Developmental Genomics of Ascidians

Preface

I have been captivated by ascidian embryology for more than 35 years, starting with Halocynthia roretzi and followed by Ciona intestinalis. There are numerous reasons why I still work with ascidians after all this time. The first and simplest reason is that ascidian embryogenesis, both temporally and spatially, is well suited to my individual research interests. While a developmental biologist's personality impacts his/her choice of experimental system, the model organisms themselves often dictate the research topics or themes. For example, vertebrates develop slowly, whereas ascidians do so quickly. Longer developmental time periods lend themselves to mechanistic investigations of detailed developmental processes; however, it is difficult for a vertebrate embryologist to follow the entire process of embryogenesis in a single study. By contrast, because I am interested in understanding the clock mechanisms that control the timing of developmental events, I selected Halocynthia as my first model organism because of its comparatively short period of embryogenesis. As I began studying the timing of differentiation marker gene expression, the length of Halocynthia embryogenesis was very reasonable—development from fertilization to the newly formed larva stage took approximately one and half days at 15 °C. Even better, in Ciona, the fertilized egg developed to the larva within 18 hours at 18 °C. This time frame of ascidian development has affected my research outlook to the point that I am less concerned with individual developmental processes, and prefer to view and investigate embryogenesis as a whole.

The second reason for my fidelity to ascidians comes from their beautiful embryos and invariant cleavage patterns. Under a stereomicroscope, we observe well-regulated cleavage, gastrulation, neurulation, and tailbud embryo formation. We can identify every blastomere up to gastrulation. All processes are very dynamic, with fewer constituent cells than vertebrate embryos. In addition, the formed tadpole larva represents the most simplified body plan of the chordates, a group to which humans also belong.

Soon after I started my research on ascidian embryos, I became convinced that ascidians would become one of the most suitable experimental systems in which to explore the cellular and molecular mechanisms of embryonic cell specification and differentiation. These were the main reasons why I published the book Developmental Biology of Ascidians, by Cambridge University Press in 1994. That book aimed to review extant ascidian embryology research, as well as highlight the advantages of the model system and encourage more people to join the field.

A dramatic change in ascidian embryology occurred in 2002, when the draft genome of C. intestinalis was sequenced after a collaborative effort of worldwide ascidian developmental biologists with the Joint Genome Institute, Department of Energy, USA. This project identified almost all of the developmentally relevant genes in the Ciona genome, which represents the basic set of gene components in chordates prior to the two genome-wide gene duplications that occurred in vertebrates. The absence of redundancy in ascidian regulatory gene functions makes it easier to elucidate the developmental roles of individual genes. In association with the genome sequencing project, a cDNA sequencing project was also carried out, providing great quantity of information on the expression profiles of regulatory genes. Whole-mount in situ hybridization reveals distinct gene expression profiles in embryos at the single cell level. We, ascidian embryologists, are proud that we can discuss mechanisms at the single cell level, rather than at the regional embryonic level that is usually discussed in vertebrate embryos.

By applying new molecular techniques to the ascidian system, we have answered questions posed by several pioneer ascidian embryologists. We now know the gene for the muscle determinant activity, first identified by Conklin. We now know the molecular mechanisms involved in self- and nonself-recognition of gametes of hermaphroditic C. intestinalis. Morgan investigated this question several times. We now know the molecular mechanisms involved in ascidian embryo notochord formation, a characteristic feature of chordates. Kowlevsky first pointed out the enormous opportunity afforded by ascidians to bridge the big evolutionary gap between invertebrates and vertebrates. The simplicity of the ascidian embryology and genome, with its basic set of chordate regulatory genes, together with the modern techniques available to researchers—including forward genetics—have made the Ciona embryo one of the best systems in which to study developmental genomics. I am confident that this system will continue to develop quickly in the years to come.

As one of the comparatively old fellows studying ascidian embryology, for me one of the greatest pleasures is to see newcomers to the field who have already enjoyed great research careers using other model systems, for example, Michael Levine and Patrick Lemaire. These established scientists are accompanied by young researchers with astonishing productivity. Most of the current, elegant experiments in the field have been carried out by these young ascidian embryologists, who are a real treasure for the community. In addition, these scientists have published good reviews on the current progress in their respective subsets of ascidian developmental genomics. I have cited their work extensively in this book. I appreciate deeply their kind consent for me to use their descriptions, and for their comments and criticisms during the draft preparation stage of this book.

Special thanks go to Drs. Hiroki Nishida, Yutaka Satou, Patrick Lemaire, Micheal Levine, Clare Hudson, Hitoyoshi Yasuo, Lionel Christiaen, Kohji Hotta, Yasunori Sasakura, Kaoru S. Imai, Brad Davidson, William R. Jeffery, Billie Swalla, William C. Smith, Di Jiang, Kazuo Kawamura, Shirae-Kurabayashi, Anthony De Tomaso, Alberto Stolfi, Shigehiro Yamada, Hiroki Takahashi, Yoshito Harada, Rixy Yamada, Hitoshi Sawada, Miho Suzuki, Robert Zeller, Keisuke Nakashima, Teruaki Nishikawa, Maki T. Kobayashi, Hidetoshi Saiga, Takehiro Kusakabe, Naohito Takatori, Honoo Satake, Masaru Nonaka, Tatsuya Ueki, Hitoshi Michibata, Eiichi Shoguchi, Takeshi Kawashima, Fuki Gyoja, Christian Sardet, Francois Prodon, Arend Sidow, Anna Di Gregorio, Jean-Stepane Joly, Kazuo Inaba, Michio Ogasawara, Kasumi Yagi, and Daniel Rokhsar.

In one sense, this book is a composite of review articles contributed by many experts in the field. However, it also contains my thoughts on ascidian embryogenesis as viewed from a long perspective. What I especially want to convey in this book is the urgent need to describe a complete (or nearly complete) set of molecular and cellular events associated with Ciona embryogenesis so that we can address the final question of embryogenesis. Specifically, with reference to Lewis Wolpert's It is not birth, marriage, or death, but gastrulation which is truly the most important time in your life, my response is Embryogenesis is not a simple series of changes to embryonic cells, but rather the place to develop phenotypic individuality as an extant organism with a long evolutionary history. We can begin to understand our development as individuals and as a species through studies of ascidian embryogenesis.

Finally, I would like to thank the members of the Molecular Developmental Biology Laboratory at Kyoto University. Kazuko Hirayama is acknowledged for her great support at Kyoto University. Thanks to Kanako Hisata for her great help in preparing the figures and tables and Shoko Yamakawa for typing the manuscript. I am especially grateful to my wife, Mikako Satoh, for her daily support of my research.

The title of this book, Developmental Genomics of Ascidians, reflects my desire for future development of this field using ascidian embryos. If readers of this book come away impressed by and attracted to the ascidians, especially the Ciona system, it is my unbidden pleasure. If they come away uninspired by the Ciona system, it is simply due to my inability to describe it adequately. At any rate, I am happy to leave this book as my last and largest contribution to ascidian developmental biology.

Noriyuki Satoh

Okinawa, Japan

Chapter 1

A Brief Introduction to Ascidians

1.1 What are Ascidians?

Ascidians, or sea squirts, are sessile marine invertebrate chordates ubiquitous throughout the world. The name ascidian originated from the Greek word askidion, meaning a small bag or vase. The class comprises approximately 2900 extant species,¹ most of which live in shallow water. Ascidians usually attach to rocks, shells, and pilings, and live by filtering tiny plankton and other nutrients from seawater. The entire adult body is invested with a thick covering, the tunic (or test), from which the subphylum name, Tunicata is derived. A major constituent of the tunic is tunicin, a type of cellulose. Tunicates or urochordates are the only animals that can synthesize cellulose independently.

An individual ascidian has two openings, an incurrent oral (branchial) siphon and an outcurrent atrial siphon (Fig. 1.1a, b). The mouth behind the oral siphon leads to a large pharynx, or branchial basket—a chamber perforated by dorsoventral rows of numerous gill slits called stigmata (Fig. 1.1b). Along the ventral margin of the branchial basket is a specialized organ called the endostyle, which secretes large quantities of mucus used for capturing food particles (Fig. 1.1b). The endostyle contains iodine, and therefore this organ has an evolutionary relationship with the vertebrate thyroid gland. The digestive tract leads to a stomach at the bottom of the U-shaped digestive loop, followed by an intestine that terminates at the anus, which opens into the atrial cavity (Fig. 1.1b).

Figure 1.1 The tunicate ascidian Ciona intestinalis. (a) An adult with oral (incurrent) and atrial (outcurrent) siphons. The white duct is the sperm duct and the orange duct paralleling it is the egg duct. (b) Diagram illustrating adult organs and tissues. (c–l) Embryogenesis. Embryos were dechorionated to clearly show their outer morphology. (c) Fertilized egg, (d) 2-cell embryo, (e) 4-cell embryo, (f) 16-cell embryo, (g) 32-cell embryo, (h) gastrula (∼150 cells), (i, j) neurulae, (k, l) tailbud embryos, and (m) tadpole larva. (n) Diagram illustrating larval organs and tissues. (o) A juvenile a few days after metamorphosis, with internal structures labeled.

The adult nervous system consists of a single cerebral ganglion lying between the two siphons and an adjacent neural gland (Fig. 1.1b). Several nerves elongate from the ganglion to various parts of the body, including the muscles, pharynx, viscera, gonad, and siphons (cover picture). By contrast, the neural gland leads through a duct to the pharynx, just behind the mouth. The open circulatory system is well developed and consists of a short, tubular heart and numerous blood vessels. The heart lies posteroventrally in the body near the stomach and behind the pharyngeal basket (Fig. 1.1b, o). The heartbeat and the direction of blood flow reverse periodically. The circulatory system contains several different types of blood cells or coelomic cells with specialized functions.

1.1.1 Taxonomy

Ascidians belong to the class Ascidiacea, subphylum Urochordata (Tunicata), and phylum Chordata (Fig. 1.2). Animals with a notochord or a rod-shaped axial organ and a dorsal hollow neural tube are classified into the phylum Chordata. Chordata consists of three subphyla, Cephalochordata, Urochordata, and Vertebrata (Fig. 1.2). Cephalochordates (lancelets or amphioxus) are headless (acraniates), and have well-segmented somites and a notochord running throughout the body. Urochordates contain a notochord in the tail during at least the larval stage, and have a well-organized larval central nervous system. Vertebrates or craniates develop vertebrae from the notochord, along with jaws, heads, and an adaptive immune system. Chordates were present on the Earth at least ∼520 million years (MYR) ago, as demonstrated by recent fossil records of Chenjan Fauna. The origin and evolution of chordates have been debated for more than 150 years. Recent molecular phylogeny and comparative genomics studies demonstrated that cephalochordates represent the most ancient and extant chordate lineage. Furthermore, urochordates and vertebrates were shown to form a sister group (Olfactants), indicating that urochordates are the invertebrates most closely related to vertebrates (Fig. 1.2).

Figure 1.2 A phylogenetic tree indicating the position of urochordates (tunicates) among deuterostomes and chordates. Several ascidians species (underlined) are listed in the corresponding phylogenetic positions. The phylogenetic relationship of Appendicularia (larvaceans) still remains to be determined (bold dotted lines).

The subphylum Urochordata or Tunicata is composed of three classes, Ascidiacea (ascidians), Thaliacea (salps), and Appendicularia (larvaceans) (Fig. 1.2). The evolutionary relationships among tunicates remain controversial because of their great variety of life cycles and rapid evolution. The class Ascidiacea comprises two subclasses, Enterogona and Pleurogona (Fig. 1.2). The Enterogona ascidians are characterized by the location of the gonad (a single ovary and a single testis; ascidians usually are hermaphrodites) within the gut loop or posterior to it. This subclass consists of Aplousobranchia (Clavelinai and others) and Phlebobranchia (Ciona intestinalis, Phallusia mammillata, and others) (Fig. 1.2). On the other hand, the Pleurogona ascidians, which have a pair of gonads inside the right and left body walls, include Molgulidae (Molgula oculuta and others), Styelidae (Styela plicata and others), and Pyuridae (Halocynthia roretzi, Botryllus schlosseri, and others) (Fig. 1.2). Some of these organisms live as individuals (solitary or simple ascidians), while others form colonies (colonial or compound ascidians). The colonial life style evolved several times independently in the orders.

1.1.2 Reproduction

Ascidians are hermaphrodites (Fig. 1.1a, b). Sexual reproduction is common in solitary ascidians, whereas colonial ascidians reproduce both sexually and asexually by budding. Colonial ascidians also have an extensive capacity for regeneration. Stereotypic embryogenesis of solitary ascidians proceeds rapidly (Fig. 1.1c–l). Bilaterally symmetrical cleavage takes place according to a highly determinate pattern (Fig. 1.1d–g). Gastrulation begins around the 120-cell stage and is followed by neurulation (Fig. 1.1h–j). Then, tailbud embryos are formed (Fig. 1.1k, l), and finally a conventional tadpole-type larva hatches from the chorion (Fig. 1.1m), usually within 12 h to a few days after fertilization. The ascidian tadpole consists of only ∼2600 cells, but has distinct tissues and organs, including the epidermis, central and peripheral nervous systems, endoderm (which gives rise to the adult digestive tract and its associated organs), notochord, muscle, and mesenchyme (from which several adult mesodermal organs are derived) (Fig. 1.1n). These organs represent the major components of any vertebrate body, including our own, and the mechanisms underlying their formation are the focal point of this book.

The ascidian tadpole larva differs from the amphibian tadpole in that it has no mouth. The nonfeeding larva swims for a few hours or more as solitary ascidians, and then metamorphoses into a juvenile (Fig. 1.1o). The growth of the juvenile is also rapid. For example, eggs of the most common and cosmopolitan species, C. intestinalis,² give rise to adults with reproductive capacity within 2–3 months, or earlier in warm waters, suggesting that under optimal conditions Ciona can pass through several generations within a year. Closed-system inland culture conditions have been established for the maintenance of these organisms for laboratory study. In Japan, C. intestinalis are provided to researchers all year round through support of the National BioResource Project (NBRP).

1.2 A Brief History of Research on Ascidian Embryos

Ascidians have been recognized since the ancient Greeks, and were described by Aristotle. Because of their soft bodies, they were long classified as a group of mollusks by Carl Linnaeus. At that time, there was a noticeable gap between vertebrates and invertebrates. After the publication of Charles Darwin's On the Origin of Species by Means of Natural Selection in 1859, evolutionary theories came into vogue to describe relationships among animals. In 1886, the great Russian embryologist Alexander Kowalevsky (Fig. 1.3a) discovered that the ascidian larva has the general appearance of a simplified vertebrate tadpole (Fig. 1.3b). This observation, together with his other finding that amphioxus possess a notochord, positioned the ascidians and amphioxus as a new group, the protochordates, which were intermediate between invertebrates and vertebrates—filling the evolutionary gap. More recently, it seems that the long debate on the origin and evolution of chordates has reached a consensus opinion that cephalochordates are basal among chordates, while urochordate ascidians are a sister group to the vertebrates.

Figure 1.3 Three great pioneers of ascidian embryology with their descriptions of ascidian embryos. (a) Alexander Kowalevsky and (b) his illustrations of ascidian embryos. (c) Lawrent Chabry and (d) his sketches depicting the manipulation of Ascidiella eggs. (e) Edwin G. Conklin and (f) his drawings of ascidian embryos.

(Photographs were obtained from Wikipedia.)

It is said that the descriptive work of van Beneden and Julin in 1884 was the first demonstration of the relationship between the egg axis and the larval body plan. The door to classical experimental embryology was opened in 1887 by the French anatomist, Laurent Chabry (Fig. 1.3c), in a study of ascidian embryos. Chabry destroyed one blastomere of a 2-cell Ascidiella aspersa embryo and found that the remaining blastomere continued to cleave as if it were half of the whole embryo, eventually forming a half-larva (instead of a complete dwarf larva) (Fig. 1.3d). After obtaining a similar result using a 4-cell embryo, he inferred that ascidian embryos could not compensate for missing parts, and thus, that the developmental pattern was mosaic. His experiment was followed by Roux's studies of amphibian embryos in 1888 and Driesch's work on sea urchin embryos in 1891. The triumph in this vein of experimental embryology was, of course, the 1924 discovery by Spemann and Mangold of the organizer, as determined by transplantation experiments on amphibian embryos.

The pioneering zoologist and geneticist, Thomas H. Morgan, carried out many seminal studies not only in Drosophila but also in other animals, including ascidians. He was especially puzzled by the self-sterility of C. intestinalis, which he first reported in 1904. Ascidians are hermaphrodites and self-fertilization is usually blocked. Morgan published ten more reports on this topic until 1945. His attempt to understand the molecular mechanisms of self-sterility has now been realized by young Japanese scientists taking full advantage of genetics, genomics, and proteomic techniques available for C. intestinalis. The molecular mechanisms involved in the ascidian self and nonself recognition are likely to be similar to those that have been recently unveiled in plants.

In 1905, in a milestone of ascidian embryology, Edwin G. Conklin (Fig. 1.3e) described the lineage of embryonic cells and the segregation of egg cytoplasmic regions into specific larval organs, suggesting that maternal factors were responsible for the later differentiation of embryonic cells (Fig. 1.3f). He found that the lineage of embryonic cells is invariant, and that most of the embryonic cells are destined to give rise to one type of tissue or organ during cleavage stages prior to the initiation of gastrulation. Conklin's insight was followed by a considerable number of experimental studies on ascidian embryogenesis, carried out between 1940–1960, particularly by Italian embryologists, including Jossepp Reverveli, Giuseppina Ortolani, and others.

In 1973, Richard Whittaker, using cleavage-arrested embryos, showed that maternally provided factors are segregated into specific lineages of embryonic cells, and that these factors trigger developmental pathways leading to cell differentiation, as Conklin proposed. Whittaker's study initiated the field of molecular developmental biology, which incorporated techniques, rules, and concepts from molecular biology. Ascidians were proposed as a model experimental system for molecular developmental biology, particularly with respect to the study of mechanisms underlying embryonic cell fate specification. This new research focus was the main impetus behind the 1994 publication of Developmental Biology of Ascidians.

A big turn in ascidian embryology came in 2002, when the draft genome of C. intestinalis was published. At that time, it was the seventh animal with sequenced genome. Importantly, the C. intestinalis draft genome revealed that the ascidian contains a basic set of developmentally relevant genes that are also used by vertebrates, including transcription factors (TFs) and cell–cell signaling pathway molecules (SPMs). Thus, Ciona continues to be an appropriate experimental system for a diverse range of biological studies.

The recent advances in the field of ascidian embryology are extraordinary. Owing to the well-described lineages, embryonic cell specification mechanisms are now understandable in view of gene regulatory networks. Imaging of embryos and juveniles has greatly advanced our understanding of the constitution of the ascidian embryo at the single cell level. Forward and reverse genetics have revealed the functions of developmental regulatory genes. With embryologic and genomic simplicity, Ciona is a model system for developmental studies of chordates. The main aim of this book is to introduce their attractiveness for future mechanistic studies.

Selected References

Brusca, R.C., Brusca, G.J. (2003) The urochodates (tunicates), in Invertebrates, 2nd edn, Sinauer Assoc. Inc, Sunderland, MA, pp. 855–864.

Chabry, L. (1887) Contribution a l'embryologie normale et teratogique des Ascidies simples. J Anat Physiol (Paris), 23, 167–319.

Cloney, R.A. (1990) Urochordata-Ascidiacea, in Reproductive Biology of Inverterbates, (eds K.G. Adiyodi and R.G. Adiyodi), Oxford and IBH, New Delhi, pp. 361–451.

Conklin, E.G. (1905) The organization and cell lineage of the ascidian egg. J Acad Natl Sci (Philadelphia), 13, 1–119.

Jeffery, W.R., Swalla, B.J. (1997) Tunicates, in Embryology, (eds S.F. Gilbert and A.M. Raunio), Sinauer, Sunderland, MA, pp. 331–364.

Kowalevsky A (1866) Entwicklungsgeshichte der einfachen Ascidien. Memory l'Academy St Petersbourg, 7 (10), 1–19.

Morgan, T.H. (1944) The genetic and the physiological problems of self-sterility in Ciona. VI. Theoretical discussion of genetic data. J Exp Zool, 95, 37–59.

Reverberi, G. (1971) Ascidians, in Experimental Embryology of Marine and Fresh-water Invertebrates (ed G. Reverberi), Amsterdam, North Holland, pp. 507–550.

Satoh, N. (1994) Developmental Biology of Ascidians, Cambridge University Press, New York.

Van Beneden, E., Julin, C.H. (1884) La segmentation chez les ascidens dans ses rapports avec l'organization de la larve, Arch Biol, 5, 111–126.

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.

¹ Based on a recent estimation of Appeltans et al. (2012) The magnitude of global marine species diversity. Curr. Biol., 22, 2189–2202.

² Recent studies demonstrate that there are at least two cryptic species of C.