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The larvacean Oikopleura dioica is a planktonic chordate and an emerging model organism with a short life cycle of 5 days that belongs toTunicata (Urochordata), the sister clade of vertebrates. It is characterized by the rapid development of a tadpole-shaped body. Organ formation in the trunk proceeds within 7 h after the hatching of the tailbud larvae at 3 h after fertilization (hpf) and is completed at 10 hpf, giving rise to fully functional juveniles as miniature adult form. Serial block face scanning electron microscopy was used to acquire ~ 2000 serial transverse section images of a 3 hpf larva and a 10 hpf juvenile to characterize the structures and cellular composition of the trunk and organs using 3D images and movies. Germ cells were found to fuse and establish a central syncytial cell in the gonad as early as 10 hpf. Larval development gave rise to functional organs after several rounds of cell division through trunk morphogenesis. The feature would make O. dioica ideal for analyzing cellular behaviors during morphogenetic processes using live imaging. The detailed descriptions of the larvae and juveniles provided in this study can be utilized as the start and end points of organ morphogenesis in this rapidly developing organism.

SummaryCentral nervous system (CNS) regeneration extent is highly diverse across the metazoans, with adult mammals demonstrating limited ability1,2. Understanding how neurons regenerate following injury remains a central challenge in regenerative medicine. Although conserved pathways associated with neural regeneration have been identified3,4, a study describing the stepwise morphogenetic changes that take place throughout a complete CNS regeneration is lacking. Utilizing the highly regenerative tunicate model Polycarpa mytiligera5, we characterized the morphological, cell proliferation, and transcriptomic dynamics that lead to entire CNS regeneration. The regenerated CNS of adult P. mytiligera expressed key neurodevelopmental markers that are not otherwise present in the adult CNS. Removal of the entire CNS resulted in high cell proliferation in the regenerated area. Transcriptome analysis revealed enhanced stem-cell related gene activity, with high expression of P53 and piRNA pathways preceding the activation of Notch, Wnt, and Nanos pathways. The CNS regeneration atlas created here depicts the transcriptomic landscape of the entire CNS regeneration process, revealing the core pathways that regulate neuronal response to injury, and the regeneration stage at which they are most pronounced. The molecular and cellular mechanisms controlling regenerative capacity that this atlas reveals could be used to develop approaches to enhancing neurogenesis in closely-related chordate species, including humans.

Glial cells play important roles in the development and homeostasis of metazoan nervous systems. However, while their involvement in the development and function in the central nervous system (CNS) of vertebrates is increasingly well understood, much less is known about invertebrate glia and the evolutionary history of glial cells more generally. An investigation into amphioxus glia is therefore timely, as this organism is the best living proxy for the last common ancestor of all chordates, and hence provides a window into the role of glial cell development and function at the transition of invertebrates and vertebrates. We report here our findings on amphioxus glia as characterized by molecular probes correlated with anatomical data at the transmission electron microscopy (TEM) level. The results show that amphioxus glial lineages express genes typical of vertebrate astroglia and radial glia, and that they segregate early in development, forming what appears to be a spatially separate cell proliferation zone positioned laterally, between the dorsal and ventral zones of neural cell proliferation. Our study provides strong evidence for the presence of vertebrate-type glial cells in amphioxus, while highlighting the role played by segregated progenitor cell pools in CNS development. There are implications also for our understanding of glial cells in a broader evolutionary context, and insights into patterns of precursor cell deployment in the chordate nerve cord.

Loss of the brain’s functional ability is a common symptom of aging and neurodegenerative diseases1,2. While the genetic and molecular mechanisms underlying human neurodegeneration are studied in-depth3–6, very little is known about the evolutionary origin of these traits and their involvement in loss of nervous system function in aged invertebrate species. Here we study evolutionarily conserved elements of brain degeneration using the colonial chordate model species Botryllus schlosseri. B. schlosseri reproduces both sexually and asexually7, with adult brains regenerating and degenerating multiple times throughout its adult life. Combining microscopy, transcriptomics and behavioral assays, we characterized adult brains from diverse stages and ages. We found that the number of neurons fluctuates each week, reaching a maximum of ∼1000 cells, and thereafter decreasing while the number of immunocytes increases. Comparing the number of neurons in the adult brains of young and old colonies, we found that older brains are smaller and contain fewer cells. Both during weekly degeneration cycles and overall with age, the decrease in neuron number correlates with reduced response to stimuli and with significant changes in the expression of genes with mammalian homologs associated with neural stem cells and neurodegenerative pathways. These results suggest persistent neural stem cell activity across ages and that cellular and molecular mechanisms of neurodegeneration are evolutionary conserved between tunicates and humans.

Background Ascidians (phylum Chordata, class Ascidiacea) represent the closest living invertebrate relatives of the vertebrates and constitute an important model for studying the evolution of chordate development. The solitary ascidian Polycarpa mytiligera exhibits a robust regeneration ability, unique among solitary chordates, thus offering a promising new model for regeneration studies. Understanding its reproductive development and establishing land-based culturing methods is pivotal for utilizing this species for experimental studies. Its reproduction cycle, spawning behavior, and developmental processes were therefore studied in both the field and the lab, and methods were developed for its culture in both open and closed water systems. Results Field surveys revealed that P. mytiligera’s natural recruitment period starts in summer (June) and ends in winter (December) when seawater temperature decreases. Laboratory experiments revealed that low temperature (21 °C) has a negative effect on its fertilization and development. Although spontaneous spawning events occur only between June and December, we were able to induce spawning under controlled conditions year-round by means of gradual changes in the environmental conditions. Spawning events, followed by larval development and metamorphosis, took place in ascidians maintained in either artificial or natural seawater facilities. P. mytiligera’s fast developmental process indicated its resemblance to other oviparous species, with the larvae initiating settlement and metamorphosis at about 12 h post-hatching, and reaching the juvenile stage 3 days later. Conclusions Polycarpa mytiligera can be induced to spawn in captivity year-round, independent of the natural reproduction season. The significant advantages of P. mytiligera as a model system for regenerative studies, combined with the detailed developmental data and culturing methods presented here, will contribute to future research addressing developmental and evolutionary questions, and promote the use of this species as an applicable model system for experimental studies.

BackgroundThe study of retroviruses dates back to the early 1900s during investigations on neoplastic diseases in chickens. Subsequently, Robert Gallo reported the first human retrovirus HLTV in 1980. What we report here is not an archetypal retrovirus, but the discovery of an oncogenic giant microbial agent with a mega-genome, where the transforming retroviral nature co-exists with multiple archaeal oncogenes.MethodsAfter their isolation from human T cells Leukaemia, these organisms were examined at electron microscopy, tested for reverse transcriptase activity, fully sequenced, used for transformation tests on NIH-3T3 cells in vitro and tumours formation in mice. Same type of particles were also isolated from Canine Transmissible Venereal Tumour (CTVT), the oldest contagious cancer in nature.ResultsEM showed the presence of giant viral particles displaying retroviral antigens. These microbial entities harbour in their mega-genome a transforming retroviral kinase, cell-based oncogenes and have reverse transcriptase activity. The purified viral particles transformed NIH-3T3 cells and induced metastatic tumours in nude mice, three weeks post infection. Ruling out the possible presence of filterable retroviruses, a filtered supernatant did not display RT activity and did not transforms.ConclusionsWe discovered an ancestral microbial agent, acutely transforming. For its giant dimensions, the ability to retain the Gram stain, the presence of a mega-genome and its retroviral nature, we tentatively named the agent Retro-Giant-Virus (RGV). However, distinct from amoeba giant Mimiviruses, this transforming human agent has a different nature and does not require for its isolation amoeba co-culture, since amoeba is not its natural host.The morphology, biology and genetic features allocate this mammalian giant microbe halfway in between a classic oncogenic virus and an infectious cancer cell. Its transforming nature goes with its constant ability to induce tumours formation in mice.

SUMMARY Colonial tunicates are the only chordate that possess two distinct developmental pathways to produce an adult body: either sexually through embryogenesis or asexually through a stem cell-mediated renewal termed blastogenesis. Using the colonial tunicate Botryllus schlosseri, we combine transcriptomics and microscopy to build an atlas of the molecular and morphological signatures at each developmental stage for both pathways. The general molecular profiles of these processes are largely distinct. However, the relative timing of organogenesis and ordering of tissue-specific gene expression are conserved. By comparing the developmental pathways of B. schlosseri with other chordates, we identify hundreds of putative transcription factors with conserved temporal expression. Our findings demonstrate that convergent morphology need not imply convergent molecular mechanisms but that it showcases the importance that tissue-specific stem cells and transcription factors play in producing the same mature body through different pathways., Graphical Abstract, In Brief Kowarsky et al. present a developmental atlas of colonial tunicates. They show how sexual and asexual reproduction processes, despite having largely distinct molecular programs, share similar timing of organ formation. Tissue-specific stem cells and transcription factors display similar genetic dynamics in different developmental pathways to produce the same organism.

SummaryAll chordates, including urochordates such as tunicates, develop through embryogenesis. The chordate larvae of colonial tunicates metamorphose to lose all chordate structures such as notochord, neural tube, segmented musculature, and then develop by asexual reproduction [blastogenesis], whereby stem cells form tissues and organs. These two developmental pathways establish the same body axis, morphogenetic patterning and organ formation. It is unknown if this convergent morphology implies convergent cellular and molecular mechanisms, and whether the stem cells that mediate these processes differ. Using the colonial tunicateBotryllus schlosseri, we combined transcriptome sequencing and multiple microscopy techniques to study the molecular and morphological signatures of cells at each developmental stage of embryogenesis and blastogenesis. This revealed that the molecular programs are distinct, but the blastogenic tissue-specific stem cells and embryonic precursor populations share similar molecular profiles. By comparing embryogenesis in other chordates we found shared developmental principles, highlighting transcription factors as key evolutionary conserved elements. This study establishes a platform for advancing the science of stem cell biology and regulation of development and regeneration.

SummaryPrevious studies on purified bovine rod outer segments (OS) disks pointed to Oxidative Phosphorylation (OXPHOS) as being the most likely mechanism involved in ATP production, as yet not fully understood, to support the first phototransduction steps. Bovine and murine rod OS disks, devoid of mitochondria, would house respiratory chain complexes I to IV and ATP synthase, similar to mitochondria. Zebrafish ( Danio rerio) is a well-suited animal model to study vertebrate embryogenesis as well as the retina, morphologically and functionally similar to its human counterpart. The present article reports fluorescence and Transmission Electron Microscopy colocalization analyses of respiratory complexes I and IV and ATP synthase with zpr3, the rod OS marker, in adult and larval zebrafish retinas. MitoTracker Deep Red 633 staining and assays of complexes I and III–IV activity suggest that those proteins are active in OS. Results show that an extramitochondrial aerobic metabolism is active in the zebrafish OS at 4 and 10 days of larval development, as well as in adults, suggesting that it is probably maintained during embryogenesis. Data support the hypothesis of an extramitochondrial aerobic metabolism in the OS of zebrafish.

Thaliaceans are pelagic tunicates that play a key role in trophic chains of the oceans. In the field of tunicate immunity, a notable gap is the lack of data on their inflammatory response. The common salp, Thalia democratica, possesses scant immunocytes, represented by a phagocytic line (hyaline amebocytes) and a mast cell-like line (granular cells). We aimed to provide the first investigation of defense reactions upon exposure to a large amount of bacteria (Bacillus clausii). We detected (i) bacterial phagocytosis by hyaline amebocytes, (ii) degradation of phagocytizing hyaline amebocytes in the tunic after transcellular diapedesis from the hemocoel, and (iii) release of heparin, histamine, and TNF-α by granular cells. Cell degranulation and phagocytosis occurred in epidermal cells lining the hemocoel, and an excess of mucus was observed in the post-branchial gut, causing a functional inhibition of cilia and microvilli. These findings indicate multi-step events comparable to an inflammation involving responses at both tissue and organismal levels.

The colonial ascidian Botryllus schlosseri is a cosmopolitan, marine filter feeder, introduced as a laboratory research organism in the 1950s. Currently, it is widely used in many laboratories to investigate a variety of biological questions. Recently, it has become a species of concern, as it is an invasive species in many coastal environments. Here, we review studies on the geographical distribution of the species, sexual and asexual reproduction in the field, tolerance to temperature, salinity and anthropogenic activity, polychromatism, enzymatic polymorphism, and the genetic basis of pigmentation. Studying the relationship between genetic polymorphism and the adaptation of B. schlosseri to environmental stress is a challenge of future research and will improve our understanding of its evolutionary success and invasive potential.

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).

Summary The colonial tunicate Botryllus schlosseri is a widespread filter-feeding ascidian that lives in shallow waters and is easily reared in aquaria. Its peculiar blastogenetic cycle, characterized by the presence of three blastogenetic generations (filtering adults, buds, and budlets) and by recurrent generation changes, has resulted in over 60 years of studies aimed at understanding how sexual and asexual reproduction are coordinated and regulated in the colony. The possibility of using different methodological approaches, from classical genetics to cell transplantation, contributed to the development of this species as a valuable model organism for the study of a variety of biological processes. Here, we review the main studies detailing rearing, staging methods, reproduction and colony growth of this species, emphasizing the asymmetry in sexual and asexual reproduction potential, sexual reproduction in the field and the laboratory, and self- and cross-fertilization. These data, opportunely matched with recent tanscriptomic and genomic outcomes, can give a valuable help to the elucidation of some important steps in chordate evolution. genesis 53:105–120, 2015. © 2014 Wiley Periodicals, Inc.

Aberrant mitochondrial dynamics contribute to disease conditions. Genetic and pharmacological inhibition of USP8 ameliorates mitochondrial phenotypes deriving from loss of function of Drosophila PINK1 and Parkin., Aberrant mitochondrial dynamics disrupts mitochondrial function and contributes to disease conditions. A targeted RNA interference screen for deubiquitinating enzymes (DUBs) affecting protein levels of multifunctional mitochondrial fusion protein Mitofusin (MFN) identified USP8 prominently influencing MFN levels. Genetic and pharmacological inhibition of USP8 normalized the elevated MFN protein levels observed in PINK1 and Parkin-deficient models. This correlated with improved mitochondrial function, locomotor performance and life span, and prevented dopaminergic neurons loss in Drosophila PINK1 KO flies. We identified a novel target antagonizing pathologically elevated MFN levels, mitochondrial dysfunction, and dopaminergic neuron loss of a Drosophila model of mitochondrial dysfunction.

Tunicates are unique animals for studying the origin and evolution of vertebrates because they are considered vertebrates' closest living relatives and share the vertebrate body plan and many specific features. Both possess neural placodes, transient thickenings of the cranial ectoderm that give rise to various types of sensory cells, including axonless secondary mechanoreceptors. In vertebrates, these are represented by the hair cells of the inner ear and the lateral line, which have an apical apparatus typically bearing cilia and stereovilli. In tunicates, they are found in the coronal organ, which is a mechanoreceptor located at the base of the oral siphon along the border of the velum and tentacles and is formed of cells bearing a row of cilia and short microvilli. The coronal organ represents the best candidate homolog for the vertebrate lateral line. To further understand the evolution of secondary sensory cells, we analysed the development and cytodifferentiation of coronal cells in the tunicate ascidian Ciona intestinalis for the first time. Here, coronal sensory cells can be identified as early as larval metamorphosis, before tentacles form, as cells with short cilia and microvilli. Sensory cells gradually differentiate, acquiring hair cell features with microvilli containing actin and myosin VIIa; in the meantime, the associated supporting cells develop. The coronal organ grows throughout the animal's lifespan, accompanying the growth of the tentacle crown. Anti-phospho Histone H3 immunostaining indicates that both hair cells and supporting cells can proliferate. This finding contributes to the understanding of the evolution of secondary sensory cells, suggesting that both ancestral cell types were able to proliferate and that this property was progressively restricted to supporting cells in vertebrates and definitively lost in mammals.

Background:Robert Gallo reported the first human retrovirus HLTV in 1980. What we report here is the first human giant virus, Mimivirus-like, with a retroviral core.Methods: The isolation of human giant viruses from human T cells Leukaemia was performed on 25% sucrose gradient. The purified viral pellet was examined using electron microscopy (EM), after immunolabelling with anti-FeLV gag p27 moAb, used for its ability to bind conserved epitopes among different mammalian retroviruses. RNA extracted from the viral particles was amplified with the Pan Retrovirus PCR technique that targets the most conserved VLPQG and YMDD in the Pol region of different retroviruses. The amplified genes were sequenced and analyzed with molecular phylogenetic tests.Results:EM showed the presence of ~400 nm giant viruses, mimivirus-like, specifically labelled by anti-FeLV gag p27 Ab. RNA extracted from the particles contained retroviral genes. Molecular phylogenetic analyses of 150 bp amplicon product, compared with the same size amplicons of the Pol gene of diverse retroviruses, showed that the retro-giant viruses are a distinct branch, missing from the current classification of retroviruses.Conclusions:Although sharing some of the morphological features with Mimiviruses, this human giant virus differs substantially from environmental DNA-giant viruses isolated so far, in that it manifests a unique mammalian transforming retroviral core and T cell tropism. The virus should not be confused with a classic human retrovirus nor even a large human retrovirus, but an ancestral human giant virus, mimivirus-like, with a mammalian retroviral core. Certainly, the oncogenic potential of the viral particle and its T cell tropism is of concern and further studies are needed to clarify the role of this giant virus in human diseases and evolution of archetypal retroviruses.

The tunicate ascidians are nonvertebrate chordates that possess mechanoreceptor cells in the coronal organ in the oral siphon, which monitor the incoming water flow. Like vertebrate hair cells, the mechanoreceptor–coronal cells are secondary sensory (axonless) cells accompanied by supporting cells and they exhibit morphological diversities of apical specialisations: they are multiciliate in ascidians of the order Enterogona, whereas they are more complex and possess one or two cilia accompanied by stereovilli, also graded in length, in ascidians of the order Pleurogona. In morphology, embryonic origin, and arrangement, coronal sensory cells closely resemble vertebrate hair cells. We describe here the coronal organs of five ascidians ( Pyura haustor (Stimpson, 1864), Pyura stolonifera (Heller, 1878), Styela gibbsii (Stimpson, 1864), Styela montereyensis (Dall, 1872), and Polyandrocarpa zorritensis (Van Name, 1931)), belonging to Pleurogona, also comprising species of one family (Pyuridae), not yet considered, and thus completing our overview of the order. Each species possesses at least two kinds of secondary sensory cells, some of them characterized by stereovilli graded in length. In some species, the coronal sensory cells exhibit secretory activity; in P. haustor, a mitotic sensory cell has also been found. We compare the coronal organ in both ascidians and with other chordate sensory organs formed of secondary sensory cells, and discuss their possible homologies.

During the metamorphosis of tunicate ascidians, the swimming larva uses its three anterior papillae to detect the substrate for settlement, reabsorbs its chordate-like tail, and becomes a sessile oozooid. In view of the crucial role played by the anterior structures and their nerve relations, we applied electron microscopy and immunocytochemistry to study the larva of the colonial ascidian Botryllus schlosseri, following differentiation of the anterior epidermis during late embryogenesis, the larval stage, and the onset of metamorphosis. Rudiments of the papillae appear in the early tail-bud stage as ectodermic protrusions, the apexes of which differentiate into central and peripheral bipolar neurons. Axons fasciculate into two nerves direct to the brain. Distally, the long, rod-like dendritic terminations extend during the larval stage, becoming exposed to sea water. After the larva selects and adheres to the substrate, these neurons retract and regress. Adjacent to the papillae, other scattered neurons insinuate dendrites into the tunic and form the net of rostral trunk epidermal neurons (RTENs) which fasciculate together with the papillary neurons. Our data indicate that the papillae are simple and coniform, the papillary neurons are mechanoreceptors, and the RTENs are chemoreceptors. The interpapillary epidermal area, by means of an apocrine secretion, provides sticky material for temporary adhesion of the larva to the substrate. J. Comp. Neurol. 518:547–566, 2010. © 2009 Wiley-Liss, Inc.

It is generally accepted that the three main chordate groups (tunicates, cephalochordates and vertebrates) originated from a common ancestor having the basic features of the chordate body plan, i.e. a neural tube and a notochord flanked by striated musculature. There is now increasing evidence that tunicates, rather than cephalochordates, are the vertebrate sister-group. Correlated with this, tunicates have sensory structures similar to those derived from placodes or neural crest in vertebrates. In this context, we discuss here whether the precursors of vertebrate hair cells, which are placodal in origin, were present in ancestral chordates. The ascidian tunicates possess a coronal organ, consisting of a row of mechanosensory cells that runs around the base of the oral siphon. Its function is to monitor the incoming water flow. The cells are secondary sensory cells, i.e. they lack axons and synapse with neurons whose somata lie in the cerebral ganglion. They are accompanied by supporting cells and, as in vertebrates, have varying morphologies in the species so far examined: in one order (Enterogona), they are multiciliate; in the other (Pleurogona), they may possess an apical apparatus, consisting of one or two cilia accompanied by stereovilli, that are graded in length. Coronal cells thus resemble vertebrate hair cells closely in their morphology, embryonic origin and arrangement, which suggests they originated early in ancestral chordates. We are continuing our study of the coronal organ in other ascidian species, and report new data here on Botrylloides leachi, which conforms with the pattern of Pleurogona and, in particular, with previously published results on other botryllid ascidians.

The continuous growth of nanotechnology and nano-industries, the considerable increase of products containing nanoparticles (NPs) and the potential release of NPs in aquatic environments suggest a need to study NP effects on aquatic organisms. In this context, in vitro assays are commonly used for evaluating or predicting the negative effects of chemicals and for understanding their mechanisms of action. In this study, a physico-chemical characterisation of titanium dioxide NPs ( n -TiO 2 ) was performed, and an in vitro approach was used to investigate the effects of n -TiO 2 on haemocytes of the clam Ruditapes philippinarum . In particular, the effects on haemocyte phagocytic activity were evaluated in two different experiments (with and without pre-treatment of haemocytes) by exposing cells to P25 n -TiO 2 (0, 1 and 10 μg/mL). In addition, the capability of n -TiO 2 to interact with clam haemocytes was evaluated with a transmission electron microscope (TEM). In this study, n -TiO 2 particles showed a mean diameter of approximately 21 nm, and both anatase (70%) and rutile (30%) phases were revealed. In both experiments, n -TiO 2 significantly decreased the phagocytic index compared with the control, suggesting that NPs are able to interfere with cell functions. The results of the TEM analysis support this hypothesis. Indeed, we observed that TiO 2 NPs interact with cell membranes and enter haemocyte cytoplasm and vacuoles after 60 min of exposure. To the best of our knowledge, this is the first study demonstrating the internalisation of TiO 2 NPs into R. philippinarum haemocytes. The present study can contribute to the understanding of the mechanisms of action of TiO 2 NPs in bivalve molluscs, at least at the haemocyte level.

Background: Mimiviruses or giant viruses that infect amoebas have the ability to retain the Gram stain, which is usually used to colour bacteria. There is some evidence suggesting that Mimiviruses can also infect human cells. Guided by these premises, we performed a routine Gram stain on a variety of human specimens to see if we could detect the same Gram positive blue granules that identify Mimiviruses in the amoebas. Methods: We analysed 24 different human specimens (liver, brain, kidney, lymph node and ovary) using Gram stain histochemistry, electron microscopy immunogold, high resolution mass spectrometry and protein identification. Results: We detected in the human cells Gram positive granules that were distinct from bacteria. The fine blue granules displayed the same pattern of the Gram positive granules that diagnose Mimiviruses in the cytoplasm of the amoebas. Electron microscopy confirmed the presence of human Mimiviruses-like structures and mass spectrometry identified histone H4 peptides, which had the same footprints as giant viruses. However, some differences were noted: the Mimivirus-like structures identified in the human cells were ubiquitous and manifested a distinct mammalian retroviral antigenicity. Conclusions: Our main hypotheses are that the structures could be either giant viruses having a retroviral antigenicity or ancestral cellular components having a viral origin. However, other possible alternatives have been proposed to explain the nature and function of the newly identified structures.

Tunicates are the closest relatives to vertebrates and include the only chordate species able to reproduce both sexually and asexually. The colonial tunicate Botryllus schlosseri is embedded in a transparent extracellular matrix (the tunic) containing the colonial circulatory system (CCS). The latter is a network of vessels external to zooids, limited by a simple, flat epithelium that originated from the epidermis. The CCS propagates and regenerates by remodelling and extending the vessel network through the mechanism of sprouting, which typically characterises vertebrate angiogenesis. In exploiting the characteristics of B. schlosseri as a laboratory model, we present a new experimental and analysis method based on the ability to obtain genetically identical subclones representing paired samples for the appropriate quantitative outcome statistical analysis. The method, tested using human VEGF and EGF to induce angiogenesis, shows that the CCS provides a useful in vivo vessel network model for testing the effects of specific injected solutes on vessel dynamics. These results show the potentiality of B. schlosseri CCS as an effective complementary model for in vivo studies on angiogenesis and anticancer therapy. We discuss this potentiality, taking into consideration the origin, nature and roles of the cellular and molecular agents involved in CCS growth.

In comparative immunology and evolution of the chordate immune system, tunicates hold an important phylogenetic position as sister group of vertebrates. However, knowledge of the tunicate immune system is limited to the class Ascidiacea, in which some species are now considered model organisms. In the class Thaliacea, represented by fragile pelagic species, the few studies on their haemocytes go back to several decades ago and do not consider comparative aspects with ascidian haemocytes. In this study, we identified various haemocyte types and their distribution in the common salp Thalia democratica by comparative observations under light and electron microscopy and by histochemical, histoenzymatic and immunohistochemical techniques. By comparing specialisations with those of ascidian haemocytes, we detected an undifferentiated cell type (lymphocyte-like cell) and three categories with four cell types, that is, (i) phagocytic line (hyaline amoebocyte and amoebocyte with large vacuoles), (ii) mast cell-like line (granular cell) and (iii) storage cells (nephrocyte). Both phagocytes and granular cells appear to migrate in the tunic. Phagocytes adhere to the tunic which internally covers the oral siphon, where they probably function as sentinel cells of the pharynx. Results show the variety of haemolymph cells in the salp similar to phlebobranch ascidians.

Background Hair cells are vertebrate secondary sensory cells located in the ear and in the lateral line organ. Until recently, these cells were considered to be mechanoreceptors exclusively found in vertebrates that evolved within this group. Evidence of secondary mechanoreceptors in some tunicates, the proposed sister group of vertebrates, has recently led to the hypothesis that vertebrate and tunicate secondary sensory cells share a common origin. Secondary sensory cells were described in detail in two tunicate groups, ascidians and thaliaceans, in which they constitute an oral sensory structure called the coronal organ. Among thaliaceans, the organ is absent in salps and it has been hypothesised that this condition is due to a different feeding system adopted by this group of animals. No information is available as to whether a comparable structure exists in the third group of tunicates, the appendicularians, although different sensory structures are known to be present in these animals. Results We studied the detailed morphology of appendicularian oral mechanoreceptors. Using light and electron microscopy we could demonstrate that the mechanosensory organ called the circumoral ring is composed of secondary sensory cells. We described the ultrastructure of the circumoral organ in two appendicularian species, Oikopleura dioica and Oikopleura albicans, and thus taxonomically completed the data collection of tunicate secondary sensory cells. To understand the evolution of secondary sensory cells in tunicates, we performed a cladistic analysis using morphological data. We constructed a matrix consisting of 19 characters derived from detailed ultrastructural studies in 16 tunicate species and used a cephalochordate and three vertebrate species as outgroups. Conclusions Our study clearly shows that the circumoral ring is the appendicularian homologue of the coronal organ of other tunicate taxa. The cladistic analysis enabled us to reconstruct the features of the putative ancestral hair cell in tunicates, represented by a simple monociliated cell. This cell successively differentiated into the current variety of oral mechanoreceptors in the various tunicate lineages. Finally, we demonstrated that the inferred evolutionary changes coincide with major transitions in the feeding strategies in each respective lineage.

In ascidians, mechanoreceptors of the oral area are involved in monitoring the incoming water flow. Sensory cells are represented by scattered, ciliated primary cells (sending their own axons to the cerebral ganglion) or secondary sensory cells (axonless cells forming afferent and efferent synapses with neurons, whose somata are located in the ganglion) of the coronal organ. Coronal cells have varying morphologies: in species of the Enterogona order, they are multiciliate, whereas those of Pleurogona possess an apical apparatus composed of one or two cilia accompanied by stereovilli, in some cases also graded in length. The coronal organ has been proposed as a homologue to the vertebrate octavo-lateralis system, because coronal cells resemble vertebrate hair cells for morphology, embryonic origin and arrangement. In the ascidian Molgula socialis (Pleurogona), we now describe the morphology of the coronal organ, which contains a few associated rows of sensory cells that run the whole length of the oral velum and the branched tentacles. Three kinds of sensory cells, accompanied by specialised supporting cells, are present. Comparisons between the coronal organ and other chordate mechanosensory structures suggest that hair cells originated in the common ancestor of chordates.

A new mechanoreceptor organ, the coronal organ, in the oral siphon of some ascidians belonging to the order Pleurogona has recently been described. In contrast to the known mechanoreceptor organs of ascidian atrium that consist of sensory neurons sending their own axons to the cerebral ganglion, coronal sensory cells are secondary mechanoreceptors, i.e., axonless cells forming afferent and efferent synapses with neurites of neurons located in the ganglion. Moreover, coronal cells exhibit an apical apparatus composed of a cilium accompanied or flanked by rod-like microvilli (stereovilli). Because of the resemblance of these cells to vertebrate hair cells, their ectodermal origin and location in a linear array bordering the bases of the oral tentacles and velum, the coronal organ has been proposed as a homologue to the vertebrate acousticolateralis system. Here we describe the morphology of the coronal organs of six ascidians belonging to the suborders Phlebobranchia and Aplousobranchia (order Enterogona). The sensory cells are ciliated, lack typical stereovilli, and at their bases form synapses with neurites. In two species, the sensory cells are accompanied by large cells involved in synthesis and secretion of protein. We hypothesize that the coronal organ with its secondary sensory cells represents a plesiomorphic feature of ascidians. We compare the coronal organ with other chordate sensory organs formed of secondary sensory cells, i.e., the ventral lip receptors of appendicularians, the oral secondary sensory cells of cephalochordates, and the acousticolateralis system of vertebrates, and we discuss their homologies at different levels of organization. J. Comp. Neurol. 495:363–373, 2006. © 2006 Wiley-Liss, Inc.

International audience; The late differentiation of the ectodermal layer is analysed in the ascidians Ciona intestinalis and Botryllus schlosseri, by means of light and electron microscopy, in order to verify the possible presence of placodal structures. Cranial placodes, ectodermal regions giving rise to nonepidermal cell types, are classically found exclusively in vertebrates; however, data are accumulating to demonstrate that the nonvertebrate chordates possess both the genetic machinery involved in placode differentiation, and ectodermal structures that are possible homologues of vertebrate placodes. Here, the term ''placode'' is used in a broad sense and defines thickenings of the ectodermal layer that can exhibit an interruption of the basal lamina where cells delaminate, and so are able to acquire a nonepidermal fate. A number of neurogenic placodes, ones capable of producing neurons, have been recognised; their derivatives have been analysed and their possible homologies with vertebrate placodes are discussed. In particular, the stomodeal placode may be considered a multiple placode, being composed of different sorts of placodes: part of it, which differentiates hair cells, is discussed as homologous to the octavo-lateralis placodes, while the remaining portion, giving rise to the ciliated duct of the neural gland, is considered homologous to the adenohypophyseal placode. The neurohypophyseal placode may include the homologues of the hypothalamus and vertebrate olfactory placode; the rostral placode, producing the sensorial papillae, may possibly be homologous to the placodes of the adhesive gland of vertebrates.