Characterization of Brachyury-Downstream Notochord

Developmental Biology 224, 69–80 (2000) doi:10.1006/dbio.2000.9765, available online at http://www.idealibrary.com on

View metadata, citation and similar papers at core.ac.uk brought to you by CORE Characterization of Brachyury-Downstream provided by Elsevier - Publisher Connector Notochord Genes in the Ciona intestinalis Embryo

Kohji Hotta,1 Hiroki Takahashi,*,1 Tomomi Asakura, Banjo Saitoh, Naohito Takatori, Yutaka Satou, and Nori Satoh2 Department of Zoology, Graduate School of Science, Kyoto University, Sakyo-ku, Kyoto 606- 8502, Japan; and *PRESTO, Japan Science and Technology Corporation, Japan

The notochord has two major roles during chordate embryogenesis, as a source of inductive signals for the patterning of neural tube and paraxial mesoderm and as a supportive organ of the larval tail. Despite the recent identification of mutations that affect the notochord development in vertebrate embryos, little is known about genes that are expressed in the differentiating notochord itself. In the urochordate ascidian Ciona intestinalis, Brachyury (Ci-Bra) plays a key role in notochord differentiation. In a previous study, we isolated cDNA clones for nearly 40 potential Ci-Bra target genes that are expressed in notochord cells (H. Takahashi et al., 1999, Genes Dev. 13, 1519–1523). Here we characterized 20 of them by determining the complete nucleotide sequences of the cDNAs. These genes encode a broad spectrum of divergent proteins associated with notochord formation and function. Two genes encode ascidian homologs of the Drosophila Prickle LIM domain proteins and another encodes the ERM protein, all 3 of which appear to be involved in the control of cytoskeletal architecture. In addition, genes for netrin, leprecan, cdc45, ATP:citrate lyase, ATP sulfurylase/APS kinase, protein tyrosine phosphatase, ␤4-galactosyltransferase, fibrinogen-like protein, divergent tropomyosin-like proteins, and Drosophila Pellino-like protein were identified. The observation of the netrin gene expression in the notochord may provide the first molecular evidence that the ascidian notochord is a source of signals as in vertebrates. In addition, the present information should be used to identify nonchordate deuterostome tissues homologous to the notochord as well as genes which are expressed in the notochord cells of vertebrate embryos. © 2000 Academic Press Key Words: ascidians; notochord; Brachyury, downstream genes; cell shape changes; netrin; leprecan; Prickle LIM domain protein; cdc45.

INTRODUCTION fication of chordamesoderm, (2) early stages of notochord differentiation, and (3) later stages of notochord differentia- The notochord has two major roles during chordate tion. In the zebrafish no tail mutant, for example, noto- embryogenesis; the first is as a source of inductive signals chord precursors exist but fail to differentiate into vacu- for the patterning of neural tube and paraxial mesoderm and olated notochord cells (Halpern et al., 1993). However, as the second is as a supportive organ of the larval tail. A pointed out by Cunliffe and Ingham (1999), the rather large-scale genetic screen in zebrafish has led to the identi- modest progress in isolating genes expressed in the noto- fication of a number of genetic loci affecting the formation chord during its differentiation has limited our understand- of the notochord (Odenthal et al., 1996; Stemple et al., ing of the molecular mechanisms underlying its structure 1996). Phenotypic analysis of the mutant embryos has and function. enabled the ordering of these genetic loci into a pathway The Brachyury gene encodes a sequence-specific activa- controlling notochord formation, comprising (1) the speci- tor that contains a T DNA-binding domain (Herrmann et al., 1990; Kispert and Herrmann, 1993; reviewed by Herr- mann and Kispert, 1994; Smith, 1999). Vertebrate 1 These authors contributed equally to this work. 2 To whom correspondence should be addressed at the Depart- Brachyury is initially expressed in the presumptive meso- ment of Zoology, Graduate School of Science, Kyoto University, derm, and during later stages the expression is gradually Sakyo-ku, Kyoto 606-8502, Japan. Fax: 81-75-705-1113. E-mail: restricted to the developing notochord and tailbud (Wilkin- [email protected]. son et al., 1990; Smith et al., 1991; Schulte-Merker et al.,

1992). This gene is critical for the posterior mesoderm screened. More than 10 candidate cDNA clones for each gene were formation and notochord differentiation in vertebrate em- subcloned, and the longest was used as a template for sequencing. bryos (reviewed by Herrmann and Kispert, 1994; Papaioan- Sequence determination and search. Nucleotide sequences nou and Silver, 1998; Smith, 1999). Ascidians (urochor- were determined for both strands with a Big-Dye Terminator Cycle dates) are one of the three chordate groups and the ascidian Sequencing Ready Reaction kit and ABI PRISM 377 DNA se- quencer (Perkin–Elmer). Each of the nucleotide sequences was used tadpole is thought to represent the most simplified and as query sequence for BLASTX programs against peptide sequence primitive chordate body plan (reviewed by Satoh, 1994; databases (nonredundant GenBank CDS translations, PIR, and Satoh and Jeffery, 1995; Di Gregorio and Levine, 1998). It PRF). contains a notochord composed of just 40 cells, of which In situ hybridization. RNA probes were prepared with the DIG the lineage has been completely described. In ascidians, it RNA labeling kit (Boehringer Mannheim). Whole-mount in situ has been found that the Brachyury genes Hr-Bra (former hybridization was performed using digoxigenin-labeled antisense As-T)ofHalocynthia roretzi (Yasuo and Satoh, 1993) and probes as described previously (Hotta et al., 1999). Control embryos Ci-Bra of Ciona intestinalis (Corbo et al., 1997) are ex- hybridized with sense probes did not show signals above back- pressed exclusively in the notochord precursor cells and ground. that the timing of the gene expression coincides with the clonal restriction of the notochord lineages at the 64-cell stage (cf. Fig. 1A). In addition, misexpression of Hr-Bra RESULTS (Yasuo and Satoh, 1998) or Ci-Bra (Takahashi et al., 1999) causes transformation of endodermal and neuronal lineages We determined the complete nucleotide sequences of into notochord cells, suggesting that the ascidian cDNAs for 20 genes downstream of Ci-Bra which are Brachyury is also critical in the specification and subse- expressed specifically or predominantly in notochord cells quent differentiation of the notochord cells. of C. intestinalis embryos. The sequence information is In a previous study, we attempted to isolate potential summarized in Table 1, and the expression pattern of each Ci-Bra target genes (Takahashi et al., 1999). Taking advan- of the genes is shown in Fig. 1. The following are descrip- tage of the fork head gene promoter of Ciona, which allows tions of predicted features of the genes. Ci-Bra misexpression in endoderm cells, and electropora- tion, which allows large quantities of Ci-Bra-misexpressing embryos to be obtained, we were able to isolate cDNA Ci-pk1 clones for 501 independent genes that were activated by Ci-Bra mis- and/or overexpression. Of them, nearly 40 The gene represented by 403e cDNA begins to be ex- genes are specifically or predominantly expressed in noto- pressed as early as the gastrula stage, about 2 h behind chord cells (Takahashi et al., 1999). Although the previous Ci-Bra expression at the 64-cell stage. The transcript is EST analysis of both 5Ј and 3Ј ends of the cDNAs suggested detected only in notochord cells up to the neural plate some features of these genes (Takahashi et al., 1999; Hotta stage, but later its expression is also evident in the posterior et al., 1999), further insight into the genes would be part of the central nervous system (CNS) (Fig. 1B). The achieved by determining the complete nucleotide se- cDNA consists of 3922 nucleotides that predict a polypep- quences of the cDNAs. Here, we show that 20 of the tide of 1066 amino acids (Table 1). A database search putative Ci-Bra target genes encode components for regu- showed that the 403e protein contains three LIM domains, lation of the cytoskeletal architecture, the extracellular which are cysteine-rich sequences with a double zinc finger matrix, enzymes, and proteins implicated in signal trans- motif (Dawid et al., 1998) (Fig. 2). However, the 403e duction, signals for axon guidance, and cell cycle control. protein contains no recognized DNA-binding, membrane- These genes are supposed to be responsible for the changes spanning, or signal peptide motifs, but instead contains a in cell shape and adhesion during the intercalation and PET (Prickle Espinas Testin) domain that has recently been extension of the notochord. identified within the prickle (pk) family proteins of Dro- sophila melanogaster by Gubb et al. (1999) (EMBL Acces- sion No. AJ243708; Fig. 2). Therefore, it is highly likely that MATERIALS AND METHODS this gene is an ascidian Prickle LIM domain protein gene, and we therefore named it Ci-pk1 (C. intestinalis prickle 1). Cloning of full-length cDNAs for Ci-Bra downstream noto- prickle (pk) is one of the “tissue polarity” genes of D. chord genes. Isolation of Ci-Bra downstream genes which are melanogaster, which control the orientation of bristles and expressed in the notochord has been reported previously (Taka- hairs in the adult cuticle (Adler, 1992). The tissue polarity hashi et al., 1999). None of the cDNAs isolated, however, con- gene products appear to be involved in the control of tained the complete 5Ј regions encoding the N-terminal portion of the gene products. To obtain nearly full-length cDNA with a cytoskeletal architecture, for example, localization of the complete open reading frame, the 5Ј-lacking cDNA was labeled as actin fibers. This suggests that Ci-pk1 is also involved in probe with [32P]dCTP using random priming and Klenow fragment. the cytoskeletal architecture associated with cell shape Approximately 1 ϫ 105 plaques of the cDNA library of Ci-Bra- changes in the process of intercalation and enlargement of overexpressing embryos made in Uni-ZAP XR (Stratagene) were the notochord cells.

TABLE 1 Characterization of cDNAs for Brachyury-Downstream Notochord Genes in the Ascidian Ciona intestinalis cDNA Accession Length of Predicted polypeptide clones Gene No. insert (bp) (No. of aa) Features Fig. 1

403e Ci-pk1 AB036840 3922 1066 Triple LIM domain, prickle (pk) of Drosophila B 407e Ci-pk2 AB036841 3871 1011 Triple LIM domain, prickle (pk) of Drosophila C 507h Ci-Noto1 AB036842 1839 446 No similarity D 006c Ci-Noto2 AB036843 1770 412 Antithrombin III-like protein E 706e Ci-netrina AB033331 2774a 650a Netrina F 903g Ci-Noto3 AB036845 1665 341 ␤3Gal-transferase-like protein G 107g Ci-tropb AB036846 1181 242 Tropomyosin-like protein H 110g Ci-ACL AB036847 4038 1101 ATP citrate-lyase I 406g Ci-cdc45 AB036848 2275 567 Cdc45 J 408h Ci-ﬁbrn AB036849 2130 652 Prothrombinase precursor (ﬁbrinogen-like protein) K 210d Ci-Noto4 AB036850 1472 307 No similarity L 212g Ci-pellino AB036851 2078 455 Pellino of Drosophila M 505a Ci-ASAK AB036852 2378 618 ATP sulfurylase/APS kinase N 402c Ci-Noto5 AB036853 1665 381 Tropomyosin-2-like protein 409d Ci-leprecan AB036854 1727 412 Leprecan O 507d Ci-Noto6 AB036855 1837 473 KIAA0990-like protein 002d Ci-ERMc AB036839 2598 609 ERM (ezrin/radixin/moesin) P 608g Ci-PTP AB036856 4542 987 Protein tyrosine phosphatase 603d Ci-Noto7 AB036857 2467 213 Mad4-like protein 309g Ci-␤4Gal-T AB036858 2796 413 ␤4Gal-transferase Q

a Analysis of the Ci-netrin cDNA was independently carried out by Takamura et al. (2000). b Di Gregorio and Levine (1999; Accession No. AAF04791). c This gene was also characterized by A. Erives and M. Levine (personal communication).

Ci-pk2 single gene, like prickle and sple, and that Ci-pk1 and Ci-pk2 are involved in a concerted fashion in the notochord The transcript of the gene represented by 407e cDNA cell shape changes. becomes evident at the neural plate stage and is restricted to notochord cells (Fig. 1C). The cDNA consists of 3871 nucleotides that predict a polypeptide of 1011 amino acids Ci-Noto1 (Table 1). Comparison of the nucleotide sequence of 407e The gene of 507h cDNA is first expressed at the gastrula with that of 403e (Ci-pk1) indicates that they share almost stage, and its expression is specific to the notochord cells Ј Ј identical 5 UTR sequences as well as 5 halves of their ORF (Fig. 1D). The cDNA consists of 1839 nucleotides that Ј sequences, although they have different 3 UTR sequences. encode a polypeptide of 446 amino acids (Table 1). The 507h The first 468 amino acid residues are identical between protein contains no specific domains or motifs, and a 407e and 403e (Fig. 2), while in the C-terminal region, the database search failed to find any sequence similarity be- two proteins differ at several amino acid residues (overall tween it and known proteins. We therefore named this gene identity between them was 97%). The 407e protein con- Ci-Noto1 (C. intestinalis notochord gene 1). tains a PET domain and three LIM domains identical with those of Ci-pk1 (Fig. 2), and therefore we named this gene Ci-pk2. Ci-pk2 is also likely to be involved in the cytoskel- Ci-Noto2 etal architecture associated with cell shape changes of the The Ci-Noto2 gene (006c cDNA) is first expressed at the notochord cells. gastrula stage. The transcript first appears in cells of the In Drosophila, it has been shown that prickle and spiny- lineages of notochord, nerve chord, and mesenchyme. Ex- legs (sple) genes have alternative 5Ј exons but share 3Ј pression in the nerve cord and mesenchyme is downregu- common exons and that the balance between isoforms (Pk lated by the initial tailbud stage, and at the early tailbud and Sple) of the two Prickle LIM domain proteins is critical stage, the transcript is found in cells of the notochord and for planar polarity in the imaginal discs (Gubb et al., 1999). part of the CNS (Fig. 1E). The 006c cDNA consists of 1770 The timing of initiation of the gene expression, patterns of nucleotides that predict a polypeptide of 412 amino acids transcript distribution, and sequences suggest that Ci-pk1 (Table 1). A database search suggested that the Ci-Noto2 and Ci-pk2 are independent genes, but it is possible that the protein has a weak similarity to the antithrombin III two transcripts are derived from alternative splicing of a precursor of mouse (Accession No. 416621) and of fugu fish

FIG. 1. Expression of Ci-Bra (A) and its downstream notochord genes in C. intestinalis tailbud embryos revealed by whole-mount in situ hybridization. Abbreviations on top of each column are 64, 64-cell stage; 110, 110-cell stage; G, gastrula stage; NP, neural plate stage; N, neurula stage; iT, initial tailbud stage; and mT, midtailbud stage. (B) Ci-pk1 (403e) gene. (C) Ci-pk2 (407e) gene. (D) Ci-Noto1 (507h) gene. (E) Ci-Noto2 (006c) gene. (F) Ci-netrin (706e) gene. (G) Ci-Noto3 (903g) gene. (H) Ci-trop (107g) gene. (I) Ci-ACL (110g) gene. (J) Ci-cdc45 (406g) gene. (K) Ci-ﬁbrn (408h) gene. (L) Ci-Noto4 (210d) gene. (M) Ci-pellino (212g) gene. (N) Ci-ASAK (505a) gene. (O) Ci-leprecan (409d) gene. (P) Ci-ERM (002d) gene. (Q) Ci-␤4Gal-T (309g) gene. N, notochord; CNS, central nervous system. Tr, trunk; Tl, tail of the embryo. Scale bar, 50 ␮m. Ci-Bra Downstream Notochord Genes 73

FIG. 2. Alignment of amino acid sequences of the PET and LIM domains of Ci-pk1 and Ci-pk2 with those of the pk family members. Sequence accession numbers are human LMO6 (EMBL AJ011654), Drosophila pk (EMBL AJ243708), mouse testin (Swiss Prot P47226), and Caenorhabditis elegans f25h5.1 (EMBL z81068).

(Accession No. BAA77461). In contrast to the high degree of spinal cord and plays an important role in axon guidance amino acid identity between the proteins of mouse and fugu during neurogenesis (e.g., Serafini et al., 1996). The obser- (about 60% over the entire sequences), the amino acid vation of the netrin gene expression in the notochord may identity between Ci-Noto2 and the vertebrate proteins was provide the first molecular evidence that the ascidian not so high (about 28% over the entire sequences). notochord is a source of signals, as in vertebrates. The Ci-netrin gene, however, is expressed predominantly in the notochord and in a part of the CNS, suggesting that the gene Ci-netrin has an additional function that is not found in the verte- The 706e gene is first expressed at the gastrula stage. The brate netrin. transcript is first detectable in A-line blastomeres including the notochord cells and anterior neural tube cells. This Ci-Noto3 expression pattern continues up to the tailbud stage; cells of the notochord and cells of the ventral region of the prospec- The Ci-Noto3 gene (903g cDNA) is first expressed at the tive sensory vesicles show distinct hybridization signals gastrula stage. The transcript appears in cells of the lineages (Fig. 1F). Determination of a part of the 706e cDNA se- of notochord, CNS, and mesenchyme. Expression in the quence resulted in a match with the sequence of the Ciona mesenchyme is downregulated by the initial tailbud stage, homolog of netrin (Ci-netrin), which has already been and at the early tailbud stage the transcript is found in cells characterized by Takamura et al. (2000). Therefore, the of the notochord and in cells of the ventral part of the CNS 706e gene is an ascidian homolog of netrin. According to (Fig. 1G). The 903g cDNA consists of 1665 nucleotides that Takamura et al. (2000), Ciona netrin consists of 650 amino predict a polypeptide of 341 amino acids (Table 1). A acids, having a well conserved laminin N-terminal domain, database search suggested that the Ci-Noto3 protein has a three laminin-like EGF repeat domains, and a netrin weak similarity to human ␤3-galactosyltransferase (Acces- C-terminal domain. sion No. 5174397), with about 20% identity over the entire In vertebrates, netrin is expressed in the floor plate of the sequences.

FIG. 3. Alignment of amino acid sequences of the Ci-ACL with those of human ATP: citrate lyase. Identical residues are shaded.

Ci-trop polypeptide of 1101 amino acids (Table 1). As shown in Fig. 3, the deduced amino acid sequence shows a high degree of The 107g gene is first expressed at the neural plate stage, identity with human ATP: citrate lyase (Accession No. and the expression is specific to the notochord cells (Fig. AAB60340), with 88% overall identity. Therefore, we con- 1H). The cDNA consists of 1181 nucleotides that predict a clude that the 110g gene is an ascidian gene for ATP: citrate polypeptide of 242 amino acids (Table 1). A database search lyase (Ci-ACL). ATP:citrate lyase is the primary enzyme showed that the 107g gene encodes a tropomyosin-like responsible for the synthesis of cytosolic acetyl-CoA in protein which is a novel member of the tropomyosin many tissues of mammals. superfamily and is most closely related to fibroblast tropo- myosins identified in rats and humans (30% overall identity). The 107g sequence has already been characterized as Ci-cdc45 the Ci-trop gene (Accession No. AAF04791) by Di Gregorio The 406g gene is expressed from the neural plate stage and Levine (1999); they showed that the expression of onward, and the expression is specific to notochord cells Ci-trop is directly regulated by Ci-Bra (Di Gregorio and (Fig. 1J). The 406g cDNA consists of 2275 nucleotides that Levine, 1999). There are, however, three amino acid residue encode a polypeptide of 567 amino acids (Table 1). As differences between the 107g and the AAF04791 proteins. shown in Fig. 4, the sequence has similarity to that of Cdc45 protein of Xenopus (Accession No. AAC67520) (about 44% overall identity) and of humans (Accession No. Ci-ACL 4502713) (about 43% overall identity). The 110g gene is first expressed at the neural plate stage, In the budding yeast Saccharomyces cerevisiae, cdc45 is and its expression is restricted to notochord cells (Fig. 1I). an essential gene required for initiation of DNA replication The cDNA consists of 4038 nucleotides that predict a (Zou et al., 1997). It seems interesting that this kind of

FIG. 4. Alignment of amino acid sequences of the Ci-cdc45 with those of Xenopus Cdc45. Identical residues are shaded. elementary gene related to cell-cycle control is expressed chord cells (Fig. 1L). The 210d cDNA consists of 1472 specifically in the ascidian notochord cells as one of the nucleotides that predict a polypeptide of 307 amino acids Ci-Bra downstream genes. (Table 1). The Ci-Noto4 protein shows very weak similarity to myb-related protein PP2 of the moss Physcomitrella patens (Accession No. 462669). Ci-fibrn The 408h gene begins to be expressed at the neural plate Ci-pellino stage and the expression is specific to notochord cells (Fig. 1K). The 408h cDNA consists of 2130 nucleotides that The transcript of the 212g gene first appears at the neural encode a polypeptide of 652 amino acids (Table 1). The plate stage, and its occurrence is restricted to the notochord deduced amino acid sequence shows about 48% identity to cells (Fig. 1M). The 212g cDNA consists of 2078 nucleotides that of mouse prothrombinase precursor (fibrinogen-like that encode a polypeptide of 455 amino acids (Table 1). As protein) (Accession No. P12804), and therefore we tenta- shown in Fig. 5, the sequence shows similarity to that of tively named this gene Ci-fibrn. Pellino of D. melanogaster (about 50% overall identity) (Accession No. AAC96298). In determination of the dorsoventral polarity of Drosoph- Ci-Noto4 ila, maternally encoded proteins Toll (a transmembrane The Ci-Noto4 (210d) gene begins to be expressed at the receptor), Pelle (a Ser/Thr protein kinase), and Tube func- neural plate stage, and the expression is specific to noto- tion intracytoplasmically to initiate the cascade that ulti-

FIG. 5. Alignment of amino acid sequences of the Ci-pellino with those of Drosophila Pellino. Identical residues are shaded.

FIG. 6. Alignment of amino acid sequences of the Ci-ASAK with those of mouse ATP sulfurylase. Identical residues are shaded.

mately activates Dorsal (Morisato and Anderson, 1995). Ci-leprecan Activation of the kinase Pelle by Tube is critical in the The 409d gene expression begins at the neural stage. Its signal transduction pathway of this process. Pellino was expression is specific to the notochord (Fig. 1O). The 409d isolated and characterized as a novel protein which associ- cDNA consists of 1727 nucleotides that predict a polypep- ates with the kinase domain of Pelle, but Pellino has also tide of 412 amino acids (Table 1). The sequence has a high been shown to be an evolutionarily conserved protein similarity to that of leprecan, a novel basement membrane- (Großhans et al., 1999). associated proteoglycan (Accession No. AAD51875). This gene was therefore named Ci-leprecan. Ci-ASAK Ci-Noto6 The 505a gene is first expressed at the neural plate stage, and the expression is specific to notochord cells (Fig. 1N). The Ci-Noto6 (507d cDNA) gene is first expressed at the The 505a cDNA consists of 2378 nucleotides that predict a neurula stage, and its expression is relatively specific to polypeptide of 618 amino acids (Table 1). The deduced notochord cells (data not shown). The 507d cDNA consists amino acid sequence shows a high degree of identity (67%) of 1837 nucleotides that predict a polypeptide of 473 amino with that of mouse ATP sulfurylase/APS kinase (Fig. 6; acids (Table 1). The Ci-Noto6 amino acid sequence shows a Accession No. AAC52328); the 505a gene may be an ascid- weak similarity to that of KIAA0990 protein, which is of ian gene for ATP sulfurylase/APS kinase. Biosynthesis of unknown function and was isolated by a human brain the activated sulfate donor, adenosine 3Ј-phosphate 5Ј- cDNA project (Accession No. BAA76834). phosphosulfate, involves the sequential action of two enzyme activities: ATP sulfurylase, which catalyzes the for- Ci-ERM mation of 5Ј-phosphosulfate (APS) from ATP and free sulfate, and APS kinase, which subsequently phosphory- The 002d gene is first expressed at the neurula stage, and lates APS to produce adenosine 3Ј-phosphate. its expression is detected in notochord cells and cells of the CNS, including the nerve cord (Fig. 1P). The 002d cDNA consists of 2598 nucleotides that predict a polypeptide of Ci-Noto5 609 amino acids (Table 1). The 002d gene is likely to encode an ascidian member of an ezrin/radixin/moesin (ERM)-like The Ci-Noto5 (402c cDNA) expression begins at the protein (60% identity to human radixin, Accession No. neural stage (data not shown). Its expression is specific to 4506467), and therefore the gene was named Ci-ERM. ERM the notochord. The 402c cDNA consists of 1665 nucleo- has been implicated in mediating changes in the cytoskel- tides that predict a polypeptide of 381 amino acids (Table 1). eton in response to signaling at the cell surface (Tsukita and The sequence has a weak similarity to that of yeast tropo- Yonemura, 1997). During ascidian embryogenesis, noto- myosin 2 (Accession No. P40414) (about 25% deduced chord cells intercalate to form an organ composed of a amino acid identity). single row of cells. Initially, each cell is columnar in shape,

FIG. 7. Alignment of amino acid sequences of the Ci-PTP with those of human PTP. Identical residues are shaded.

but gradually becomes cuboidal. This transition in shape is the human Mad4 homolog (Accession No. 5453734) (44% directly responsible for axial extension and tail morphogen- deduced amino acid identity). Mouse Mad3 and Mad4 were esis. Ci-ERM is likely to be involved in these processes. characterized as novel Max-interacting transcriptional repressors that suppress c-myc-dependent transformation and are expressed during neural and epidermal differentiation Ci-PTP (Hurlin et al., 1995). They are basic helix-loop-helix-leucine The 608g gene expression commences at the neurula zipper proteins, and it has been suggested that the regulated stage, and the expression is detected not only in notochord expression of Myc and Mad family proteins in a concerted cells but also in cells of the CNS and mesenchyme cells fashion regulates cell proliferation in differentiating tissues. (data not shown). The cDNA consists of 4542 nucleotides Because the notochord cells cease to divide after the neu- that encode a polypeptide of 987 amino acids (Table 1). As rula stage, Ci-Noto7 is likely to be involved in this prolif- shown in Fig. 7, the sequence has fairly high similarity to eration process. mouse testis-enriched protein tyrosine phosphatase (Acces- sion No. AF106702) (55% deduced amino acid identity) and was therefore named Ci-PTP. Ci-ß4Gal-T

The 309g transcript is ﬁrst evident in notochord cells as Ci-Noto7 well as a few neuronal cells in the early tailbud embryo (Fig. The Ci-Noto7 (603d cDNA) gene is ﬁrst expressed at the 1Q). The cDNA consists of 2796 nucleotides that predict a neural plate stage, predominantly in notochord cells and polypeptide of 413 amino acids (Table 1). The 309g protein weakly in the epidermis cells of the tail region as well as a shows fairly high sequence similarity to human ␤4- few cells of the CNS (data not shown). The 603d cDNA galactosyltransferase (Accession No. 4502349). Therefore, consists of 2467 nucleotides that predict a polypeptide of this gene (Ci-␤4Gal-T) may be an ascidian homolog of 213 amino acids (Table 1). The sequence has similarity to ␤4-galactosyltransferase.

arrangement, shape changes, and alteration of the extracellular environment occur as the notochord is transformed from a loose plate or mass of cells during neurulation and initial tailbud formation into an elongated rod surrounded by a sheath of ﬁbrous materials during larval formation (Miyamoto and Crowther, 1985). The basal surfaces of the notochord cells rest on a basal lamina termed the notochordal sheath that completely surrounds the notochord (e.g., Cloney, 1969). The notochordal sheath contains many circumferentially oriented and longitudinally oriented ﬁla- ments. Most of the genes characterized here appear to be associated with the formation and function of the notochord cells.

Significance of the Present Characterization of Notochord Genes in the Ascidian Embryo The evolution of chordate body plans is one of the major research subjects of the field of evolutionary developmental biology (Satoh and Jeffery, 1995; Gee, 1996; Di Gregorio and FIG. 8. Diagrammatic representation of expression patterns of Levine, 1998). The notochord is a defining characteristic of Ci-Bra-downstream notochord genes in relation to their inferred the chordate embryo. It is a dorsally located rod of tensile functions. See the text for details. CNS, central nervous system; ES, mesodermal tissue that lies immediately beneath the neu- endodermal strand; Mu, muscle; Noto, notochord; NS, notochordal ral tube. The notochord functions as a skeletal element sheath; Nu, nucleus; Va, intracellular vacuole. during early chordate embryogenesis. Each notochord cell produces a large central vacuole, which displaces the nucleus to the periphery of the cell. These characteristic DISCUSSION features are common in all of the chordate groups, although the amphioxus notochord cells are unique in developing myofilaments in the cytoplasm (e.g., Flood, 1975; Suzuki Features of Brachyury-Downstream Notochord and Satoh, 2000). In addition, in vertebrates, the notochord Genes in the Ascidian Embryo functions as a source of signals that pattern the neural tube The 20 notochord genes of the ascidian embryo charac- and paraxial mesoderm. Cunliffe and Ingham (1999) have terized in the present study are categorized into several pointed out that, despite the recent molecular identifica- groups depending on their inferred functions (Fig. 8). The tion of mutations affecting notochord development in mice first group of genes is involved in the control of cytoskeletal (Herrmann et al., 1990) and in zebrafish (Odenthal et al., architecture and therefore responsible for cell shape 1996; Stemple et al., 1996), the rather modest progress in changes. This group includes Ci-pk1, Ci-pk2, and Ci-ERM. isolating genes expressed in the notochord during its differ- Ci-trop and Ci-Noto5, encoding tropomyosin-like proteins, entiation has limited our understanding of the molecular are also likely to be associated with this kind of function. mechanisms underpinning its structure and function. The The second group of genes encodes components of the present characterization of Ci-Bra-downstream notochord extracellular matrix, involved in the notochordal sheath genes addresses this problem and will therefore provide formation. This group includes Ci-leprecan, Ci-fibrn, and clues for isolating and characterizing genes expressed in the Ci-Noto2, which encodes an antithrombin III-like protein. notochord cells of vertebrate embryos. Ci-␤4Gal-T and Ci-Noto3 may belong to this group. The The present characterization of the notochord genes of third group of genes encodes proteins with enzymatic urochordate ascidians also provides information key to activity. This group includes Ci-ACL, Ci-ASAK, and Ci- examining evolutionary links between the notochord and PTP. Ci-cdc45 is essential for cell cycle control, whereas the nonchordate deuterostome tissues and/or organs sus- Ci-netrin and Ci-pellino may be associated with signals for pected to be homologous to the notochord. For example, in axon guidance and cell signaling pathways, respectively. hemichordate acorn worms, the stomochord is formed as an Ci-Noto7 encodes a protein with a Mad4-like sequence. In outgrowth of the dorsal wall of the foregut, as in the case of addition, Ci-Noto1 and Ci-Noto4 encode proteins with no the amphioxus notochord. The evolutionary link between similarity to other known proteins, and Ci-Noto6 encodes a the notochord and the stomochord has been debated for a protein related to a human protein with unknown function. long time (Bateson, 1885). Although the Brachyury gene of Studies of the formation of the notochord in living C. the acorn worm Ptychodera flava is not expressed in the intestinalis embryos with time-lapse video photography stomochord (Peterson et al., 1999), comparison of genes demonstrate that cell proliferation, growth, migration, re- that are expressed in the stomochord and those character-

Copyright © 2000 by Academic Press. All rights of reproduction in any form reserved. Ci-Bra Downstream Notochord Genes 79 ized in the present study will lead to better understanding stream of both VegT (another T-box gene of Xenopus) and of the genetic circuitry underlying notochord formation Xbra to induce formation of mesoderm and endoderm. Xbra during chordate evolution, because our long-range goal is to also directly regulates expression of eFGF by binding to an determine the gene circuits underlying notochord forma- element 5Ј upstream of the eFGF transcription start site tion in ascidians and use this information to explore the (Casey et al., 1998). In zebrafish, no tail (Brachyury) acts evolutionary origins of the notochord among lower deuter- with other T-box genes (Griffin et al., 1998). In this animal, ostomes, including echinoderms and hemichordates. FGF regulates no tail. Tail and notochord development is dependent upon no tail, which regulates another T-box gene, Tbx6. These results indicate that, in vertebrates, Target Genes of Brachyury Brachyury first regulates another transcriptional factor One important question to be answered is whether Ci- gene(s), which is likely to lead to the eventual activation of Bra-downstream notochord genes characterized here are genes responsible for notochord formation and function. direct targets of Ci-Bra or Ci-Bra regulates another tran- Therefore, the vertebrate pathway in which Brachyury scriptional factor gene which in turn activates these noto- activates and/or acts with other transcriptional factor genes chord genes. The original subtractive library was made from may have been an innovation during the evolution of Ci-Bra-overexpressing embryos at the neurula and initial vertebrates. tailbud stages (Takahashi et al., 1999). Therefore, it is possible that our library lacks genes that are immediate and direct targets of Ci-Bra. The analysis of staged embryos ACKNOWLEDGMENTS suggests that the genes exhibit different temporal patterns of expression (Hotta et al., 1999; the present study). Tran- We thank Dr. Mike Levine for his interest and comments on this research. We also thank Kazuko Hirayama for technical help. This scripts of Ci-pk1, Ci-Noto1, Ci-Noto2, Ci-netrin, and Ci- research was supported by a grant from the Human Frontier Noto3 are detectable as early as the gastrula stage, a few Science Program (RG212/1997) and by a Grant-in-Aid for Specially hours after the initiation of Ci-Bra expression. Expression Promoted Research (No. 07102012) from the Monbusho, Japan, to of Ci-pk2, Ci-trop, Ci-ACL, Ci-cdc45, Ci-fibrn, Ci-Noto4, N.S. Ci-pellino, Ci-ASAK, and Ci-Noto7 is evident at the neural plate stage, while Ci-Noto5, Ci-leprecan, Ci-Noto6, Ci- ERM, and Ci-PTP begin to be expressed at the neurula stage, REFERENCES and Ci-␤4Gal-T transcript is first detected at the tailbud stage. 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