Unusual number and genomic organization of Hox genes in the tunicate Ciona intestinalis

Hox genes are organized in genomic clusters. In all organisms where their role has been studied, Hox genes determine developmental fate along the antero-posterior axis. Hence, these genes represent an ideal system for the understanding of relationships between the number and expression of genes and body organization. We report in this paper that the ascidian Ciona intestinalis genome appears to contain a single Hox gene complex which shows absence of some of the members found in all chordates investigated up to now. Furthermore, the complex appears to be either unusually long or split in different subunits. We speculate that such an arrangement of Hox genes does not correspond to the chordate primordial cluster but occurred independently in the ascidian lineage.     

Ion current activity and molecules modulating maturation and growth stages of ascidian (Ciona intestinalis) oocytes

Electrophysiological techniques were used to study ion currents in the ascidian Ciona intestinalis oocyte plasma membranes during different stages of growth and meiosis. Three stages (A, B, C) of immature oocytes were discriminated in the ovary, with the germinal vesicle (GV) showing specific different features of growth and maturation. Stage A (pre‐vitellogenic) oocytes exhibited the highest L‐type Ca²⁺current activity, and were incompetent for meiosis resumption. Stage B (vitellogenic) oocytes showed Na⁺ currents that remained high during the maturation, up to the post‐vitellogenic stage C oocytes. The latter had acquired meiotic competence, undergoing spontaneous maturation and interacting with the spermatozoon. However, fertilized oocytes did not produce normal larvae, suggesting that cytoplasmic maturation plays a specific role in embryo development. Spontaneous maturation was inhibited at low pH whereas trypsin was able to trigger germinal vesicle breakdown (GVBD) regardless of pH; in addition spontaneous maturation was not affected by removal of follicle cells or by inhibiting junctional communication between oocyte and follicle cells. Taken together these results imply: (i) Ca²⁺ and Na⁺ currents are involved in meiotic progression, growth, and acquisition of meiotic competence; (ii) trypsin‐like molecules may have a role as candidates for providing the physiological stimulus to resume meiosis. Finally, we provide evidence that follicle cells in Ciona are not involved in triggering GVBD as it occurs in other ascidians. Mol. Reprod. Dev. 76: 1084–1093, 2009. © 2009 Wiley‐Liss, Inc.     

Porcine relaxin, a 500 million-year-old hormone? the tunicate Ciona intestinalis has porcine relaxin.

The fossil record of tunicates reaches back to the upper Cambrian period. Ascidians have mobile, tadpole-like juvenile forms with a notochord, which inspired the classification of tunicates as Urochordata, i.e., predecessors of vertebrates. The genome of the tunicate Ciona intestinalis contains a relaxin coding region that is organized like a mammalian gene, i.e., signal peptide, B-chain domain, connecting peptide domain, followed by the A-chain domain with a stop codon after cysteine A-22. RNA-derived cDNA encodes a relaxin that is identical to the circulating form of the porcine hormone. In contrast to the porcine gene, the ascidian gene has no intron in the C-peptide domain, and in that respect is similar to the bombyxin gene of the silkworm. During the spawning period, only enough relaxin could be extracted and isolated from gonads of C. intestinalis for a partial sequence analysis. Remarkable as it may be, these findings suggest that relaxin is identical in pigs, whales, and the tunicate C. intestinalis.     

Proteome map of the neural complex of the tunicate Ciona intestinalis,the closest living relative to vertebrates

Ciona intestinalis (the common sea squirt) is the closest living chordate relative to vertebrates with cosmopolitan presence worldwide. It has a relatively simple nervous system and development, making it a widely studied alternative model system in neuroscience and developmental biology. The use of Ciona as a model organism has increased significantly after the draft genome was published. In this study, we describe the first proteome map of the neural complex of C. intestinalis. A total of 544 proteins were identified based on 1DE and 2DE FTMS/ITMSMS analyses. Proteins were annotated against the Ciona database and analyzed to predict their molecular functions, roles in biological processes, and position in constructed network pathways. The identified Ciona neural complex proteome was found to map onto vertebrate nervous system pathways, including cytoskeleton remodeling neurofilaments, cell adhesion through the histamine receptor signaling pathway, γ‐aminobutyric acid‐A receptor life cycle neurophysiological process, glycolysis, and amino acid metabolism. The proteome map of the Ciona neural complex is the first step toward a better understanding of several important processes, including the evolution and regeneration capacity of the Ciona nervous system.     

Specific induction of self-discrimination by follicle cells in Ciona intestinalis oocytes

Self-incompatibility, a mechanism that prevents self-fertilization in ascidians, is based on the ability of the oocyte vitelline coat to distinguish and accept only heterologous spermatozoa. In Ciona intestinalis self-discrimination is established during late oogenesis and is contributed or controlled by products of the overlying follicle cells. In this study we have further investigated the role of the follicle cells in the onset of self-discrimination by using in vitro maturation of ovarian oocytes deprived of the follicle cells and incubated with either autologous or heterologous follicle cells. Fertilization assays demonstrate that the action of the follicle cells is exerted even when they are detached from the vitelline coat and that only autologous follicle cells can promote the induction of self-sterility on the egg coat. Electron microscopy of the oocytes during maturation reveals that the switch from self-fertility to self-sterility is accompanied by the appearance of a thin electron-dense layer on the outer surface of the vitelline coat. We suggest that the formation of this layer is the result of the interaction between products of the follicle cells and the autologous vitelline coat.     

Morphological and physiological properties of non-striated muscle from the tunicate, Ciona intestinalis: parallels with vertebrate skeletal muscle

Non-striated muscle from the longitudinal body wall muscle in an adult ascidian (tunicate) is characterized morphologically and physiologically. These muscles are unique among chordate non-striated (i.e. 'smooth') muscles in that they are composed of discrete bundles of several small diameter (4-10 microns) muscle cells (fibers) arranged in parallel functional units. Each bundle is wrapped by a basal lamina, and at least some appear to be directly innervated at a neuromuscular junction similar to an end plate. In these regards, a bundle of Ciona smooth muscle cells is analogous to a skeletal muscle fiber of a vertebrate. This analogy also extends to physiological properties. Ciona muscle generates a rapid all-or-none Ca action potential which gives rise to a brisk twitch with brief latency. These anatomical and physiological adaptations are discussed in terms of the evolution of vertebrate skeletal muscle.     

Protochordate immunity. I. Primary immune response of the tunicate Ciona intestinalis to vertebrate erythrocytes

Serum from the tunicate Ciona intestinalis contains a low titer, natural agglutinin for a variety of vertebrate erythrocytes. Primary injections of 8.5 × 10³ human erythrocytes or 7.0 × 10² duck erythrocytes into the tunic tissues or perivisceral cavity were agglutinated and then cleared by phagocytosis within 24 hr. Higher concentrations of human or duck erythrocytes injected into the tunic tissues were encapsulated. Concomitant with clearance or encapsulation, serum agglutinin levels decreased and then returned to normal within 24 hr. Tunicates are classified taxonomically with the vertebrates in the phylum Chordata. Since phagocytosis and encapsulation reactions are typical of most invertebrates, the results of this study suggest that tunicate internal defense mechanisms are more closely allied to invertebrates than to vertebrates.     

Ion currents involved in oocyte maturation, fertilization and early developmental stages of the ascidian Ciona intestinalis

Electrophysiological techniques were used to study the role of ion currents in the ascidian Ciona intestinalis oocyte plasma membrane during different stages of growth, meiosis, fertilization and early development. Three stages of immature oocytes were discriminated in the ovary, with the germinal vesicle showing specific different features of growth and maturation. Stage-A (pre-vitellogenic) oocytes exhibited the highest L-type calcium current activity and were incompetent for meiosis resumption. Stage-B (vitellogenic) oocytes showed a progressive disappearance of calcium currents and the first appearance of sodium currents that remained high during the maturation process, up to the post-vitellogenic stage-C oocytes. The latter had acquired meiotic competence, undergoing spontaneous in vitro maturation and interacting with the spermatozoon. However, fertilized oocytes did not produce normal larvae, suggesting that cytoplasmic maturation may affect embryo development. In mature oocytes at the metaphase I stage, sodium currents were present and remained high up to the zygote stage. Oocytes fertilized in the absence of sodium showed significant reduction of the fertilization current amplitude and high development of anomalous "rosette" embryos. Current amplitudes became negligible in embryos at the 2- and 4-cell stage, whereas resumption of all the current activities occurred at the 8-cell embryo. Taken together, these results suggest: (i) an involvement of L-type calcium currents in initial oocyte meiotic progression and growth; (ii) a role of sodium currents at fertilization; (iii) a role of the fertilization current in ensuring normal embryo development.     

Studies on the receptors in Ciona intestinalis

Summary A photoreceptor type structure not previously described has been found in the dorsal wall of the cerebral vesicle of the tadpole larva of Ciona intestinalis. The membranes of this receptor are organised as tubules some 60–100 nm in diameter and up to 1.5 m long. The tubules are confined in bundles about 1.5 m in diameter, which extend from the cell surface into the cavity of the cerebral vesicle. These tubules are similar to those in the rhabdomeric type of photoreceptor. However, in the cells from which the tubule processes arise are structures typical of the bases of cilia, and found in ciliary type photoreceptors.I should like to thank Professor J. Z. Young, F. R. S. for his continuing encouragement and help, and Dr. R. Bellairs for the use of electron microscope facilities. Mr. R. Moss and Mrs. J. Hamilton gave excellent technical assistance.     

Molecular evidence from Ciona intestinalis for the evolutionary origin of vertebrate sensory placodes

Cranial sensory placodes are focused areas of the head ectoderm of vertebrates that contribute to the development of the cranial sense organs and their associated ganglia. Placodes have long been considered a key character of vertebrates, and their evolution is proposed to have been essential for the evolution of an active predatory lifestyle by early vertebrates. Despite their importance for understanding vertebrate origins, the evolutionary origin of placodes has remained obscure. Here, we use a panel of molecular markers from the Six, Eya, Pax, Dach, FoxI, COE and POUIV gene families to examine the tunicate Ciona intestinalis for evidence of structures homologous to vertebrate placodes. Our results identify two domains of Ciona ectoderm that are marked by the genetic cascade that regulates vertebrate placode formation. The first is just anterior to the brain, and we suggest this territory is equivalent to the olfactory/adenohypophyseal placodes of vertebrates. The second is a bilateral domain adjacent to the posterior brain and includes cells fated to form the atrium and atrial siphon of adult Ciona. We show this bares most similarity to placodes fated to form the vertebrate acoustico-lateralis system. We interpret these data as support for the hypothesis that sensory placodes did not arise de novo in vertebrates, but evolved from pre-existing specialised areas of ectoderm that contributed to sensory organs in the common ancestor of vertebrates and tunicates.     

Electron microscope studies on Tomentella

Summary Tomentella bombycina andT. fuscoferruginosa were examined by routine techniques of light and electron microscopy. Special attention was paid to the ultrastructure and inter-relationships of the generative subicular hyphae and subhymenial hyphae. The cell wall consisted of three layers in the generative subicular hyphae and only one layer in the subhymenial hyphae; this corresponded to the inner layer of the first and originated from it. Cells with two nuclei, normal mitochondria, large vacuoles, sparse endoplasmic reticulum and lomasomes were observed. Sometimes the nuclear membrane had unusually large pores and larger open gaps extending over 1/4 of the circumference. Septa with central dolipore-type pores were present. Clamp connections were not frequent. Finally, certain cells of the subhymenial hyphae appeared full of globular material, presumably lipids.     

Electron microscope studies on collagen

Summary The present paper describes the effect of intensive tryptic digestion of native tropocollagen (TC) macromolecules in solution. Contrary to earlier investigations it has been found that the trypsin treatment results in a fragmentation of the TC molecules. The addition of ATP to solutions exposed to the enzyme yields SLS fragments, 2250 Å in length. Comparison between these and normal SLS type aggregates shows that the scission occurs in a well-defined locus adjacent to the -1/2-line seen after positive staining. The significance of this finding is discussed.This study was supported by grant NB-02215-04 from the National Institute of Neurological Diseases and Blindness, Public Health Service, U.S.A. and a Student research fellowship from the Norwegian Research Council for Science and the Humanities. This aid is gratefully acknowledged.I am indebted to Mrs. J. Line Vaaland and Mr. B. V. Johansen for technical assistance.