Ascidian embryos as a model system to analyze expression and function of developmental genes

C Blackwell Wissenschafts-Verlag 2001

Differentiation (2001) 68:1–12

REVIEW ARTICLE

Nori Satoh

Ascidian embryos as a model system to analyze expression and function of developmental genes

Accepted in revised form: 1 May 2001

Abstract Ascidians have served as an appropriate experimental system in developmental biology for more than a century. The fertilized egg develops quickly into a tadpole larva, which consists of a small number of organs including epidermis, central nervous system with two sensory organs, endoderm and mesenchyme in the trunk, and notochord and muscle in the tail. This configuration of the ascidian tadpole is thought to represent the most simplified and primitive chordate body plan. Their embryogenesis is simple, and lineage of embryonic cells is well documented. The ascidian genome contains a basic set of genes with less redundancy compared to the vertebrate genome. Cloning and characterization of developmental genes indicate that each gene is expressed under discrete spatio-temporal pattern within their lineage. In addition, the use of various molecular techniques in the ascidian embryo system highlights its advantages as a future experimental system to explore the molecular mechanisms underlying the expression and function of developmental genes as well as genetic circuitry responsible for the establishment of the basic chordate body plan. This review is aimed to highlight the recent advances in ascidian embryology. Key words ascidian embryos ¡ primitive chordate body plan ¡ lineage ¡ developmental genes function ¡ largescale gene expression profiles ¡ mutagenesis screen

What are ascidians? Ascidians are commonly called ‘sea squirts’. They are marine invertebrate animals ubiquitous throughout the world and are sessile on decks or rocks in the sea (Fig. 1A). They possess incurrent and outcurrent siphons and are specialized for filter feeding (Brusca and Brusca, 1990). Ascidians were originally thought to be molluscs. However, the description by Kowalevsky (1866) of the occurrence of ascidian tadpole-type larvae with a notochord and nerve cord indicated that this animal group belongs to a taxonomic position close to vertebrates. Namely, we human beings are vertebrates, and, based on recent classification of animals, vertebrates, together with urochordates (ascidians) and cephalochordates (amphioxus), are a group of the phylum Chordata. These three groups share a notochord, dorsal hollow neural tube, and gill slits in larvae or in adults, and they evolved from a common ancestor more than 500 MYR ago (Schaeffer, 1987; Wada and Satoh, 1994; Cameron et al., 2000). Therefore, ascidians are our distant cousins (Gee, 1996).

Why do we use ascidian embryos in study of developmental mechanisms? There are several reasons why we choose ascidians as an experimental system in embryology (Satoh, 1994, 1999). The main reasons are stated below.

Ascidian tadpole larvae represent the most simplified and primitive chordate body plan N. Satoh Department of Zoology, Graduate School of Science, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan e-mail: satoh/ascidian.zool.kyoto-u.ac.jp Tel: π 81 75 753 4081, Fax: π 81 75 705 1113 U. S. Copyright Clearance Center Code Statement:

Figure 1B shows an ascidian tadpole larva, which consists of only approximately 2,600 cells. The tadpole is organized into a trunk and tail (see also Fig. 2K–M). The trunk contains a dorsal central nervous system

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Fig. 1 A cosmopolitan ascidian, Ciona intestinalis. A Adults are hermaphroditic and they have a white sperm duct (arrow) and pinkish oviduct (arrowhead). B Larva developed from dechor-

ionated egg. End, endoderm; Epi, epidermis; N, notochord; Oc, ocellus, Ot, otolith; Tr, trunk; Tl, tail.

(CNS) with two sensory organs (otolith and ocellus), endoderm, mesenchyme, trunk lateral cells (TLCs), and trunk ventral cells (TVCs). The tail contains a notochord flanked dorsally by the nerve cord, ventrally by the endodermal strand, and bilaterally by three rows of muscle cells (Fig. 2M). The entire surface of the larva is covered by an epidermis. This configuration of the ascidian tadpole is thought to represent the most simplified and primitive chordate body plan (Satoh, 1994, 1999; Satoh and Jeffery, 1995; Di Gregorio and Levine, 1998; Satou and Satoh, 1999; Wada and Satoh, 2001). Table 1 summarizes cell types and numbers of constituting cells of the ascidian larva, specification pattern of each cell-type, genes for molecular differentiation markers, and genes with important roles in the formation of each cell-type. We may investigate cellular and molecular mechanisms underlying the formation of chordate body plan using this very simple system of ascidians.

toh, 1978). Following neurulation the tailbud embryo is formed, which eventually develops into a tadpole larva. It takes only about 18 hours for Ciona intestinalis fertilized eggs to reach the larval form.

Embryogenesis of ascidians is simple Reflecting the small number of cells in an ascidian larva, their embryogenesis itself is comparatively simple and easily understandable. The cleavage pattern is invariant, and cleavage is bilaterally symmetrical (Fig. 2). Because every division of the embryonic cells is recognizable, developmental stages of early ascidian embryos are called the 8-, 16-, 32-, 64-, and 110-cell stages, instead of the morula and blastula stages (Fig. 2). Gastrulation is initiated around the 118-cell stage, and it involves epibolic movements of ectodermal cells and migration of endodermal and mesodermal cells inside the embryo (Satoh, 1978). Neurulation is accomplished by folding of the presumptive neural cells, as do vertebrate embryos (Sa-

The lineage of embryonic cells is well documented and developmental fate is restricted early in embryogenesis Every blastomere of the early embryo up to the early gastrula stage is distinguishable from the outside. They are named according to the nomenclature of Conklin (1905) (Fig. 2). The lineage of embryonic cells is completely documented until the early gastrula stage. In addition, the lineage leading to the formation of epidermis, CNS, endoderm, mesenchyme, TLCs, muscle, or notochord is well characterized by detailed descriptions (Conklin, 1905; Ortolani, 1955; Nishida, 1987; Nicol and Meinertzhagen 1988, 1991). During ascidian embryogenesis, as in other animals, the developmental fates of embryonic cells are gradually restricted to give rise to a single type of tissue. The fate restriction in ascidian embryos takes place relatively early (Fig. 2). As early as the 16-cell stage, a pair of epidermis-restricted cells (b5.4 pair) appear in the animal hemisphere (Fig. 2C). Then, at the 32-cell stage, a pair of cells (A6.1 pair) in the vegetal hemisphere become restricted to endoderm (Fig. 2F). At the 64-cell stage, blastomeres appear that are restricted to various individual fates: notochord, muscle, nerve cord, mesenchyme and TLCs (Fig. 2G, H). Reflecting such an early fate restriction, the ascidian embryo shows a highly determinate mode of development (Table 1). In many cell-types, if precursor cells are lost, they cannot be replaced by other cells during development. In addition to classic descriptive and experi-

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mental studies (Reverberi, 1971), recent studies have provided convincing evidence for maternal factors or determinants responsible for autonomous differentiation of muscle, endoderm and epidermis, factors for the establishment of antero-posterior axis of the embryo, and those for initiation of gastrulation (Satoh, 1994, 1999; Nishida, 1997; Jeffery, 2001). In particular, the posterior-vegetal cytoplasm of the fertilized egg or the socalled myoplasm contains muscle determinants, factors for the antero-posterior axis establishment, and those for initiation of gastrulation. In addition, recent as well as classic experiments have provided evidence for requirement of cell-cell interaction in the differentiation process of CNS, notochord, mesenchyme, and TLCs (Table 1) (Nishida, 1997).

Distinct expression of developmental genes

Fig. 2 Cleavage pattern, nomenclature of blastomeres, lineage, and gradual restriction of developmental fates of ascidian embryonic cells. The name of each blastomere is indicated. Each blastomere of the early ascidian embryo is distinguishable, and they are named according to the nomenclature of Conklin (1905), such as a4.2, b5.3, A6.1, and B7.4. The letter a denotes descendents of two (or pair of) anterior animal blastomeres of the 8-cell embryo; b shows those of the posterior animal blastomeres; A, those of the anterior vegetal; and B, those of the posterior vegetal. The first numerical digit denotes cell generation, counting the unsegmented eggs as the first. The second digit gives the cell its own number, which doubles at each division (e.g., A5.2 divides into A6.3 and A6.4). Cells that lie near the vegetal pole are assigned the lower number. Underlining is used to indicate the blastomeres on the right side of the bilaterally symmetrical embryo. The uncleaved zygote is named A1. Blastomeres of the 2-cell embryo are called the left AB2 and the right AB2. The anterior and posterior blastomeres of the 4-cell embryo are named A3 and B3 pair, respectively. Blastomeres are colored when the developmental fate is restricted to give rise to cells of a single type of tissue. A A 4-cell embryo, animal pole view. Anterior is up and posterior is down. B An 8-cell embryo, lateral view. Animal pole is up and vegetal pole is down. Anterior is to the left and posterior is to the right. C, D A 16- cell embryo, viewed from animal (C) and vegetal pole (D). Anterior is up and posterior is down. E, F A 32-cell embryo, animal (E) and vegetal (F) views, respectively. G, H A 64-cell embryo, animal (G) and vegetal (H) views, respectively. I, J A 110-cell embryos, animal (I) and vegetal

In situ hybridization of whole-mount specimens of ascidian embryos shows that signals for zygotic expression of developmental genes are first seen in nuclei of embryonic cells, and as development proceeds, signals are distributed over the entire cytoplasm. Because every blastomere of the embryo is distinguishable, we can judge exactly which cells are expressing the gene, when gene expression is initiated, and to which lineage gene expression is inherited. In other words, we can discuss the developmental gene expression in terms of ‘embryonic cells’ rather than ‘embryonic regions’. For example, the ascidian Brachyury gene is expressed exclusively in notochord cells and the timing of the gene expression coincides with that of developmental fate restriction of blastomeres to give rise to notochord (Yasuo and Satoh, 1993; Corbo et al., 1997a).

Manipulation of embryonic cells is easy In 1887, Chabry carried out blastomere-destruction experiments with ascidian eggs, the first such effort in the history of embryology. Since then, extensive studies of blastomere isolation, destruction and combination were carried out to explore the development potential of blastomeres at various stages (Reverberi, 1971). When the technique of embryonic cell manipulation is combined with distinct molecular markers for differentiation, results of experiments become more meaningful.

(J) views, respectively. K, L, M Schematic drawing showing tissues and organs of the tailbud embryo. Midsagittal section (K) and sagittal section (L) of the embryo, and transverse section of the tail (M). Br, brain (marked by purple); End, endoderm (yellow); Epi, epidermis (green); ES, endodermal strand (yellow); Mch, mesenchyme (dark green); Mu, muscle (red); N, notochord (pink); NC, nerve cord (light purple); TLC, trunk lateral cells (light blue).

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For example, a recent study of Wada et al. (1999) used probes for four homeobox genes, Hrdll-1, Hroth, HrHox-1, and Hrcad, which are expressed in epidermal cells step by step from the anterior to posterior end of Halocynthia roretzi tailbud embryos. By isolating the animal a4.2 or b4.2 blastomeres (epidermal lineage) and by combining the isolated animal blastomeres with vegetal B4.l or A4.1 blastomeres, they clearly showed that the anteroposterior patterning of the epidermis is established by interaction with the vegetal blastomeres. One of the interesting systems in manipulation of ascidian embryos is cleavage-arrest. Whittaker (1973) showed that when early ascidian embryos are immersed in seawater containing cytochalasin B, cytokinesis but not nuclear division of blastomeres is blocked. However, blastomeres of these cleavage-arrested embryos eventually express differentiation markers depending on their lineage. This study not only provides experimental evidence for the presence and segregation of maternal determinants for muscle and endoderm but it also is a prototype for a convenient system used in numerous experiments. For example, we may use this system in experiments in which we inject synthesized mRNA of a certain muscle-related transcription factor gene to analyze effects of its overexpression. When the injected embryos are arrested at the 110-cell stage and examined for differentiation markers, we may easily judge which cells of non-muscle lineage are affected by the overexpression. In addition, the cleavage-arrest provides a simple experimental system to explore the molecular mechanisms involved in the neural induction. Namely, when a4.2 cells are isolated and cleavage-arrested immediately after isolation, they eventually show differentiating markers for epidermis. On the other hand, when isolated a4.2 is combined with isolated A4.1 and then arrested, a4.2 cells eventually show differentiation markers for neuronal cells (Okado and Takahashi, 1988). Therefore, the two-celled combination of a4.2 and A4.1 provides an experimental system to explore the signal molecules for neuronal induction and subsequent differentiation of neuronal cell features (Okamura et al., 1993).

gene duplication, whereas the ascidian has a non-duplicated and basic set of the chordate-type genome. Due to this small genome size, it is relatively easy to isolate genomic clones which cover an entire gene. Analyses of the 5ø regulatory regions with the minimal promoter required for correct spatial expression of developmental genes are also not so difficult. Usually the minimal promoter is located within ∂ 500 bp upstream of the transcription start site of the gene (Satou and Satoh, 1996; Corbo et al., 1997a; Takahashi et al., 1999a).

A short life span and possible mutagenesis screen Ciona intestinalis is a cosmopolitan species used by researchers throughout the world, and C. savignyi is common in the northern Pacific coast. They spawn all year round and the generation time is about 3 to 4 months. Ascidians are hermaphroditic and Ciona eggs are selffertile. These conditions allow us to screen mutants that affect developmental processes. This will be explained later.

Cellular and molecular mechanisms underlying embryonic cell differentiation As mentioned above, the lineage is well documented on each tissue so that we can follow its developmental process precisely. In situ hybridization of whole-mount specimens shows that signals for zygotic expression of developmental genes are first seen in nuclei of embryonic cells with distinct lineage, and therefore we can judge exactly which cells are expressing the gene and when the gene expression is initiated. These and other features of ascidian embryos allow us to investigate the entire process of embryonic cell differentiation from fertilization until the final stage of specific structural gene expression. This may be one of the most advantageous features of the use of ascidian embryos in the field of developmental biology. Here are two examples illustrating primary muscle cell differentiation and endoderm-notochord differentiation.

A compact genome and a small number of genes The solitary ascidian Ciona intestinalis has a small, compact genome of about 1.6 ¿ 108 bp/haploid and the number of genes approximately 15,500 (Simmen et al., 1998), although the compound ascidian Botryllus schrosseri has a comparatively large genome of about 7.3 ¿ 108 bp/haploid (De Tomaso et al., 1998). The Ciona genome size and gene number is comparable to that of Drosophila. It is thought that two large-scale gene duplications, most likely genome duplications, occurred in an evolutionary lineage giving rise to vertebrates (Holland et al., 1994; Sidow, 1996). Vertebrates may evolve the novel and increasingly complex developmental process through this

Primary muscle cells During embryogenesis of the ascidian H. roretzi, exactly 42 unicellular and striated muscle cells are formed in the tail of the tadpole larva (Table 1, Fig. 2). The lineage of these cells is completely documented: 28 of the anterior and middle part of the tail are derived from B4.1-line (B-line), 4 of the posterior part of the tail from A4.1line (A-line), and 10 of the caudal tip from b4.2-line (bline) blastomeres (Nishida, 1987). Extensive descriptive and experimental studies have provided convincing evidence that the B-line (primary) muscle cells are able to

5 Table 1 The ascidian larva: cell-types, number of constituting cells, specification pattern, specific marker genes, and related genes. Cell-types

No. of constituting cells

Specification pattern*1

Specific marker genes

epidermis CNS

800 ∂ 265

autonomous conditional

sensory pigment cells nerve cord endoderm

2 ∂ 65 ∂ 500

conditional conditional autonomous

mesenchyme TLCs TVCs notochord

∂ 900 ∂ 32 ∂ 20 40

conditional conditional – conditional

many epidermis-specific genes TuNa1, HrTBB2 many other marker genes tyrosinase specific marker genes alkalin phosphatase, many other marker genes many marker genes Hr-TLC1, Hr-TLC2

muscle B-line A- and b-line

∂ 42 (28) (10, 4)

autonomous conditional

*1

Related genes

BMP, otx, lim Notch forkhead/HNF-3b, lhx3, ttf1

Ci-pk1, Ci-noto1, many other marker genes

Brachyury

actin, myosin HC many other marker genes

machio-1, Snail, MyoD, Tbx6

For more information, see review of Nishida (1997)

differentiate autonomously without interaction with cells of other lineages. This autonomous differentiation depends on maternal information localized to the myoplasm of the egg (Satoh, 1994; Nishida, 1997; Jeffery, 2001). Therefore, the molecular identification of muscle determinants, the exploration of the mode of action of the factors, and the maintenance of the state of differentiation have been key research subjects for the elucidation of molecular mechanisms underlying the cell fate determination of ascidian embryos. A candidate gene named machio-1 which could act as a muscle determinant was discovered in a recent study of Nishida and Sawada (2001). They isolated this gene in a subtractive hybridization screen of mRNAs between animal and vegetal blastomeres of the 8-cell satge Halocynthia embryos. The machio-1 mRNA encodes a zincfinger protein, and the mRNA is localized to the myoplasm. Depletion of the mRNA specifically results in loss of primary muscle cells, and ectopic expression of machio-1 in epidermal precursor cells evokes differentiation of muscle cells. Unexpectedly, expression of muscle-specific structural genes such as muscle actin gene (HrMA4) and myosin heavy chain gene (HrMHC) is initiated at the 32-cell stage (Satou et al., 1995). Therefore, it is possible that machio-1 mRNA is translated after fertilization and the gene product is segregated into the B-line muscle cells to trigger directly or indirectly transcription activation of HrMA4 and HrMHC. The 5ø upstream sequence of HrMA4 required for its muscle-specific expression has been examined in detail (Hikosaka et al., 1994; Satou and Satoh, 1996). Close to the transcription start site of HrMA4 there are several consensus sequences, which include a TATA box at position -30, an E-box (CAACTG) at -71, a CArG box at -116, and a T-binding motif (AAGTGTAGAA) at -180. Analyses of the de-

letion constructs suggested that the 103-bp upstream region of HrMA4, but not the 66-bp upstream region, is sufficient for the appropriate spatial expression of the reporter gene (Satou and Satoh, 1996). Also, there are two short sequences between -103 and -66, which are essential to the muscle-specificity of HrMA4 promoter; one is 9 bp long (5ø-TCGCACTTC-3ø) and the other is 13 bp long (5ø-GTGATAACAACTG-3ø) (Satou and Satoh, 1996). The two sequences are conserved within 100 bp of the 5ø flanking sequence of Ciona muscle actin gene (Satou and Satoh, unpublished). Therefore, it would be interesting to determine if Machio-1 protein would bind to either of the essential sequences of HrMA4. The myogenic bHLH transcription factor gene AMD1 (Araki et al., 1994; Satoh et al., 1996) and T-box transcription factor gene HrTbx6 (As-T2) (Yasuo et al., 1996) are expressed slightly later (around 64-cell stage) than that of the muscle-specific structural genes. However, loss of trascriptional activity of HrTbx6 results in downregulation of HrMA4 and HrMHC transcription (Mitani et al., submitted). In addition, mis-expression of HrTbx6 promotes ectopic expression of HrMA4 and HrMHC (Mitani et al., 1999). These results together with other experimental results suggest that the transcriptions of muscle-specific structural genes HrMA4 and HrMHC are controlled by two phases of regulation, its initiation with Machio-1 and maintenance by HrTbx6.

Endoderm and notochord Another example to show the advantage of ascidian embryos to explore developmental mechanisms is in the endoderm and notochord. As in the case of vertebrate em-

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bryos, the endoderm of ascidian embryos is specified autonomously, and specified endoderm induces specification of notochord. As shown in Fig. 2F, as early as the 32-cell stage, a pair of vegetal blastomeres (A6.1) becomes restricted to generate endoderm only, and at the 64-cell stage, five pairs of vegetal blastomeres (A7.1, A7.2, A7.5, B7.1, and B7.2) become endoderm-restricted (Fig. 2H). Reflecting such an early fate restriction, presumptive endodermal blastomeres show a high potential for autonomous differentiation when they are isolated from early embryos (Whittaker, 1990; Nishida, 1992). This autonomy is dependent on maternal factors or determinants that are prelocalized in the endoplasm of eggs and early embryos (Nishida, 1993). Recently, convincing evidence has accumulated showing an involvement of b-catenin in the axis determination and embryonic cell specification of widespread phyla from cnidarians to vertebrates (Cadigan and Nusse, 1997; Moon and Kimelman, 1998; Sokol, 1999). To exert its function, b-catenin enters the nucleus and activates downstream genes together with TCF/LEF1 (Willert and Nusse, 1998; Sharpe et al., 2001). In Ciona embryos, the nuclear accumulation of b-catenin is most likely the first step of endoderm specification (Imai et al., 2000). During cleavages, b-catenin accumulates in the nuclei of vegetal blastomeres. Mis- and/or overexpression of b-catenin induces the development of an endoderm-specific alkaline phosphatase in presumptive notochord cells and epidermal cells without affecting differentiation of primary lineage muscle cells (Imai et al., 2000). Downregulation of b-catenin nuclear accumulation induced by the overexpression of cadherin results in the suppression of endodermal cell differentiation. This suppression was accompanied by the differentiation of extra epidermal cells. The nuclear accumulation of bcatenin in Ciona presumptive endodermal cells regulates many downstream genes as will be mentioned later. In the 32-cell embryo, A6.1 is a primordial endodermal cell, A6.3 is an endoderm/TLC precursor cell, while their neighbors A6.2 and A6.4 are presumptive notochord/nerve cord cells (Fig. 2F, H). At the second half of the 32-cell stage, A6.1 and A6.3 emanate signals to A6.2 and A6.4 to induce the specification of notochord (Nakatani and Nishida, 1994). After the division to form the 64-cell stage, A7.3 and A7.7 are destined to differentiate into notochord cells. It is suggested that bFGF in Halocynthia embryos act as signaling molecules in this process (Nakatani et al., 1996) and Notch in Ciona embryos (Corbo et al., 1998). This induction in turn activates the Brachyury gene (Hr-Bra or previously As-T in H. roretzi and Ci-Bra in C. intestinalis) in notochord cells at the 64-cell stage (Yasuo and Satoh, 1993; Corbo et al., 1997a). Interestingly, the ascidian Brachyury is expressed exclusively in notochord cells, and the timing of the gene expression at the 64-cell stage coincides with that of the developmental fate restriction (Yasuo and Satoh, 1993). In Ciona em-

bryos, expression of snail gene in muscle and mesenchyme cells suppresses Ci-Bra expression in these nonnotochord mesodermal cells (Fujiwara et al., 1998). Analyses of various deletion constructs of Ci-Bra (Corbo et al., 1997a) and Hr-Bra (Takahashi et al., 1999a) demonstrates an evolutionary change in the regulation of the 5ø flanking sequences required for notochord-specific expression of the ascidian Brachyury genes. The ascidian Brachyury genes play an essential role in notochord differentiation (Yasuo and Satoh, 1998; Takahashi et al., 1999b), and Ci-Bra activate many downstream genes as described below (Takahashi et al., 1999b; Hotta et al., 2000).

Isolation of novel genes with developmental functions Partially due to the smaller number of genes with less redundancy and partially due to characteristic features of the embryo, ascidians provide an appropriate system to isolate novel genes with possible developmental function. Namely, differential hybridization screens and subtractive hybridization screens have yielded successful isolation of novel developmental genes. For example, as mentioned above, a cDNA for machio-1, a muscle determinant gene, has been isolated by a subtractive hybridization screen of mRNAs between animal and vegetal blastomeres of the 8-cell stage Halocynthia embryos (Nishida and Sawada, 2001). Besides machio-1, four representative and interesting examples will be described below.

pem gene The egg of Ciona savignyi has brownish myoplasm and reddish endoplasm. Centrifugation of the unfertilized egg yielded four types of fragments: a large nucleated red fragment, and small enucleated black, clear and brown fragments (Marikawa et al., 1994). When inseminated, only red fragments cleave and develop, but they form so-called permanent blastulae in which only epidermal cell differentiation is evident. However, when red fragments are fused with black fragments and fusion products are fertilized, nearly all of the fusion products develop muscle cells, endodermal cells, and sometimes they give rise to morphologically normal tadpole larvae (Marikawa et al., 1994). Because clear and brown fragments have no such abilities, it is likely that maternal information responsible for the differentiation of muscle, endoderm, and establishment of embryonic axis are preferentially separated into the black fragments. Yoshida et al. (1996) took advantage of this experimental system to isolate cDNA clones for mRNAs specific to the black fragments, and they succeeded in the isolation of a novel maternal gene named posterior end mark (pem). pem encodes a protein with a Graucho-like

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repressor motif in the C-terminus. The pem transcript is initially distributed in the peripheral cytoplasm of the unfertilized egg, and after fertilization, the pem transcript is concentrated in the posterior-vegetal cytoplasm of the fertilized egg. Later the distribution of the transcript marks the posterior end of developing embryos. This was the first report of a gene with localized maternal mRNA in ascidian eggs. Microinjection of synthetic capped pem mRNA into fertilized eggs results in development of larvae with deficiencies of the anteriormost adhesive organ, dorsal brain and sensory pigment cells, suggesting that pem is involved in anterior and dorsal pattern formation of the embryo.

manx gene Molgula oculata develops a conventional tailed (urodele) larvae, while its closely related species M. occulta develops a tailless (anural) larvae (Jeffery and Swalla, 1990). Subtractive screening of mRNAs in the ovary of the two species yielded cDNA clones for three different genes that are expressed only in the urodele species (Swalla et al., 1993). One of the genes, named manx, encodes a zinc finger nuclear protein. Treatment with antisense oligonucleotides inhibits manx transcriptional activity. This inhibition suppresses the formation of chordate features, suggesting that manx has an important function for development of the chordate larva phenotype in ascidians (Swalla and Jeffery, 1996).

b-catenin downstream genes As mentioned above, b-catenin plays pivotal roles in embryonic axis determination and embryonic cell specification. Therefore, identification of b-catenin target genes and elucidation of their functional cascade are one of the current research subjects of developmental biology. As mentioned above, in Ciona embryos b-catenin nuclear accumulation in vegetal blastomeres is most likely the first step of endoderm specification (Imai et al., 2000). If b-catenin is mis- and/or overexpressed, presumptive notochord cells and epidermis cells change their fates into endodermal cells. If b-catenin nuclear localization is downregulated by the overexpression of cadherin, endodermal cell differentiation is suppressed (Imai et al., 2000). Subtractive hybridization screens of mRNAs between b-catenin overexpressed embryos and cadherin overexpressed embryos in C. savignyi have yielded ten and more potential b-catenin target genes, which include Cs-FoxA5 (forkhead/HNF-3b), a LIM-homeobox gene Cs-lhx3, an orthodenticle homologue Csotx, an NK2 homeobox gene Cs-ttf1, cadherin-related genes Cs-cadherinII and Cs-protocadherin, Cs-netrin and Cs-EPH (Imai et al., submitted). Functional analysis of these genes is now underway.

Ci-Bra target notochord-specific genes In Ciona eggs, fusion gene constructs are very easily introduced by conventional electroporation (Corbo et al., 1997a). The forkhead/HNF-3b gene of C. intestinalis (Cifkh) is expressed in the endoderm, endodermal strand, notochord, and ventral ependymal cells of the neural tube (Corbo et al., 1997b). A 2.6 kb 5ø-flanking region of Ci-fkh is sufficient to direct the expression of a lacZ reporter gene in these tissues after electroporation into 1-cell embryos. The Ci-Bra gene is misexpressed in ectopic tissues by attaching the Ci-Bra coding sequence to the Ci-fkh promoter region, and this misexpression causes an extensive transformation of the endodermal cells into notochord cells. The efficiency of the electroporation method allowed us to obtain large quantities of mutant embryos, thereby facilitating subtractive hybridization reactions using mRNAs extracted from wildtype and mutant embryos (Takahashi et al., 1999b). The subtractive library contains 923 cDNA clones. Sequence analysis of about 500 bps of both 5ø and 3ø regions of the cDNAs suggests that 599 of the clones represent different genes. Whole-mount in situ hybridization demonstrates that nearly 40 of these genes are expressed specifically or predominantly in the notochord cells (Takahashi et al., 1999b). Twenty of the newly cloned genes have been characterized by determining the complete nucleotide sequences of the cDNAs (Hotta et al., 2000). These genes encode a broad spectrum of divergent proteins associated with notochord formation and function. Two genes encode ascidian homologs of Drosophila Prickle LIM domain proteins and another encodes ERM protein, all three 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, b4-galactosyltransferase, fibrinogen-like protein, divergent tropomyosin-like proteins, and Drosophila Pellino-like protein are identified. These information could be used to identify genes which are expressed in the notochord cells of vertebrate embryos as well as non-chordate deuterostome tissues homologous to the notochord. As some of the above examples show, in a sense, the ascidian embryo provides an in vivo macroarray system, from which many target genes are able to be isolated by taking advantages of their characteristic features. We may expect that ascidian embryos are used in future studies to isolate novel genes with developmental functions and also to identify target genes downstream of transcription factor genes or signal transduction molecules. Large-scale cDNA analyses for gene expression profiles Ciona intestinalis has a small, compact genome of about 1.6 ¿ 108 bp/haploid and the number of genes approxi-

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mately 15,500 (Simmen et al., 1998). Therefore, large scale cDNA analyses for gene expression profiles may facilitate investigation of developmental gene expression during ascidian embryogenesis. The first such effort was conducted to investigate the molecular nature of the maternal genetic information stored in the Halocynthia egg (Makabe et al., personal communication). In this study, sequences of both the 5ø- and 3ø- ends of a total of 4,240 cDNAs were determined, and the localization of individual mRNA was examined in staged embryos by whole-mount in situ hybridization [the data obtained are stored in the database MAGEST (Kawashima et al., 2000) (http://www.genome.ad.jp/magest). These analyses demonstrate that the 4,240 cDNA clones represent 2,221 gene transcripts, including at least 934 different proteincoding sequences. The mRNA population of the egg consists of a low prevalence, high complexity sequence set. The majority of the clones are of the rare sequence class, and of these, 42 % of the clones show significant matches with known peptides, mainly consisting of proteins with housekeeping functions such as metabolism and cell division. They also found cDNAs encoding components involved in different signal transduction pathways and for nucleotide-binding proteins. In addition, large-scale analyses of the distribution of the RNA corresponding to each cDNA reveal that a small fraction of the maternal RNAs are localized in the 8-cell stage embryo and that 7.9 % of the clones are exclusively maternal, while 40.6 % of the maternal clones show zygotic expression in the later stages. In collaboration with the Ciona genome project consortium members, similar and more comprehensive studies of gene expression profiles are now being conducted on fertilized eggs, 32 ∂ 110-cell stage embryos, tailbud embryos, larvae, and young adults of C. intestinalis. For example, a set of 3,423 expressed sequence tags (ESTs) derived from the C. intestinalis tailbud embryos was categorized into 1,213 independent clusters (Satou et al., submitted). When compared against DDBJ/GenBank/EMBL database, 502 clusters of them show significant matches to reported proteins with distinct function, while 527 have no significant similarities to known proteins. Sequence similarity analyses of the 502 clusters in relation to the biosynthetic function as well as the structure of the message population at the tailbud stage demonstrate that 390 of them are associated with functions that many kinds of cells use, 85 with cell-cell communication, and 27 with transcription factors and other gene regulatory proteins. Whole-mount in situ hybridization analysis reveals that a total of 387 clusters show expression specific to a certain tissue or organ; 149 shows epidermis-specific expression, 34 are specific to the nervous system, 29 to endoderm, 112 to mesenchyme, 32 to notochord, and 31 to muscle. Some examples are shown in Fig. 3. As of the end of March 2001, 16,000 ESTs have been determined for C. intestinalis tailbud embryos, and se-

quences of more than 10,000 clones are accessible with DDBJ (GenBank/EMBL) database. In addition, 15,000, 11,000, 12,000, and 5,000 ESTs have been determined for Ciona fertilized eggs, 32 ∂ 110-cell stage embryos, larvae, and young adults, respectively (sequencing is carried out at the Academia Sequencing Center directed by Dr. Yuji Kohara, National Institute of Genetics, Japan). In situ hybridization analyses of gene expression profiles of randomly selected 1,000 clusters for each of the stages will be finished by next 3 ∂ 4 months. In addition, similar large scale cDNA projects are also conducting on adult ganglion, blood cells (coelomic cells) and hepatic pancreas. Altogether, these data will be used in future studies including introduction of micro-array system.

Functional analyses of developmental genes A variety of developmental genes have been isolated from ascidian embryos, and the expression of each gene is recognizable under very limited pattern of expression in relation to the embryonic cell lineage. Therefore, a key issue regarding the utility of the ascidian embryo depends on methods to explore gene function. This problem has been addressed by introduction or development of new techniques as described below. Ectopic and/or overexpression of genes An easy way to deduce function of a certain gene is misor overexpress the gene by microinjection of its synthetic capped mRNA into fertilized eggs. For example, microinjection of in vitro transcribed pem mRNA results in development of tadpole larvae with deficiency of the anterior most adhesive organ, dorsal brain and sensory pigment cells, suggesting that this gene plays an important role in the establishment of anterior and dorsal patterning of the embryo (Yoshida et al., 1996). Microinjection of synthetic mRNA of the notochord-specific HrBra (As-T) gene evokes ectopic differentiation of notochord cells in non-notochord lineage blastomeres, suggesting a pivotal role of Hr-Bra in the notochord differentiation (Yasuo and Satoh, 1998). Combination of this technique together with another experimental procedure such as blastomere isolation makes an interpretation of results much more clear-cut. For example, machio-1 is a maternally expressed gene with a pivotal role in differentiation of primary lineage muscle cells. a4.2 blastomeres of the 8-cell stage embryo do not have the potential to form muscle cells. Injection of synthesized machio-1 mRNA into isolated a4.2 blastomeres results in the expression of muscle differentiation markers in resultant partial embryos, providing strong evidence for the maternally expressed muscle determinant gene (Nishida and Sawada, 2001). Another and more mild way of ectopic gene expression is achieved with the aid of a promoter of a specific

9

Fig. 3 Whole-mount in situ hybridization showing the gene expression specific to (A) epidermis, (B) nervous system, (C) brain, (D) nerve cord, (E) endoderm, (F) mesenchyme and TLCs, (G) notochord and (H) muscle of Ciona intestinalis tailbud embryos. Arrow

heads in (B) indicate epithelial sensory cells. Abbreviations: br, brain; mes, mesenchyme; nc, nerve cord; pap, palps; TLC, trunk lateral cells. Scale bar in (A) is 100 mm and is applicable to all photographs.

gene. For example, as mentioned above, Ci-fkh is expressed in the endoderm, notochord, and ventral ependymal cells of the neural tube (Corbo et al., 1997b). A 2.6-kb 5ø-flanking region of Ci-fkh is sufficient to direct the expression of a lacZ reporter gene in these tissues after electroporation into 1-cell embryos. The Ci-Bra gene is thus misexpressed in ectopic tissues by attaching the Ci-Bra coding sequence to the Ci-fkh promoter region. The resulting fusion gene causes an extensive transformation of the endoderm into notochord. These mutant tailbud embryos were used to isolate Ci-Bra downstream gene, as mentioned above (Takahashi et al., 1999b). It is obvious that further development of such promoters, including general promoters which allow ectopic gene expression in a wide range of embryonic cells and those for ectopic expression in a specific type of cells, will provide appropriate system of ectopic gene expression and facilitate the analysis of gene function in ascidian embryos.

otides are widely used to block ascidian gene function. For example, an inevitable role of manx in the tail formation of Molgula embryos can be deduced by inhibition of its function by injection of antisense oligos (Swalla and Jeffery, 1996). The muscle determinant function of machio-1 is also ascertained by suppression of its function with antisense oligos (Nishida and Sawada, 2001). However, generally speaking, we know that antisense oligos are toxic to embryos and their effects are not always conceivable. Recent studies have shown that morpholino oligos act with less-toxicity and effectively as antisense oligos in Xenopus embryos (Summerton, 1999; Heasman et al., 2000). This is the case in ascidian embryos, and with very much reduced toxicity morpholino oligos suppress certain gene function in ascidian embryos. In addition, it is applicable to most of genes so far tested (Satou et al., 2001). For example, a LIM-homeobox gene Cs-lhx3 is one of the b-catenin downstream genes in C. savignyi embryos, and injection of Cs-lhx3 morpholino oligos completely inhibits endodermal cell differentiation (Satou et al., submitted). In addition, injection of cadherin mRNA into C. savignyi eggs results in the suppression of endodermal cell development, however, injection of cadherin mRNA along with Cs-lhx3 mRNA rescues the endodermal cell development. These results strongly suggest that the early expression of Cs-lhx3 in embry-

Disruption of gene function by antisense oligonucleotides However, the interpretation of results of over and/or ectopic expression of transcription factor genes or signal transduction genes is not always easy and is rather problematic. Loss of function analysis may more accurately reflect the function of the gene. Antisense oligonucle-

10

onic endodermal cells is responsible for their differentiation. Morpholino oligos may provide a powerful tool as a loss-of-function analysis of the function of ascidian developmental genes. Dominant negative forms or dominant positive forms of certain molecules may also function in ascidian embryos. As discussed previously, the bFGF-like signal is likely involved in the induction of notochord cells in Halocynthia embryos (Nakatani et al., 1996). The disruption of this signal by injection of mRNA for the dominant negative form of the FGF receptor, in which the intracellular tyrosine kinase domain is deleted, results in the downregulation of notochord-specific Brachyury expression and failure of notochord cell differentiation (Shimauchi et al., submitted).

Mutagenesis screens Ciona intestinalis is a cosmopolitan species used by researchers throughout the world. C. savignyi is common in the northern Pacific coast. They spawn all year round and the generation time is about 3 ∂ 4 months. Ascidians are hermaphroditic and Ciona eggs are self-fertile. These conditions allow us to screen mutants that affect developmental processes. Two laboratories have already attempted mutagenesis screening of the ascidians by treatment of adults with the chemical point mutagen N-ethyl-N-nitrosourea (ENU). Dr. W. Smith’s lab of the University of California at Santa Barbara (USA) have isolated several mutants of C. savignyi (Moody et al., 1999; Nakatani et al., 1999). For example, a screen for ENU-induced mutations that affect early development results in the isolation of a number of mutants including the complementing notochord mutants chongmague and chobi (Nakatani et al., 1999). In chongmague embryos the notochord fails to develop, and the notochord cells instead adopt a mesenchyme-like fate, while in chobi embryos the early development of the notochord appears normal and defects occur later as the notochord attempts to extend and direct elongation of the tail. Researchers including Drs. P. Sordino and R. DeSantis of the Stazione Zoologica at Naples (Italy) have also conducted mutagenesis screening using C. intestinalis with ENU. They have isolated several mutants, including one in which two sensory pigment cells completely fail to develop (Sordino et al., 2000). Scaling up of mutant screening is necessary to fully take advantage of the approach. In addition to chemical mutagen ENU or trimethylpsoralen, efforts of insertion mutagenesis are also required. Also, establishment of genetic background including the establishment of inbred Ciona strains must be initiated (Kano et al., 2001). All these efforts, together with Ciona genome project, will make the ascidian embryo an excellent model experimental system to analyze the expression and func-

tion of developmental genes with known or unknown function.

Conclusions The ascidian embryo has several advantages as an experimental system to study the genetic circuitry underlying development of the basic chordate body plan. An advantage is that the ascidian tadpole consists of a very small number of distinct types of tissues, and the cell lineage is completely described up to the gastrula stage. We are able to identify every blastomere in early embryos, and thus are able to analyze mechanisms of cell fate determination at the single cell level. This advantage is particularly pronounced when we determine the expression of specific genes; namely we can assess the timing of gene expression with respect to that of developmental fate restriction and in relation to the developmental potentials of blastomeres. These approaches are not feasible in vertebrate embryos in which the lineage is not rigidly determined. Recent efforts of large-scale cDNA projects for gene expression profiles and the genome project to determine the entire sequence of Ciona genome may facilitate the development of a Ciona gene catalog in very near future. Introduction of new techniques including mutagenesis screens and morpholino oligos provides powerful tools to explore gene function. Altogether, ascidian embryos may be one of the most appropriate future experimental systems to explore expression and function of developmental genes. Acknowledgements Work from the author’s laboratory was supported by grants from the Ministry of Education, Science, Sports and Culture, Japan and Human Frontier Science Program (RG212/ 1997, RG0290/2000-MR). I would like to thank Dr. Laurence D. Etkin for helpful comments on the manuscript and members of my laboratory for their contribution.

References Araki, I., Saiga, H., Makabe, K.W. and Satoh, N. (1994) Expression of AMD1, a gene for a MyoD1-related factor in the ascidian Halocynthia roretzi. Roux’s Arch Dev Biol 203:320–327. Brusca R.C. and Brusca, G.J. (1990) Invertebrates. Sinauer Associates Inc., Sunderland MA, USA. Cadigan, K.M. and Nusse, R. (1997) Wnt signaling: a common theme in animal development. Genes Dev 11:3286–3305. Cameron, C.B., Garey, J.R. and Swalla, B.J. (2000) Evolution of the chordate body plan: New insights from phylogenetic analyses of deuterostome phyla. Proc Natl Acad Sci USA 97:4469–4474. Chabry, L. (1887) Contribution a l’embryologie normale et teratologique des Ascidies simples. J Anat Physiol (Paris) 23:167–319. Conklin, E.G. (1905) The organization and cell lineage of the ascidian egg. J Acad Nat Sci (Philadelphia) 13:1–119. Corbo, J.C., Levine, M. and Zeller, R.W. (1997a) Characterization of a notochord-specific enhancer from the Brachyury promoter region of the ascidian, Ciona intestinalis. Development 124:589– 602.

11 Corbo, J.C., Erives, A., Di Gregorio, A., Chang, A. and Levine, M. (1997b) Dorsoventral patterning of the vertebrate neural tube is conserved in a protochordate. Development 124:2335–2344. Corbo, J.C., Fujiwara, S., Levine, M. and Di Gregorio, A. (1998) Suppressor of Hairless activates Brachyury expression in the Ciona embryo. Dev Biol 203:358–368. De Tomaso, A.W., Saito, Y., Ishizuka, K.J., Palmeri, K.J. and Weissman, I.L. (1998) Mapping the genome of a model protochordate. I. A low resolution genetic map encompassing the fusion/histocompatibility (Fu/HC) locus of Botryllus schlosseri. Genetics 149:277–287. Di Gregorio, A. and Levine, M. (1998) Ascidian embryogenesis and the origins of the chordate body plan. Curr Opin Genet Dev 8:457–463. Fujiwara, S., Corbo, J.C. and Levine, M. (1998) The Snail repressor establishes a muscle/notochord boundary in the Ciona embryo. Development 125:2511–2520. Gee H. (1996) Before the Backbone. Views on the origin of the vertebrates. Chapman & Hall, London, UK. Heasman, J., Kofron, M. and Wylie, C. (2000) b-catenin signaling activity dissected in the early Xenopus embryo: A novel antisense approach. Dev Biol 222:124–134. Hikosaka, A., Kusakabe, T. and Satoh, N. (1994) Short upstream sequences associated with the muscle-specific expression of an actin gene in ascidian embryos. Dev Biol 166:763–769. Holland P.W.H., Garcia-Ferna`ndez, J., Williams, N.A. and Sidow, A. (1994) Gene duplications and the origins of vertebrate development. Development Suppl:125–133. Hotta, K., Takahashi, H., Asakura, T., Saitoh, B., Takatori, N., Satou, Y. and Satoh, N. (2000) Characterization of Brachyurydownstream notochord genes in the Ciona intestinalis embryo. Dev Biol 224:69–80. Imai, K., Takada, N., Satoh, N. and Satou, Y. (2000) b-catenin mediates the specification of endoderm cells in ascidian embryos. Development 127:3009–3020. Jeffery, W.R. (2001) Determinants of cell and positional fate in ascidian embryos. Int Rev Cytol 203:3–62. Jeffery, W.R. and Swalla, B.J. (1990) Anural development in ascidians: evolutionary modification and elimination of the tadpole larva. Semin Dev Biol 1:253–261. Kano, S., Chiba, S. and Satoh, N. (2001) Genetic relatedness and variability in inbred and wild populations of the solitary ascidian Ciona intestinalis revealed by arbitrarily primed polymerase chain reaction. Marine Biotechnol 3:58–67. Kawashima, T., Kawashima, S., Kanehisa, M., Nishida, H. and Makabe, K.W. (2000) MAGEST: MAboya Gene Expression patterns and Sequence Tags. Nucl Acids Res 28:133–135. Kowalevsky, A. (1866) Entwicklungsgeschichte der einfachen Ascidien. Mem l’Acad St Petersbourg, Ser 7 10:1–19. Marikawa, Y., Yoshida, S. and Satoh, N. (1994) Development of egg fragments of the ascidian Ciona savignyi: the cytoplasmic factors responsible for muscle differentiation are separated into a specific fragment. Dev Biol 162:134–142. Mitani, Y., Takahashi, H. and Satoh, N. (1999) An ascidian T-box gene As-T2 is related to the Tbx6 subfamily and is associated with embryonic muscle cell differentiation. Dev Dyn 215:62– 68. Moody, R., Davis, S., Cubas, F. and Smith, W.C. (1999) Isolation of developmental mutants of the ascidian Ciona savignyi. Mol Gen Genet 262:199–206. Moon, R.T. and Kimelman, D. (1998) From cortical rotation to organizer gene expression: toward a molecular explanation of axis specification in Xenopus. BioEssays 20:536–545. Nakatani, Y. and Nishida, H. (1994) Induction of notochord during ascidian embryogenesis. Dev Biol 166:289–299. Nakatani, Y., Yasuo, H., Satoh, N. and Nishida, H. (1996) Basic fibroblast growth factor induces notochord formation and the expression of As-T, a Brachyury homolog, during ascidian embryogenesis. Development 122:2023–2031.

Nakatani, Y., Moody, R. and Smith, W.C. (1999) Mutations affecting tail and notochord development in the ascidian Ciona savignyi. Development 126:3293–3301. Nicol, D.R. 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 130:737–766. Nicol, D. and Meinertzhagen, I.A. (1991) Cell counts and maps in the larval central nervous system of the ascidian Ciona intestinalis (L.). J Comp Neurol 309:415–429. Nishida, H. (1987) Cell lineage analysis in ascidian embryos by intracellular injection of a tracer enzyme. III. Up to the tissue restricted stage. Dev Biol 121:526–541. Nishida, H. (1992) Developmental potential for tissue differentiation of fully dissociated cells of the ascidian embryo. Roux’s Arch Dev Biol 201:81–87. Nishida, H. (1993) Localized regions of egg cytoplasm that promote expression of endoderm-specific alkaline phosphatase in embryos of the ascidian Halocynthia roretzi. Development 118:1–7. Nishida, H. (1997) Cell fate specification by localized cytoplasmic determinants and cell interactions in ascidian embryos. Int Rev Cytol 176:245–306. Nishida, H. and Sawada, K. (2001) macho-1 encodes a localized mRNA in ascidian eggs that specifies muscle fate during embryogenesis. Nature 409:724–729. Okado, H. and Takahashi, K. (1988) A simple ‘‘neural induction’’ model with two interacting cleavage-arrested ascidian blastomeres. Proc Natl Acad Sci USA 85:6197–6201. Okamura, Y., Okado, H. and Takahashi, K. (1993) The ascidian embryo as a prototype of vertebrate neurogenesis. BioEssays 15:723–730. Ortolani, G. (1955) The presumptive territory of the mesoderm in the ascidian germ. Experientia 11:445–446. Reverberi, G. (1971) Ascidians. In: Reverberi G (ed.) Experimental Embryology of Marine and Fresh-Water Invertebrates, North Holland, Amsterdam, pp 507–550. Satoh, N. (1978) Cellular morphology and architecture during early morphogenesis of the ascidian egg: an SEM study. Biol Bull 155:608–614. Satoh N. (1994) Developmental Biology of Ascidians. Cambridge University Press, New York. Satoh, N. (1999) Cell Fate Determination in the Ascidian Embryo. In: Moody SA (ed.) Cell Lineage and Fate Determination, Academic Press, pp 59–74. Satoh, N. and Jeffery, W.R. (1995) Chasing tails in ascidians: developmental insights into the origin and evolution of chordates. Trends Genet 11:354–359. Satoh, N., Araki, I. and Satou, Y. (1996) An intrinsic genetic program for autonomous differentiation of muscle cells in the ascidian embryo. Proc Natl Acad Sci USA 93:9315–9321. Satou, Y., Kusakabe, T., Araki, I. and Satoh, N. (1995) Timing of initiation of muscle-specific gene expression in the ascidian embryo precedes that of developmental fate restriction in lineage cells. Dev Growth Differ 37:319–327. Satou, Y. and Satoh, N. (1996) Two cis-regulatory elements are essential for the muscle-specific expression of an actin gene in the ascidian embryo. Dev Growth Differ 38:565–573. Satou, Y. and Satoh, N. (1999) Development gene activities in ascidian embryos. Curr Opin Genet Dev 9:542–547. Satou Y., Imai-Satou, K. and Satoh, N. (2001) Action of morpholinos in Ciona embryos. Genesis (in press). Schaeffer, B. (1987) Deuterostome monophyly and phylogeny. Evolutionary Biology 21:179–235. Sharpe, C., Lawrence, N. and Martinez Arias, A. (2001) Wnt signalling: a theme with nuclear variations. BioEssays 23:311–318. Sidow, A. (1996) Gen(om)e duplications in the evolution of early vertebrates. Curr Opin Genet Dev 6:715–722. Simmen, M.W., Leitgeb, S., Clark, V.H., Jones, S.J.M. and Bird, A.

12 (1998) Gene number in an invertebrate chordate, Ciona intestinalis. Proc Natl Acad Sci USA 95:4437–4440. Sokol, S.Y. (1999) Wnt signaling and dorso-ventral axis specification in vertebrates. Curr Opin Genet Dev 9:405–410. Sordino, P., Heisenberg, C-P., Cirino, P., Toscano, A., Giuliano, P., Marino, R., Pinto, M.R. and De Santis, R. (2000) A mutational approach to the study of development of the protochordate Ciona intestinalis (Tunicata, Chordata). Sarsia 85:173–176. Summerton, J. (1999) Morpholino antisense oligomers: the case for an RNase H-independent structural type. Biochim Biophys Acta 1489:141–158. Swalla, B.J., Makabe, K.W., Satoh, N. and Jeffery, W.R. (1993) Novel genes expressed differentially in ascidians with alternate modes of development. Development 119:307–318. Swalla, B.J. and Jeffery, W.R. (1996) Requirement of the Manx gene for expression of chordate features in a tailless ascidian larva. Science 274:1205–1208. Takahashi, H., Mitani, Y., Satoh, G. and Satoh, N. (1999a) Evolutionary alterations of the minimal promoter for notochord-specific Brachyury expression in ascidian embryos. Development 126:3725–3734. Takahashi, H., Hotta, K., Erives, A., Di Gregorio, A., Zeller, R.W., Levine, M. and Satoh, N. (1999b) Brachyury downstream notochord differentiation in the ascidian embryo. Genes Dev 13:1519–1523. Wada, H. and Satoh, N. (1994) Details of the evolutionary history from invertebrates to vertebrates, as deduced from the sequences of 18S rDNA. Proc Natl Acad Sci USA 91:1801–1804.

Wada, H. and Satoh, N. (2001) Patterning the protochordate neural tube. Curr Opin Neurobiol 11:16–21. Wada, S., Katsuyama, Y. and Saiga, H. (1999) Anteroposterior patterning of the epidermis by inductive influences from the vegetal hemisphere cells in the ascidian embryo. Development 126:4955–4963. Whittaker, J.R. (1973) Segregation during ascidian embryogenesis of egg cytoplasmic information for tissue-specific enzyme development. Proc Natl Acad Sci USA 70:2096–2100. Whittaker, J.R. (1990) Determination of alkaline phosphatase expression in endodermal cell lineages of an ascidian embryo. Biol Bull 178:222–230. Willert, K. and Nusse, R. (1998) b-catenin: a key mediator of Wnt signaling. Curr Opin Genet Dev 8:95–102. Yasuo, H. and Satoh, N. (1993) Function of vertebrate T gene. Nature 364:582–583. Yasuo, H., Kobayashi, M., Shimauchi, Y. and Satoh, N. (1996) The ascidian genome contains another T-domain gene that is expressed in differentiating muscle and the tip of the tail of the embryo. Dev Biol 180:773–779. Yasuo, H. and Satoh, N. (1998) Conservation of the developmental role of Brachyury in notochord formation in a urochordate, the ascidian Halocynthia roretzi. Dev Biol 200:158–170. Yoshida, S., Marikawa, Y. and Satoh, N. (1996) posterior end mark, a novel maternal gene encoding a localized factor in the ascidian embryo. Development 122:2005–2012.