Identification of a retinoic acid‐responsive neural enhancer in the Ciona intestinalis Hox1 gene

The Hox1 gene in the urochordate ascidian Ciona intestinalis (Ci‐Hox1) is expressed in the nerve cord and epidermis. We identified a nerve cord enhancer in the second intron of Ci‐Hox1, and demonstrated that retinoic acid (RA) plays a major role in activating this enhancer. The enhancer contained a putative retinoic acid‐response element (RARE). Mutation of the RARE in the Ci‐Hox1 nerve cord enhancer only partially abolished the enhancer activity. Genes encoding RA synthase and the RA receptor were knocked down using specific antisense morpholino oligos (MOs), and injection of embryos with these MOs resulted in the complete disappearance of epidermal expression of Ci‐Hox1 and reduction of neural expression. However, nerve cord expression was not completely repressed. These results suggest that the nerve cord enhancer is activated by two partially redundant pathways; one RA‐dependent and one RA‐independent.     

The central nervous system of the ascidian larva: mitotic history of cells forming the neural tube in late embryonic Ciona intestinalis

Ascidian larvae develop after an invariant pattern of embryonic cleavage. Fewer than 400 cells constitute the larval central nervous system (CNS), which forms without either extensive migration or cell death. We catalogue the mitotic history of these cells in Ciona intestinalis, using confocal microscopy of whole-mount embryos at stages from neurulation until hatching. The positions of cells contributing to the CNS were reconstructed from confocal image stacks of embryonic nuclei, and maps of successive stages were used to chart the mitotic descent, thereby creating a cell lineage for each cell. The entire CNS is formed from 10th- to 14th-generation cells. Although minor differences exist in cell position, lineage is invariant in cells derived from A-line blastomeres, which form the caudal nerve cord and visceral ganglion. We document the lineage of five pairs of presumed motor neurons within the visceral ganglion: one pair arises from A/A 10.57, and four from progeny of A/A 9.30. The remaining cells of the visceral ganglion are in their 13th and 14th generations at hatching, with most mitotic activity ceasing around 85% of embryonic development. Of the approximately 330 larval cells previously reported in the CNS of Ciona, we document the lineage of 226 that derive predominantly from A-line blastomeres.     

Chondroitin 4‐O‐sulfotransferases are required for cell adhesion and morphogenesis in the Ciona intestinalis embryo

Chondroitin sulfate (CS) is a carbohydrate component of proteoglycans. Several types of sulfotransferases determine the pattern of CS sulfation, and thus regulate the biological functions of proteoglycans. The protochordate ascidians are the closest relatives of vertebrates, but the functions of their sulfotransferases have not been investigated. Here, we show that two chondroitin 4‐O‐sulfotransferases (C4STs) play important roles in the embryonic morphogenesis of the ascidian Ciona intestinalis. Ci‐C4ST‐like1 is predominantly expressed in the epidermis and muscle. Epidermal and muscle cells became spherical upon the injection of a Ci‐C4ST‐like1‐specific morpholino oligo (MO), thus suggesting weakened cell adhesion. Co‐injection of a Ci‐C4ST‐like1‐expressing transgene rescued the phenotype, suggesting that the effects of the MO were specific. Ci‐C4ST‐like3 was expressed in the central nervous system, muscle, and mesenchyme. A specific MO appeared to affect cell adhesion in the epidermis and muscle. Convergent extension movement of notochordal cells was also impaired. Forced expression of Ci‐C4ST‐like3 restored normal morphogenesis, suggesting that the effects of the MO were specific. The present study suggests that Ci‐C4ST‐like1 and Ci‐C4ST‐like3 are required for cell adhesion mainly in the epidermis and muscle.     

Efficient targeted mutagenesis of the chordate Ciona intestinalis genome with zinc-finger nucleases

Zinc‐finger nucleases (ZFNs) are engineered nucleases that induce DNA double‐strand breaks (DSBs) at target sequences. They have been used as tools for generating targeted mutations in the genomes of multiple organisms in both animals and plants. The DSB induced by ZFNs is repaired by non‐homologous end joining (NHEJ) or by homologous recombination (HR) mechanisms. Non‐homologous end joining induces some errors because it is independent of a reference DNA sequence. Through the NHEJ mechanism, ZFNs generate insertional or deletional mutations at the target sequence. We examined the usability, specificity and toxicity of ZFNs in the basal chordate Ciona intestinalis. As the target of ZFNs, we chose an enhanced green fluorescent protein (EGFP) gene artificially inserted in the C. intestinalis genome because this locus is neutral for the development and growth of C. intestinalis, and the efficiency of mutagenesis with ZFNs can thus be determined without any bias. We introduced EGFP ‐ZFN mRNAs into the embryos of an EGFP ‐transgenic line and observed the mutation frequency in the target site of EGFP . We also examined the effects of the EGFP ‐ZFNs at off‐target sites resembling the EGFP target sequence in the C. intestinalis genome in order to examine the specificity of ZFNs. We further investigated the influence of ZFNs on embryogenesis, and showed that adequate amounts of ZFNs, which do not disrupt embryogenesis, can efficiently induce mutations on the on‐target site with less effect on the off‐target sites. This suggests that target mutagenesis with ZFNs will be a powerful technique in C. intestinalis.     

Structure of ocellus photoreceptors in the ascidian Ciona intestinalis larva as revealed by an anti‐arrestin antibody

Although there have been several studies on the structure of the ocellus photoreceptors in ascidian tadpole larvae using electron microscopy, the overall structure of these photoreceptor cells, especially the projection sites of the axons, has not been revealed completely. The number of photoreceptor cells is also controversial. Here, the whole structure of the ocellus photoreceptors in the larvae of the ascidian Ciona intestinalis was revealed by using an anti‐arrestin (anti–Ci‐Arr) antibody. The cell bodies of 30 photoreceptor cells covered the right side of the ocellus pigment cell and their outer segments extended through the pigment cell into the pigment cup. The axons of the photoreceptor cells were bundled together ventro‐posteriorly in a single tract extending towards the midline. The nerve terminals diverged antero‐posteriorly at the midline of the posterior sensory vesicle (SV). The Ci‐arr gene was expressed throughout the SV at the embryonic mid‐tailbud stage and it became restricted to the neighborhood of the ocellus pigment when ocellus pigmentation occurred. At the same time, the Ci‐Arr protein was first detected, suggesting that the photoreceptor cells began to differentiate. The development of photoreceptor cells after hatching was also investigated using the anti–Ci‐Arr antibody. Three hours after hatching, the photoreceptor terminals began to ramify and then expanded. Previous behavioral analysis showed that the larvae did not respond to the step‐down of light until 2 h after hatching and then the photoresponse became robust. Accordingly, our results suggest that growth of the photoreceptor terminal is critical for the larvae to become photoresponsive. © 2005 Wiley Periodicals, Inc. J Neurobiol, 2005     

Cupular organs in two species of Corella (Tunicata: Ascidiacea)

Abstract. Simple cupular organs similar to those described in Ciona intestinalis were observed in Corella eumyota. They consist of a macula containing the cell bodies of 20–30 primary sensory neurons whose cilia project into a dome‐ or finger‐shaped structure, the cupula. Rather than being found in the mantle lining as in C. intestinalis, the organs were located on the atrial surface of the branchial sac. The sensory innervation was examined in whole‐mount preparations using anti‐tubulin immunohistochemistry. Sensory neurons in C. eumyota showed no immunoreactivity with antisera raised against gonadotropin‐releasing hormone (GnRH). A novel, elongated sense organ termed the cupular strand was found in Corella inflata. It has the same basic components as the simple type of cupular organ but consists of a single, long structure containing ～1500 sensory cells. Located on the atrial surface of the branchial sac, it extends along the midline of the dorsal fold, from the gonoduct openings almost as far as the brain. Preparations were examined using optical and electron microscopy. Nerves and cilia were visualized by anti‐tubulin immunofluorescence microscopy. It was possible to follow the sensory axons from the macula of the cupular strand to points where they joined branches of the visceral nerve, which enters a nerve root at the back of the brain. In C. inflata the sensory cell bodies and their axons were immunoreactive not only with anti‐tubulin but also with an antiserum raised against Tunicate I GnRH. There was no immunoreactivity, however, with Chicken II and catfish GnRH antisera. All three GnRH antisera labeled the dorsal strand plexus, a structure associated with production of GnRH in its role as a reproductive hormone. We concluded that the GnRH‐like molecule labeled in sensory neurons differs from the form of GnRH found in the dorsal strand plexus, and may have a different function, perhaps in the neural control of ciliary activity. The function of the cupular organs in species of Corella has not yet been investigated physiologically, but by analogy with such structures in other metazoans, cupular organs are probably hydrodynamic sensors registering local disturbances or changes in water flow through the atrial cavity.     

Toxicity assessment of the antifouling compound zinc pyrithione using early developmental stages of the ascidian Ciona intestinalis

Abstract

This study investigated the toxicity of zinc pyrithione (Zpt) on the early stages of development of the ascidian Ciona intestinalis. Larval morphological abnormalities were studied after the exposure of C. intestinalis embryos at different stages of development. The median effective concentrations (EC₅₀) ranged from 226–590 nM. The larval settlement stage was the most sensitive to Zpt. Toxic effects of Zpt on larval settlement were detected at 9 nM (EC₁₀). The inhibition of C. intestinalis embryonic development was also used to study the loss of toxicity in Zpt solutions exposed to direct sunlight and laboratory UV light. The results showed that the toxicity of Zpt solutions decreased but did not disappear after 4 h exposure to direct sunlight (EC₅₀ = 484 nM) or UV light (EC₅₀ = 453 nM), compared to control Zpt solutions prepared in dark conditions. On the basis of the present data, predicted no effect concentrations of Zpt to C. intestinalis larvae are lower than predicted environmental concentrations of Zpt in certain polluted areas and therefore, may pose a risk to C. intestinalis populations.     

Neuronal form in the central nervous system of the tadpole larva of the ascidian Ciona intestinalis.

The dorsal tubular central nervous system (CNS) of the ascidian tadpole larva is a diagnostic feature by which the chordate affinities of this group, as a whole, are recognized. We have used two methods to identify larval neurons of Ciona intestinalis. The first is serial electron microscopy (EM), as part of a dedicated study of the visceral ganglion (1), and the second is the transient transfection of neural plate progeny with green fluorescent protein (GFP) (2), to visualize the soma and its neurites of individual neurons in whole-mounted larvae of C. intestinalis. Our observations reveal that ascidian larval neurons are simple inform, with a single axonal neurite arising from a soma that is either monopolar or has only very few, relatively simple neurites arising from it, as part of a presumed dendritic arbor. Somata in the visceral ganglion giving rise to axons descending in the caudal nerve cord are presumed to be those of motor neurons.     

A conserved role for the MEK signalling pathway in neural tissue specification and posteriorisation in the invertebrate chordate,the ascidian Ciona intestinalis

Ascidians are invertebrate chordates with a larval body plan similar to that of vertebrates. The ascidian larval CNS is divided along the anteroposterior axis into sensory vesicle, neck, visceral ganglion and tail nerve cord. The anterior part of the sensory vesicle comes from the a-line animal blastomeres, whereas the remaining CNS is largely derived from the A-line vegetal blastomeres. We have analysed the role of the Ras/MEK/ERK signalling pathway in the formation of the larval CNS in the ascidian, Ciona intestinalis. We show evidence that this pathway is required, during the cleavage stages, for the acquisition of: (1) neural fates in otherwise epidermal cells (in a-line cells); and (2) the posterior identity of tail nerve cord precursors that otherwise adopt a more anterior neural character (in A-line cells). Altogether, the MEK signalling pathway appears to play evolutionary conserved roles in these processes in ascidians and vertebrates, suggesting that this may represent an ancestral chordate strategy.     

Trunk lateral cells are neural crest-like cells in the ascidian Ciona intestinalis: insights into the ancestry and evolution of the neural crest

Neural crest-like cells (NCLC) that express the HNK-1 antigen and form body pigment cells were previously identified in diverse ascidian species. Here we investigate the embryonic origin, migratory activity, and neural crest related gene expression patterns of NCLC in the ascidian Ciona intestinalis. HNK-1 expression first appeared at about the time of larval hatching in dorsal cells of the posterior trunk. In swimming tadpoles, HNK-1 positive cells began to migrate, and after metamorphosis they were localized in the oral and atrial siphons, branchial gill slits, endostyle, and gut. Cleavage arrest experiments showed that NCLC are derived from the A7.6 cells, the precursors of trunk lateral cells (TLC), one of the three types of migratory mesenchymal cells in ascidian embryos. In cleavage arrested embryos, HNK-1 positive TLC were present on the lateral margins of the neural plate and later became localized adjacent to the posterior sensory vesicle, a staging zone for their migration after larval hatching. The Ciona orthologues of seven of sixteen genes that function in the vertebrate neural crest gene regulatory network are expressed in the A7.6/TLC lineage. The vertebrate counterparts of these genes function downstream of neural plate border specification in the regulatory network leading to neural crest development. The results suggest that NCLC and neural crest cells may be homologous cell types originating in the common ancestor of tunicates and vertebrates and support the possibility that a putative regulatory network governing NCLC development was co-opted to produce neural crest cells during vertebrate evolution.     

Developmental expression of tryptophan hydroxylase gene in <Emphasis Type="Italic">Ciona intestinalis</Emphasis>

To describe the serotonergic system in a tunicate larva, we cloned a gene encoding for tryptophan hydroxylase (TPH), the rate-limiting enzyme in serotonin synthesis, in the ascidian Ciona intestinalis and studied its expression pattern during development. Ci-TPH expression was found from tailbud stage in the precursor cells of the visceral ganglion and in the tail. In the larva, TPH-expressing neurons formed two clusters in the anterior central nervous system at the level of the visceral ganglion. Moreover, we found Ci-TPH expression at the level of the muscle cells of the tail and suggested that this localisation might be at the level of neuro-muscolar junctions. Moreover, we discussed the involvement of serotonin in the control of larval locomotory activity.     

The non-LTR retrotransposons in Ciona intestinalis: new insights into the evolution of chordate genomes

Background  

Non-long terminal repeat (non-LTR) retrotransposons have contributed to shaping the structure and function of genomes. In silico and experimental approaches have been used to identify the non-LTR elements of the urochordate Ciona intestinalis. Knowledge of the types and abundance of non-LTR elements in urochordates is a key step in understanding their contribution to the structure and function of vertebrate genomes.     

Morphological evidence that the molecularly determined Ciona intestinalis type A and type B are different species: Ciona robusta and Ciona intestinalis

Ciona intestinalis is considered a widespread and easily recognizable tunicate, the sister group of vertebrates. In recent years, molecular studies suggested that C. intestinalis includes at least two cryptic species, named ‘type A’ and ‘type B’, morphologically indistinguishable. It is dramatic to certify that two different species may be hidden under the name of a species widely used as a model species in biological researches. This raised the problem of identifying diagnostic morphological characters capable of distinguishing these types. We compared the morphology of specimens belonging to the two types and found that only type A specimens possess tunic tubercular prominences, allowing unambiguous discrimination. Remarkably, these structures were already described as distinctive of the Japanese species Ciona robusta, Hoshino and Tokioka, 1967; later synonymized under C. intestinalis (sensu Millar, 1953). In this study, we have confirmed that C. intestinalis type A corresponds to C. robusta. Based on the geographic distribution of C. intestinalis type B, and considering that the original C. intestinalis species was described from North European waters, we determined that C. intestinalis type B corresponds to C. intestinalis as described by Millar in 1953 and possibly to Linnaeus' Ascidia intestinalis L., 1767 for which we have deposited a neotype (from Roscoff, France) and for which we retain the name Ciona intestinalis (Linnaeus, 1767).     

Evidence that talin alternative splice variants from Ciona intestinalis have different roles in cell adhesion

Background  

Talins are large, modular cytoskeletal proteins found in animals and amoebozoans such as Dictyostelium discoideum. Since the identification of a second talin gene in vertebrates, it has become increasingly clear that vertebrate Talin1 and Talin2 have non-redundant roles as essential links between integrins and the actin cytoskeleton in distinct plasma membrane-associated adhesion complexes. The conserved C-terminal I/LWEQ module is important for talin function. This structural element mediates the interaction of talins with F-actin. The I/LWEQ module also targets mammalian Talin1 to focal adhesion complexes, which are dynamic multicomponent assemblies required for cell adhesion and cell motility. Although Talin1 is essential for focal adhesion function, Talin2 is not targeted to focal adhesions. The nonvertebrate chordate Ciona intestinalis has only one talin gene, but alternative splicing of the talin mRNA produces two proteins with different C-terminal I/LWEQ modules. Thus, C. intestinalis contains two talins, Talin-a and Talin-b, with potentially different activities, despite having only one talin gene.     

Ephrin-mediated restriction of ERK1/2 activity delimits the number of pigment cells in the Ciona CNS

Recent evidence suggests that ascidian pigment cells are related to neural crest-derived melanocytes of vertebrates. Using live-imaging, we determine a revised cell lineage of the pigment cells in Ciona intestinalis embryos. The neural precursors undergo successive rounds of anterior–posterior (A–P) oriented cell divisions, starting at the blastula 64-cell stage. A previously unrecognized fourth A–P oriented cell division in the pigment cell lineage leads to the generation of the post-mitotic pigment cell precursors. We provide evidence that MEK/ERK signals are required for pigment cell specification until approximately 30 min after the final cell division has taken place. Following each of the four A–P oriented cell divisions, ERK1/2 is differentially activated in the posterior sister cells, into which the pigment cell lineage segregates. Eph/ephrin signals are critical during the third A–P oriented cell division to spatially restrict ERK1/2 activation to the posterior daughter cell. Targeted inhibition of Eph/ephrin signals results in, at neurula stages, anterior expansion of both ERK1/2 activation and a pigment cell lineage marker and subsequently, at larval stages, supernumerary pigment cells. We discuss the implications of these findings with respect to the evolution of the vertebrate neural crest.     

Identification of testis‐specific ubiquitin‐conjugating enzyme in the ascidian Ciona intestinalis

The ubiquitin–proteasome system is known to play a key role in fertilization in ascidians, sea urchins, and mammals. To obtain insights into the ubiquitin‐conjugating enzymes (Ube2) involved in reproductive systems, we systematically explored Ube2 enzymes expressed in the testis of the ascidian Ciona intestinalis. Here, we report cDNA cloning and characterization of a novel type of Ube2r (Ci0100152677) that is capable of making a thiolester bond with ubiquitin. Northern analysis, whole‐mount in situ hybridization and immunocytochemistry indicate that this enzyme is exclusively expressed in the testis, mainly in the germ cells during the late stage of spermatogenesis, and is localized in the sperm head and tail, suggesting possible participation in fertilization or spermatogenesis/spermiogenesis. Mol. Reprod. Dev. 77: 640–647, 2010. © 2010 Wiley‐Liss, Inc.