC in specification of the brain and palps, and that ZicL regulates Six3/6, but not FoxC and MYTF (Imai et al., 2006; Wagner and Levine, 2012). EphrinA-d is also regulated by ZicL, because EphrinA-d expression is expanded to the brain lineage in ZicL morphants (supplementary material Fig. S3A). Next, we made a construct in which the ZicL coding sequence was linked to the DMRT1 upstream sequence. With this construct, ZicL is expected to be expressed in the brain/palp lineage from the 64-cell to late gastrula stages. When this construct is injected into fertilized eggs, EphrinA-d, islet and ZF220 are lost, and Six3/6 is ectopically expressed in the palp lineage (supplementary material Fig. S3B,C; Fig. 2Q,R). Thus, overexpression of ZicL generates a similar phenotype to knockdown of BZ1 and BZ2, suggesting that repression of ZicL might be the primary function of BZ1 and BZ2. BZ1 and BZ2 temporally control ZicL expression To understand how BZ1 and BZ2 regulate ZicL expression, we made constructs in which either of the BZ1 or BZ2 coding sequences was linked to the DMRT1 upstream sequence. Following injection of these constructs into an anterior animal cell at the eight-cell stage, ZicL expression is completely or partially lost in the injected octant (Fig. 3A,B). Thus, BZ1 and BZ2 repress ZicL expression in the brain/palp lineage. In BZ1/2 morphants, ZicL is precociously expressed at the 64-cell stage in the bi-potential progenitors a7.9 and a7.10 (Fig. 3C,D). FGF signaling is required for this precocious expression, because ectopic ZicL expression is rarely observed in BZ1/2 morphants treated with the MEK inhibitor U0126 at the 44-cell stage (Fig. 3E). In addition, BZ1/2 morphant embryos treated with recombinant human basic fibroblast growth factor (bfgf) at the 44- cell stage ectopically express ZicL in a7.9/a7.10 and in neighboring epidermal/brain progenitor cells (a7.13/a7.14), whereas similarly treated uninjected embryos do not show ectopic ZicL expression (Fig. 3F,G). Therefore, FGF signaling acts positively on a7.9/a7.10 to induce ZicL expression at the 64-cell stage, and BZ1 and BZ2 repress the precocious expression of ZicL in normal embryos at this stage. The brain/palp progenitors at the 64-cell stage are derived from a pair of anterior animal cells (a6.5) in the 32-cell embryo. Although no precocious ZicL expression was detected in BZ1/2 morphants at the 32-cell stage, we found ectopic and precocious ZicL expression in the anterior animal cells of BZ1, BZ2 and Hes-a triple morphants (Fig. 3H-J). Hes-a transcriptional repressor gene is normally expressed in all of the blastomeres except for the posterior-most pair of cells at the 16-cell stage, and the expression in the animal hemisphere diminishes by the 32-cell stage (Imai et al., 2004). By contrast, in embryos injected with only the Hes-a MO, ectopic expression is observed only weakly in a small number of cells 4704

3 RESEARCH REPORT Fig. 2. BZ1 and BZ2 are required for proper brain and palp formation. (A-B ) Tadpole larvae developed from eggs injected with a lacz MO (A,A ), and BZ1 and BZ2 MOs (B,B ). Palps and the brain region are shown by red and blue arrowheads. A and B show higher magnification views of the trunk region of larvae (different individuals from those shown in A and B). (C-P) The expression of islet (C,D), ZF220 (E,F), EphrinA-d (G,H), FoxC (I,J), Six3/6 (K,L), MYTF (M,N) and ZicL (O,P) in control (C,E,G,I,K,M,O) and BZ1/2 morphant (D,F,H,J,L,N,P) embryos at the tailbud stage (C-F), at the late gastrula stage (G-N) and at the early gastrula stage (O,P). (G-P) The expression is illustrated schematically in the lower right by red (palps) and blue (brain). (Q,R) The expression of islet (Q) and ZF220 (R) in embryos expressing ZicL under the DMRT1 upstream sequence at the tailbud stage. The number of embryos examined and the proportion of embryos that each panel represents are shown within the panels. BZ1 and BZ2 expression is transient, owing to a negativefeedback loop The results described above indicate that transient expression of BZ1 and BZ2 is important for ZicL expression in the brain lineage. This transient expression is controlled mainly by BZ1, because BZ1 and BZ2 expression is strongly upregulated in BZ1 morphants (Fig. 4AD), but weakly in BZ2 morphants (Fig. 4E,F). We injected synthetic BZ1 and BZ2 mrnas into embryos. Because the synthetic BZ1 and BZ2 mrnas lack the endogenous 3 -UTRs, we were able to measure the amount of the endogenous mrnas of BZ1 and BZ2 by RT-qPCR with primers designed to their 3 -UTRs. Whereas levels of the maternal control mrna macho-1 are unchanged by injection of BZ1 and BZ2 mrnas, BZ1 and BZ2 expression levels are greatly reduced by injection of synthetic BZ1 mrna (Fig. 4G). By contrast, injection of BZ2 mrna rarely reduces BZ1 and BZ2 expression. Therefore, feedback repression of BZ1 and BZ2 expression by BZ1 results in transient expression of these two repressor genes. Conclusions FGF9/16/20 is expressed as early as the 16-cell stage, and is used repeatedly to induce different genes in different cells at different times by functioning in combination with different transcription factors (Imai et al., 2002; Bertrand et al., 2003; Imai et al., 2003; Yagi et al., 2004; Imai et al., 2006; Kumano et al., 2006; Lamy et al., 2006; Hudson et al., 2007; Picco et al., 2007; Shi and Levine, 2008; Hashimoto et al., 2011). The present study shows that the same signaling pathway can evoke differential outputs (ZicL expression or not) in a single lineage of cells at different times by functioning in combination with Hes-a and BZ1/BZ2 repressors (supplementary material Fig. S4). Without these repressors, the brain/palp progenitors precociously express ZicL, and therefore the palps are not properly specified. After the brain/palp progenitors divide, the palp precursor cells do not abut on FGF9/16/20expressing cells (Wagner and Levine, 2012), and therefore ZicL is not induced in the palp lineage. The duration of this repression is probably determined by a negative-feedback circuit of BZ1/2, the kinetics of which are likely to depend on mrna and protein synthesis and turnover rates. Our results suggest that this duration coincides with cell division of the brain/palp progenitors, and thus that the feedback loop acts as a delay timer to temporally control ZicL expression in the brain lineage without regulating the cell division directly. This situation contrasts with instances in which cell cycle genes are directly controlled by regulatory gene circuits. In vertebrates, the key 4705 (Fig. 3K). Therefore, at the 32-cell stage, ZicL is repressed by Hesa, BZ1 and BZ2. Ectopic expression of ZicL in BZ1/2/Hes-a morphants at the 32cell stage is dependent on FGF signaling, because this ectopic expression is lost in BZ1/2/Hes-a morphants treated with U0126 (Fig. 3L) and because bfgf treatment evokes ectopic expression in all anterior ectodermal cells of BZ1/2/Hes-a morphants (Fig. 3M,N).

4 RESEARCH REPORT Fig. 3. Precocious expression of ZicL is repressed by BZ1, BZ2 and Hes-a. (A,B) ZicL expression at the early gastrula stage in embryos expressing BZ1 (A) and BZ2 (B) under the DMRT1 upstream sequence. The overexpression constructs were injected into one anterior animal cell of eight-cell embryos. Descendants of the blastomere injected with the constructs are encircled by pink dashed lines. The expression is illustrated schematically at the bottom right in blue (brain). (C-G) ZicL expression at the 64-cell stage in control (C,F) and BZ1/2 morphant (D,E,G) embryos (shown in anterior views; animal pole up). The embryo shown in E is treated with U0126, and the embryos in F and G are treated with bfgf. (H-N) ZicL expression at the 32-cell stage in control (H,M), BZ1/2 morphant (I), BZ1/2/Hes-a morphant (J,L,N) and Hes-a morphant (K) embryos (shown in animal views). The embryo shown in L is treated with U0126, and the embryos in M and N are treated with bfgf. Ectopic expression is shown by yellow arrowheads. Black arrowheads indicate the expression of ZicL. transcription factors MyoD and Myf5 control cell cycle withdrawal and induction of differentiation in skeletal muscle cells (Kitzmann et al., 1998). This type of regulation is likely to be more robust. Nevertheless, evolutionary processes might favor delay timers in early embryos, probably because gene regulatory networks in early animal embryos typically have a cascade-like structure (Davidson et al., 2002; Levine and Davidson, 2005; Imai et al., 2006). Although we do not have solid evidence that BZ1 and BZ2 are Blimp-1 orthologs, it should be noted that Blimp-1 is required for proper anterior neural tissues in a variety of animals including amphioxus, zebrafish and Xenopus (de Souza et al., 1999; Roy and Ng, 2004; Onai et al., 2010). In zebrafish, blimp-1 (prdm1a Zebrafish Information Network) is expressed at the boundary of the neural plate and non-neural ectoderm. Loss of blimp-1 activity inhibits specification of neural crest cells and the primary sensory neurons, whereas misexpression of blimp-1 generates supernumerary primary sensory neurons. It has been suggested that the ascidian palps represent a rudimentary placode and might be derived from an ancestral structure that evolved into separate anterior placodes in the vertebrate lineage (Manni et al., 2004; Mazet et al., 2005; Wagner and Levine, 2012). Addition of the BZ1/2 delay timer circuit to the gene regulatory network responsible for brain formation might represent a key event in the acquisition of the primitive palps/placodes in an ancestral animal, because this circuit makes room within the neural plate for the palps to be specified by a FoxC-dependent mechanism. MATERIALS AND METHODS Animals Ciona intestinalis adults were obtained from the National Bio-Resource Project of MEXT. An inhibitor of MEK signaling (U0126, Sigma) (Hudson et al., 2003) and human recombinant bfgf (Sigma) were used at 10 μm, and 100 ng/ml, respectively. Microarray screen We manually isolated individual blastomeres from 16-cell embryos, and obtained gene expression profiles at single-cell resolution with microarrays (Matsuoka et al., 2013). We injected synthetic mrnas for genes that were activated in specific cells at the 16-cell stage, and found that injection of BZ1 mrna severely disrupted morphology. We therefore examined the function of BZ1 in the present study. Data from the microarray screen will be available in Gene Expression Omnibus under accession number GSE Knockdown and overexpression studies We used the same MOs (Gene Tools) for Hes-a [E(spl)/Hairy-a] and ZicL that we used in a previous study (Imai et al., 2006). We designed two MOs for BZ1 (BZ1a: 5 -CAGAAGATCGTAATTACCTCGATGT-3 ; and BZ1b: 5 -CGTAACTTTCGCGGTGATTCCTCAT-3 ) and one MO for BZ2 (5 - GTCTGAACACACATGATTCCGACAT-3 ). Both combinations of BZ1a/BZ2 MOs and BZ1b/BZ2 MOs gave the same morphological phenotype and elicited precocious expression of ZicL in the neural lineage. Although we could not design additional MOs for BZ2 because of its short 5 -UTR, the BZ2 MO gave specific phenotypes only when it was injected with the BZ1a or BZ1b MO, strongly suggesting specificity of this BZ2 MO. We describe results obtained from injection of the BZ1a and BZ2 MOs. 4706

5 RESEARCH REPORT Fig. 4. Negative-feedback loops of BZ1 and BZ2 expression. (A-F) Expression of BZ1 (A,C,C,E) and BZ2 (B,D,D,F) at the early gastrula stage in normal embryos (A,B), BZ1 morphants (C-D ) and BZ2 morphants (E,F). Embryos in A-F are shown in lateral views (animal pole right). C and D show animal views of the embryos in C and D, respectively. Arrowheads in C-F indicate upregulated expression. (G) The amount of endogenous BZ1 and BZ2 mrnas is measured by qpcr in embryos injected with GFP (control), BZ1 and BZ2 mrnas. The relative amount of mrna in the experimental embryos compared with control embryos is shown. A maternal mrna, macho-1, is used as a control. Error bars indicate standard errors between two biological duplicates. Whole-mount in situ hybridization The detailed procedure for whole-mount in situ hybridization was described previously (Imai et al., 2004). To discriminate between BZ1 and BZ2 transcripts, we used 437-bp and 658-bp portions of the 5 -regions of cdnas for BZ1 and BZ2 cdnas. RT-qPCR For RT-qPCR, we extracted RNA from wild-type embryos and embryos injected with BZ1, BZ2 and Gfp mrnas. The RNA was reverse-transcribed with an oligo-dt primer. The obtained cdna samples were then analyzed by quantitative PCR with the SYBR green method. For each qpcr, the amount of cdna used was equivalent to two-thirds of an embryo. Primers used were: BZ1, 5 -GAATTGGACATGAGAATAAAACAACTACA-3 and 5 -TGCTTTGAAGATGCGCATAAA-3 ; BZ2, 5 -AAAAACACGTCAGAAGTAGCTCTCTAAA-3 and 5 -CCCAGGCACTTGAAAGTTAACAGT3 ; Macho-1, 5 -CCCAGTATGCACCAAATTCAGA-3 and 5 -TGGTGAGAAAACGGGTGAAAC-3. Acknowledgements We thank Reiko Yoshida and Chikako Imaizumi and all staff members of the Maizuru Fisheries Research Station of Kyoto University for collecting and cultivating Ciona intestinalis under the National Bio-Resource Project (NBRP) of MEXT, Japan. Competing interests The authors declare no competing financial interests. Author contributions T.I. designed and performed the experiments. T.M. performed the experiment to identify BZ1 and BZ2 genes. Y.S. designed and supervised the study, and wrote the paper. Funding This research was supported by Grants-in-Aid from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) and the Japan Society for the Promotion of Science (JSPS) [ to Y.S.]. Supplementary material Supplementary material available online at References Bertrand, V., Hudson, C., Caillol, D., Popovici, C. and Lemaire, P. (2003). Neural tissue in ascidian embryos is induced by FGF9/16/20, acting via a combination of maternal GATA and Ets transcription factors. Cell 115, Davidson, E. H., Rast, J. P., Oliveri, P., Ransick, A., Calestani, C., Yuh, C. H., Minokawa, T., Amore, G., Hinman, V., Arenas-Mena, C. et al. (2002). A genomic regulatory network for development. Science 295, de Souza, F. S., Gawantka, V., Gómez, A. P., Delius, H., Ang, S. L. and Niehrs, C. (1999). The zinc finger gene Xblimp1 controls anterior endomesodermal cell fate in Spemann s organizer. EMBO J. 18, Hashimoto, H., Enomoto, T., Kumano, G. and Nishida, H. (2011). The transcription factor FoxB mediates temporal loss of cellular competence for notochord induction in ascidian embryos. Development 138, Hudson, C. and Lemaire, P. (2001). Induction of anterior neural fates in the ascidian Ciona intestinalis. Mech. Dev. 100, Hudson, C., Darras, S., Caillol, D., Yasuo, H. and Lemaire, P. (2003). A conserved role for the MEK signalling pathway in neural tissue specification and posteriorisation in the invertebrate chordate, the ascidian Ciona intestinalis. Development 130, Hudson, C., Lotito, S. and Yasuo, H. (2007). Sequential and combinatorial inputs from Nodal, Delta2/Notch and FGF/MEK/ERK signalling pathways establish a gridlike organisation of distinct cell identities in the ascidian neural plate. Development 134, Imai, K. S., Satoh, N. and Satou, Y. (2002). Early embryonic expression of FGF4/6/9 gene and its role in the induction of mesenchyme and notochord in Ciona savignyi embryos. Development 129, Imai, K. S., Satoh, N. and Satou, Y. (2003). A Twist-like bhlh gene is a downstream factor of an endogenous FGF and determines mesenchymal fate in the ascidian embryos. Development 130, Imai, K. S., Hino, K., Yagi, K., Satoh, N. and Satou, Y. (2004). Gene expression profiles of transcription factors and signaling molecules in the ascidian embryo: towards a comprehensive understanding of gene networks. Development 131, Imai, K. S., Levine, M., Satoh, N. and Satou, Y. (2006). Regulatory blueprint for a chordate embryo. Science 312, Kitzmann, M., Carnac, G., Vandromme, M., Primig, M., Lamb, N. J. and Fernandez, A. (1998). The muscle regulatory factors MyoD and myf-5 undergo distinct cell cyclespecific expression in muscle cells. J. Cell Biol. 142, Kumano, G., Yamaguchi, S. and Nishida, H. (2006). Overlapping expression of FoxA and Zic confers responsiveness to FGF signaling to specify notochord in ascidian embryos. Dev. Biol. 300, Synthetic transcripts of BZ1, BZ2 and Gfp were prepared from cdna cloned into the pbluescript RN3 vector (Lemaire et al., 1995) using the mmessage mmachine T3 Kit (Ambion), and injected into fertilized eggs (1 mg/ml). To overexpress ZicL, BZ1 and BZ2 under the DMRT1 upstream sequence, we injected DNA constructs (20 μg/ml). We amplified the DMRT1 enhancer with the following primers: 5 -ATGGGTACCCTTCGCCGGAGAGTTAAAATC-3 and 5 -ATGGAGCTCCATGCCAGTTAAACGAACTGTTTG-3 (restriction sites used for cloning are underlined). The amplified region was slightly longer than that used in a previous report (Wagner and Levine, 2012). All knockdown and overexpression phenotypes were confirmed in at least two independent injections.

6 RESEARCH REPORT Lamy, C., Rothbächer, U., Caillol, D. and Lemaire, P. (2006). Ci-FoxA-a is the earliest zygotic determinant of the ascidian anterior ectoderm and directly activates Ci-sFRP1/5. Development 133, Lemaire, P., Garrett, N. and Gurdon, J. B. (1995). Expression cloning of Siamois, a Xenopus homeobox gene expressed in dorsal-vegetal cells of blastulae and able to induce a complete secondary axis. Cell 81, Levine, M. and Davidson, E. H. (2005). Gene regulatory networks for development. Proc. Natl. Acad. Sci. USA 102, Longabaugh, W. J. (2012). BioTapestry: a tool to visualize the dynamic properties of gene regulatory networks. Methods Mol. Biol. 786, Manni, L., Lane, N. J., Joly, J. S., Gasparini, F., Tiozzo, S., Caicci, F., Zaniolo, G. and Burighel, P. (2004). Neurogenic and non-neurogenic placodes in ascidians. J. Exp. Zool. 302B, Matsuoka, T., Ikeda, T., Fujimaki, K. and Satou, Y. (2013). Transcriptome dynamics in early embryos of the ascidian, Ciona intestinalis. Dev. Biol. (in press). Mazet, F., Hutt, J. A., Milloz, J., Millard, J., Graham, A. and Shimeld, S. M. (2005). Molecular evidence from Ciona intestinalis for the evolutionary origin of vertebrate sensory placodes. Dev. Biol. 282, Moret, F., Christiaen, L., Deyts, C., Blin, M., Vernier, P. and Joly, J. S. (2005). Regulatory gene expressions in the ascidian ventral sensory vesicle: evolutionary relationships with the vertebrate hypothalamus. Dev. Biol. 277, Nicol, D. and Meinertzhagen, I. A. (1988). Development of the central nervous system of the larva of the ascidian, Ciona intestinalis L. II. Neural plate morphogenesis and cell lineages during neurulation. Dev. Biol. 130, Nishida, H. and Satoh, N. (1983). Cell lineage analysis in ascidian embryos by intracellular injection of a tracer enzyme. I. Up to the eight-cell stage. Dev. Biol. 99, Onai, T., Yu, J. K., Blitz, I. L., Cho, K. W. and Holland, L. Z. (2010). Opposing Nodal/Vg1 and BMP signals mediate axial patterning in embryos of the basal chordate amphioxus. Dev. Biol. 344, Picco, V., Hudson, C. and Yasuo, H. (2007). Ephrin-Eph signalling drives the asymmetric division of notochord/neural precursors in Ciona embryos. Development 134, Roy, S. and Ng, T. (2004). Blimp-1 specifies neural crest and sensory neuron progenitors in the zebrafish embryo. Curr. Biol. 14, Shi, W. and Levine, M. (2008). Ephrin signaling establishes asymmetric cell fates in an endomesoderm lineage of the Ciona embryo. Development 135, Tresser, J., Chiba, S., Veeman, M., El-Nachef, D., Newman-Smith, E., Horie, T., Tsuda, M. and Smith, W. C. (2010). doublesex/mab3 related-1 (dmrt1) is essential for development of anterior neural plate derivatives in Ciona. Development 137, Wagner, E. and Levine, M. (2012). FGF signaling establishes the anterior border of the Ciona neural tube. Development 139, Yagi, K., Satou, Y. and Satoh, N. (2004). A zinc finger transcription factor, ZicL, is a direct activator of Brachyury in the notochord specification of Ciona intestinalis. Development 131,