Toward a new paradigm for studying ascidian developmental dynamics


Major animal groups have their own characteristic features and/or advantages for studies of developmental biology. Among them, urochordate ascidians, especially Ciona intestinalis, provide special advantages with respect to future studies of developmental genomics and developmental systems biology (Satoh et al.,2003). First, C. intestinalis was the seventh assembled animal genome, which is composed of just 160 Mb and contains approximately 16,000 protein-coding genes (Dehal et al.,2002). This genome size and gene number is quite compact as compared with those of vertebrates. In addition, a large quantity of cDNA and EST (expressed sequence tag) information substantiates the gene models, especially those encoding transcription factors and signaling molecules. A freely distributed package of cDNA clones named “Ciona gene collection” also facilitates studies of expression and function of developmentally relevant genes.

Second, the simplicity of Ciona embryogenesis permits in-depth analyses of the complex mechanisms involved in the establishment of the chordate body plan. Embryos and tadpole-larvae consist of comparatively small numbers of constituent cells that form tissues including notochord, nervous system, muscle, mesenchyme, endoderm, and epidermis. Most of these cell types are specified at or before the 110-cell stage (Nishida,2005). Due to the invariant cell division patterns, basically all molecular mechanisms can be analyzed at the single-cell level (Imai et al.,2006). More importantly, Ciona is a chordate, and recent molecular phylogenetic studies suggest that urochordates but not cephalochordates are a sister group of vertebrates (Delsuc et al.,2006). This means that the basic body plan common to the chordates can be studied much more easily in the ascidian system than in vertebrates (Satoh,2003).

Highlighting the importance of ascidian developmental biology, this special focus of Developmental Dynamics includes seven review articles, three research articles, and one technique article on this field. Two articles review the significant roles of maternally provided cytoplasmic information in formation of the body axis. François Prodon et al. discuss recent data on the cellular and molecular mechanisms responsible for maternal mRNA localization. In ascidian eggs, more than 40 members of localized maternal mRNAs have been reported; they are called postplasmic/PEM RNAs and are subgrouped into two types (Type I and Type II) depending on different localization patterns from fertilization to the eight-cell stage. After the eight-cell stage, a macromolecular cortical structure called CAB (for Centrosome Attracting Body) in the posterior-vegetal (B4.1) blastomeres is responsible for asymmetric divisions and the partitioning of postplasmic/PEM RNAs at the posterior pole of embryos. Prodon et al. make an extensive classification of embryonic localization profiles of the mRNAs and discuss their functions revealed by knockdown and/or overexpression experiments. They also discuss the role of the 3′-untranslated region controlling both their localization and translation, and the relationship between postplasmic/PEM RNAs, posterior specification, and germ cell formation in ascidians.

Christian Sardet et al. discuss cellular and molecular mechanisms involved in the establishment of dorsoventral and anteroposterior axes of the ascidian embryo. Oocytes of ascidians acquire a primary animal–vegetal (a-v) axis during meiotic maturation, when a subcortical mitochondria-rich domain (myoplasm) and a domain rich in cortical endoplasmic reticulum (cER) and maternal postplasmic/PEM RNAs (cER-mRNA domain) become polarized and asymmetrically enriched in the vegetal hemisphere. Fertilization evokes a series of dynamic cytoplasmic and cortical reorganizations of the zygote, which occur in two major phases. In the second phase, sperm aster microtubules and then cortical microfilaments cause the cER-mRNA domain and myoplasm to become positioned toward the posterior of the zygote. At the first cleavage, both cER-mRNA and myoplasm domains in the posterior region are partitioned equally between the first two blastomeres and then asymmetrically over the next two cleavages. At the eight-cell stage the cER-mRNA domain compacts and gives rise to CAB, which is responsible for a series of unequal divisions in posterior vegetal blastomeres. The postplasmic/PEM RNAs contained in CAB are involved in patterning the posterior region of the embryo. Sardet et al. discuss these multiple events and phases of reorganizations in detail and their relationship to physiological, cell cycle, and cytoskeletal events.

In recent studies, confocal laser scanning microscopy (CLSM) has been used as a powerful tool to reveal dynamic movements and rearrangement of cytoplasmic components, and this imaging technique is successfully used to trace the composition of cells in the Ciona embryo. Specifically, Kohji Hotta et al. reconstruct images of embryos at the newly defined 26 distinct developmental stages (stages 126) from more than 3,000 high-resolution real images collected by CLSM. Then they make a standard Web-based image resource called ABA (Ascidian Body Atlas), which includes three-dimensional and cross-sectional images of Ciona embryos through the developmental time course. Their data set will be very helpful in standardizing developmental stages for morphology comparison and in studying gene expression and function at the single cell level.

As mentioned above, ascidian embryos provide a model system for studying cellular and molecular mechanisms of developmental fate specification and differentiation. There have been good reviews on this subject including recent ones by Passamaneck and Di Gregorio (2005) and Nishida (2005), which were published in Developmental Dynamics. In this special issue, Gaku Kumano and Hiroki Nishida have reviewed more recent works on this subject. Discussion of results obtained from ascidians with those from vertebrates highlights similarities and dissimilarities of molecular mechanisms underlying early developmental modes of the two sister groups of chordate.

The notochord is the most prominent feature of the chordates, as reflected by their name. Elucidation of molecular mechanisms responsible for formation of this organ is of special interest not only to developmental biologists but also to evolutionary biologists (Satoh,2003). In this special issue, two research groups pay attention to this subject. In ascidians, a T-box transcription factor Brachyury (Ci-Bra) plays a pivotal role in the notochord formation. Ci-multidom is one of Ci-Bra downstream target genes expressed in differentiating notochord cells. Izumi Oda-Ishii and Anna Di Gregorio report that, although Ci-Bra is homogeneously expressed in all the notochord cells, Ci-multidom is transcribed at detectable levels only in a random subset of these cells, suggesting that, despite its morphological simplicity and invariant cell-lineage, the ancestral notochord is a mosaic of cells in which the gene cascade downstream of Ci-Bra is differentially modulated. Di Jiang and William Smith discuss the formation of notochord from cellular behaviors. Convergent extension is responsible for the intercalation of notochord cells and to some degree for notochord elongation, whereas a second phase of elongation occurs as the notochord narrows medially and increases in volume. Although the mechanism by which the volume of the notochord increases differs between ascidian species, the resulting notochord serves as a hydrostatic skeleton allowing for the locomotion of the swimming larva. Jiang and Smith emphasize that the process of notochord morphogenesis contains several basic cell behaviors such as cell shape changes, cell rearrangement, establishment of cell polarity, and alteration of the extracellular environment, and that modern analysis of ascidian notochord morphogenesis promises to contribute to our understanding of these fundamental biological processes.

An expectation of ascidian biology is an elucidation of developmentally relevant genes with novel function, being revealed by introducing genome-wide reverse genetics and forward genetics. Mayuko Hamada et al. report their efforts using reverse genetics, whereas Yasunori Sasakura reports their progress using forward genetics. The sequenced genome of C. intestinalis suggests the presence of nearly 2,500 genes that have vertebrate homologues, but their functions are as yet unknown. To identify genes with novel function, they screen 504 genes overall by knockdown experiments using specific morpholino antisense oligonucleotides. They find that suppression of the translation of a total of 111 genes results in some morphological defects in manipulated embryos. This type of large-scale screening is a powerful approach to identify novel genes with significant developmental functions, although the details of which will be determined in future studies. Sasakura reviews recent advances in ascidian forward genetics, which have mainly been established in his own laboratory. Namely, transposon-mediated transformation using a Tc1/mariner transposable element Minos has been introduced into the Ciona system. He reports that as much as 37% of Minos-injected Ciona transmit transposon insertions to the subsequent generation. This kind of stable transgenesis is a splendid technique for the creation of useful marker lines, enhancer/gene trappings, and insertional mutagenesis. Taking advantage of the Ciona system, further expansion of developmental genetics is expected.

An increasing usage of Ciona intestinalis as an object of developmental biology research demands further sophistication of this species as an experimental system. This includes the establishment of laboratory strains and construction of a fine genetic map. Shungo Kano, who has contributed in this field for more than a decade, summarizes recent advance in the Ciona genetics. A high degree of polymorphism is useful for genetic mapping if one considers particular combinations of genetic backgrounds and techniques. To do that, it is necessary to establish laboratory strains instead of using samples with various genetic backgrounds. Rich genomic resources should facilitate the next stage of genetic map construction based on type I markers using coding sequences. The meiotic events that occur in crossing experiments carried out for mapping purposes should shed light on population genetics and speciation issues. The results of such investigations may provide feedback for comparative genomics and developmental genetics in the near future.

The characterization of the function, localization, and molecular interaction of cellular proteins results in a more direct description of the molecular mechanism underlying important developmental processes. The disclosure of genomic information has ushered in the postgenomic era, spearheaded by extensive protein analysis. To date, most gene expression and function studies of ascidians have been done at the gene level but not at the protein level. Again, decoding the Ciona genome provides a unique experimental system for proteomics in a variety of fields, including reproductive biology, developmental biology, neurobiology, immunology and evolutional biology. Proteomics in ascidians, however, has just recently appeared, and in this special issue, Kazuo Inaba et al. give us an outline of the technical processes used in proteomics and review the recent status of ascidian proteomics.

Finally Jean-Stéphane Joly et al. report improvements in closed-system culturing methods for Ciona intestinalis, because in the near future this cosmopolitan species will be used by a wider population of researches as a model organism in various fields of biology, including embryology and genetics. They describe the establishment of conditions for Ciona in closed systems, located close to or far from the sea. Especially at Gif-sur-Yvette in France, an inland site, they determine the optimal conditions for conserving artificial seawater, minimizing water pollution, and improved handling operations. A mixture of at least two types of live algae is better than any single-organism diet. With these maintenance protocols, they are now able to obtain several generations of Ciona in compact rearing devices. This system should make it easier to rear Ciona in laboratories, increasing the potential for the use of this organism in research.

This special focus could not include all possible contributions in the realm of developmental biology of ascidians; further contributions on this topic for submission to Developmental Dynamics are welcomed. A selection of 11 articles from currently expanding fields are included here to reflect an increasing interest of Ciona intestinalis as an experimental system of developmental biology. The simplicity of Ciona embryogenesis and its sequenced genome promise that the biology common to the chordates can be studied much more easily in the ascidian system than in vertebrates.