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In the colonial ascidian Botryllus schlosseri, phagocytes are involved in the clearance of apoptotic cells and corpses during the periodical generation changes or takeovers (TOs) that assure the renewal of the colonial zooids. The persistent respiratory burst associated with efferocytosis, leads to the induction of senescence. Indeed, giant, senescent phagocytes are abundant in the colonial circulation at TO, whereas, in the other phases of the colonial blastogenetic cycle, they colonise the ventral islands (VIs), a series of mesenchymal niches located in the lateral lacunae of the mantle, on both sides of the subendostylar sinus. VI phagocytes produce reactive oxygen species probably as a consequence of the massive phagocytosis of effete cells. VIs are progressively dismantled with the progress of the blastogenetic phases and phagocytes are released in the peribranchial chamber via transepithelial expulsion to be definitely expelled with the outflowing water through the cloacal siphon., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

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., Competing Interests: Declarations of interests The authors declare no competing interests., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

In the second half of the eighteenth century, Schlosser and Ellis described the colonial ascidian Botryllus schlosseri garnering the interest of scientists around the world. In the 1950's scientists began to study B. schlosseri and soon recognized it as an important model organism for the study of developmental biology and comparative immunology. In this review, we summarize the history of B. schlosseri studies and experiments performed to characterize the colony life cycle and bud development. We describe experiments performed to analyze variations in bud productivity, zooid growth and bilateral asymmetry (i.e., the situs viscerum), and discuss zooid and bud removal experiments that were used to study the cross-talk between consecutive blastogenetic generations and vascular budding. We also summarize experiments that demonstrated that the ability of two distinct colonies to fuse or reject is controlled by a single polymorphic gene locus (BHF) with multiple, codominantly expressed alleles. Finally, we describe how the ability to fuse and create chimeras was used to show that within a chimera somatic and germline stem cells compete to populate niches and regenerate tissue or germline organs. Starting from the results of these 60 years of study, we can now use new technological advances to expand the study of B. schlosseri traits and understand functional relationships between its genome and life history phenotypes., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Tunicates, the sister group of vertebrates, possess a mechanoreceptor organ, the coronal organ, which is considered the best candidate to address the controversial issue of vertebrate hair cell evolution. The organ, located at the base of the oral siphon, controls the flow of seawater into the organism and can drive the "squirting" reaction, i.e., the rapid body muscle contraction used to eject dangerous particles during filtration. Coronal sensory cells are secondary mechanoreceptors and share morphological, developmental, and molecular traits with vertebrate hair cells. In the colonial tunicate Botryllus schlosseri, we described coronal organ differentiation during asexual development. Moreover, we showed that the ototoxic aminoglycoside gentamicin caused morphological and mechanosensorial impairment in coronal cells. Finally, fenofibrate had a strong protective effect on coronal sensory cells due to gentamicin-induced toxicity, as occurs in vertebrate hair cells. Our results reinforce the hypothesis of homology between vertebrate hair cells and tunicate coronal sensory cells.

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., (© 2014 Wiley Periodicals, Inc.)

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., (Copyright © 2013 Elsevier B.V. All rights reserved.)

We analyzed the mouth of three species, representative of the three orders of the class Thaliacea (Tunicata)--Pyrosoma atlanticum (Pyrosomatida), Doliolum nationalis (Doliolida), and Thalia democratica (Salpida)--to verify the presence of mechanoreceptors, particularly hair cells. In vertebrates, hair cells are well-known mechanoreceptors of the inner ear and lateral line, typically exhibiting an apical hair bundle composed of a cilium and stereovilli but lacking an axon. For a long time, hair cells were thought to be exclusive to vertebrates. However, evidence of a mechanosensory organ (the coronal organ) employing hair cells in the mouth of tunicates, considered the sister group of vertebrates, suggests that tunicate and vertebrate hair cells may share a common origin. This study on thaliaceans, a tunicate group not yet investigated, shows that both P. atlanticum and D. nationalis possess a coronal organ, in addition to sensory structures containing peripheral neurons (i.e., cupular organs and triads of sensory cells). In contrast, in T. democratica, we did not recognize any oral multicellular sensory organ. We hypothesize that in T. democratica, hair cells were secondarily lost, concomitantly with the loss of branchial fissures, the acquisition of a feeding mechanism based on muscle activity, and a mechanosensory apparatus based on excitable epithelia. Our data are consistent with the hypothesis that hair cells were present in the common ancestor of tunicates and vertebrates, from which hair cells progressively evolved., (Copyright © 2013 Wiley Periodicals, Inc., A Wiley Company.)

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.

Background: An important question behind vertebrate evolution is whether the cranial placodes originated de novo, or if their precursors were present in the ancestor of chordates. In this respect, tunicates are of particular interest as they are considered the closest relatives to vertebrates. They are also the only chordate group possessing species that reproduce both sexually and asexually, allowing both types of development to be studied to address whether embryonic pathways have been co-opted during budding to build the same structures., Results: We studied the expression of members of the transcriptional network associated with vertebrate placodal formation (Six, Eya, and FoxI) in the colonial tunicate Botryllus schlosseri. During both sexual and asexual development, each transcript is expressed in branchial fissures and in two discrete regions proposed to be homologues to groups of vertebrate placodes., Discussion: Results reinforce the idea that placode origin predates the origin of vertebrates and that the molecular network involving these genes was co-opted in the evolution of asexual reproduction. Considering that gill slit formation in deuterostomes is based on similar expression patterns, we discuss possible alternative evolutionary scenarios depicting gene co-option as critical step in placode and pharynx evolution., (Copyright © 2013 Wiley Periodicals, Inc.)

Tunicates are the unique chordates to possess species reproducing sexually and asexually. Among them, the colonial ascidian Botryllus schlosseri is a reference model for the study of similarities and differences in these two developmental pathways. We here illustrate the characterization and expression pattern during both pathways of a transcript for a gene orthologous to Dazap1. Dazap1 genes encode for RNA-binding proteins and fall into the Musashi-like (Msi-like) group. Our phylogenetic analysis shows that these are related to other RNA-binding proteins (Tardbp and several heterogeneous nuclear ribonucleoproteins types) that share the same modular domain structure of conserved tandem RNA Recognition Motifs (RRMs). We also classify the whole group as derived from a single ancient duplication of the RRM. Our results also show that Dazap1 is expressed with discrete spatiotemporal pattern during embryogenesis and blastogenesis of B. schlosseri. It is never expressed in wholly differentiated tissues, but it is located in all bud tissues and in different spatiotemporally defined territories of embryos and larva. These expression patterns could indicate different roles in the two processes, but an intriguing relationship appears if aspects of cell division dynamics are taken into account, suggesting that it is related to the proliferative phases in all tissues, and raising a similarity with known Dazap1 orthologs in other metazoans., (© 2011 Wiley Periodicals, Inc.)

The study of hair cells in invertebrates is important, because it can shed light on the debated question about the evolutionary origin of vertebrate hair cells. Here, we review the morphology and significance of hair cells in two groups of invertebrates, the lower chordates (tunicates and cephalochordates) and the molluscs. These taxa possess complex mechanoreceptor organs based on both primary (sensory neurons) and/or secondary, axonless, sensory cells, bearing various apical specializations. Compared with vertebrates, these taxa show interesting examples of convergent evolution and possible homologies of sensory systems. For example, the "lateral line organ" of Octopoda and Decapoda, composed of primary sensory cells aligned on the arms and the head, is considered a classic example of convergent evolution to mechanoreception. Similarly, in ascidians, the cupular organ, formed of primary sensory cells embedded in a gelatinous cupula, is seen as an analog of neuromasts in vertebrates. However, the coronal organ of the oral siphon of ascidians, represented by a line of secondary sensory cells with a hair bundle also comprising graded stereovilli, is currently the best candidate for tracing the evolutionary origin of the vertebrate octavo-lateralis system. Several features, such as embryological origin, position, gene expression and morphology, support this hypothesis., (Copyright © 2010 Elsevier B.V. All rights reserved.)

The morphology of ascidian gonad is very similar among species. The testis consists of variable number of testicular follicles; the ovary consists of ovarian tubes that are thickened forming the germinal epithelium with stem cells for female germ cells with the exception of botryllid ascidians. Peculiar accessory cells that would be germline in origin accompany the oocytes. Using vasa homologues as a molecular marker, germline precursor cells can be traced back to the embryonic posterior-most blastomeres and are found in the tail of tailbud embryo in some solitary and colonial ascidians. In Ciona, they are subsequently located in the larval tail, while in colonial botryllid ascidians vasa-expressing cells become obscure in the tail. Recent evidence suggests that ascidian germ cells can regenerate from cells other than embryonic germline. An ensemble of the embryonic stringency of germ cell lineage and the postembryonic flexibility of gonad formation is discussed., (Copyright © 2010 Wiley-Liss, Inc.)

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., (2009 Wiley-Liss, Inc.)

Background: Ascidians are tunicates, the taxon recently proposed as sister group to the vertebrates. They possess a chordate-like swimming larva, which metamorphoses into a sessile adult. Several ascidian species form colonies of clonal individuals by asexual reproduction. During their life cycle, ascidians present three muscle types: striated in larval tail, striated in the heart, and unstriated in the adult body-wall., Results: In the colonial ascidian Botryllus schlosseri, we investigated organisation, differentiation and gene expression of muscle beginning from early buds to adults and during zooid regression. We characterised transcripts for troponin T (BsTnT-c), adult muscle-type (BsMA2) and cytoplasmic-type (BsCA1) actins, followed by in situ hybridisation (ISH) on sections to establish the spatio-temporal expression of BsTnT-c and BsMA2 during asexual reproduction and in the larva. Moreover, we characterised actin genomic sequences, which by comparison with other metazoans revealed conserved intron patterns., Conclusion: Integration of data from ISH, phalloidin staining and TEM allowed us to follow the phases of differentiation of the three muscle kinds, which differ in expression pattern of the two transcripts. Moreover, phylogenetic analyses provided evidence for the close relationship between tunicate and vertebrate muscle genes. The characteristics and plasticity of muscles in tunicates are discussed.

Tunicates are useful models for comparing differing developmental processes such as embryogenesis, asexual reproduction, and regeneration, because they are the closest relatives to vertebrates and are the only chordates to reproduce both sexually and asexually. Among them, the ascidian Botryllus schlosseri displays high regenerative potential of the colonial circulatory system (CCS). The CCS runs in the common tunic, forming an anastomized network of vessels defined by simple epithelia and connected to the open circulatory system of the zooids. During asexual propagation, new vessels form by means of a tubular-sprouting mechanism, resembling that occurring in other metazoans, particularly during vertebrate angiogenesis. We studied the regeneration of experimentally ablated CCS by analyzing the general dynamics of reorganization of vessels and tunic, their ultrastructure, cell proliferation, and the immunohistology of regenerating structures using antibodies against vertebrate angiogenic factors-vascular endothelial growth factor (VEGF), fibroblast growth factor-2 (FGF-2), epidermal growth factor (EGF), and receptors: VEGFR-1, VEGFR-2, and EGFR. Results show that the regenerative process of CCS occurs by a sprouting mechanism, with participation of angiogenic factors. They also show correspondence between the CCS sprouting of B. schlosseri and angiogenic sprouting in vertebrates, during both normal development and regeneration, and support the idea that this morphogenetic mechanism was co-opted during the evolution of various developmental processes in different taxa.

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.

A recurrent blastogenetic cycle characterizes colonies of the ascidian Botryllus schlosseri. This cycle starts when a new zooid generation opens its siphons and ends with take-over, when adult zooids cease filtering and are progressively resorbed and replaced by a new generation of buds, reaching functional maturity. During the generation change, massive apoptosis occurs in the colony, mainly in the tissues of old zooids. In the present study, we have investigated the behaviour of haemocytes during the colonial blastogenetic cycle, in terms of the occurrence of cell death and the expression of molecules involved in the induction of apoptosis. Our results indicate that, during take-over, caspase-3 activity in haemocyte lysates increases. In addition, about 20%-30% of haemocytes express phosphatidylserine on the outer leaflet of their plasma membrane, show DNA fragmentation and are immunopositive for caspase-3. Senescent cells are quickly ingested by circulating phagocytes that frequently, having once engulfed effete cells, in turn enter apoptosis. Dying cells and corpses are replaced by a new generation of cells that appear in the circulation during the generation change.

The colonial ascidian Botryllus schlosseri forms new zooids by blastogenesis, through the formation of palleal buds which progressively grow and mature until adults are formed. At a temperature of 19 degrees C, adult zooids remain active for about one week; then they contract, close their siphons and are gradually resorbed, being replaced by buds which reach functional maturity, open their siphons and begin their filtering activity as adult zooids. This recurrent generation change, known as take-over, is characterised by the occurrence of diffuse programmed cell death by apoptosis. Immediately before the take-over, an increase in the expression of molecules recognised by anti-Bax antibodies and a parallel decrease in the expression of molecules immunopositive to anti-Bcl-2 antibodies were observed in zooid tissues, suggesting a mitochondrion-dependent apoptotic pathway. During the take-over, circulating phagocytes infiltrate the zooid tissues and engulf apoptotic cells; in addition, the frequency of haemocytes showing nuclear condensation and annexin-V labelling significantly increases. Previous experiments showed the involvement of phosphatidylserine and CD36 in the recognition of effete cell. The resorption of old zooids is closely related to the rejuvenation of the colony occurring at the take-over. The death of adult zooids puts a quantity of material at the colony disposal. This material is represented by senescent cells, which, once ingested and digested by phagocytes, can be recycled and used to sustain the burden of blastogenesis: this involves a cross-talk between old tissues, phagocytes and developing buds. Therefore, B. schlosseri can be considered a new and promising model organism for the study of natural apoptosis.

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.

Although phylogenetically related to vertebrates, invertebrate chordate tunicates possess an open circulatory system, with blood flowing in lacunae among organs. However, the colonial circulatory system (CCS) of the ascidian Botryllus schlosseri runs in the common tunic and forms an anastomized network of vessels, defined by simple epithelium, connected to the open circulatory system of the zooids. The CCS originates from epidermal evagination, grows, and increases its network accompanying colony propagation. New vessels are formed by means of mechanisms of tubular sprouting which, in their morphogenesis and molecular regulation, are very similar to those occurring in other metazoans, particularly during vertebrate angiogenesis. From the apex of new vessels, epithelial cells detach and migrate into the tunic, while exploring filopodia extend toward the tunic and possibly guide vessel growth. Immunohistology showed that growth factors fibroblast growth factor-2 and vascular endothelial growth factor and the receptor vascular endothelial growth factor receptor-1 participate in sprouting, associated with cell proliferation. As in vertebrates, these factors may regulate cell migration, proliferation, sprouting, and tube formation. Our data indicate that similar, conserved signals were co-opted in the sprouting processes of two nonhomologous circulatory systems, that of ascidian CCS, and vertebrate circulatory systems, by recruitment of the same signaling pathway.

Botryllus schlosseri, a cosmopolitan colonial ascidian reared in the laboratory for more than 50 years, reproduces both sexually and asexually and is used as a model organism for studying a variety of biological problems. Colonies are formed of numerous, genetically identical individuals (zooids) and undergo cyclical generation changes in which the adult zooids die and are replaced by their maturing buds. Because the progression of the colonial life cycle is intimately correlated with blastogenesis, a shared staging method of bud development is required to compare data coming from different laboratories. With the present review, we aim (1) to introduce B. schlosseri as a valuable chordate model to study various biological problems and, especially, sexual and asexual development; (2) to offer a detailed description of bud development up to adulthood and the attainment of sexual maturity; (3) to re-examine Sabbadin's (1955) staging method and re-propose it as a simple tool for in vivo recognition of the main morphogenetic events and recurrent changes in the blastogenetic cycle, as it refers to the developmental stages of buds and adults.

Colonial ascidians offer opportunities to investigate how developmental events are integrated to generate the animal form, since they can develop similar individuals (oozooids from eggs, blastozooids from pluripotent somatic cells) through very different reproductive processes, i.e. embryogenesis and blastogenesis. Moreover, thanks to their key phylogenetic position, they can help in the understanding of the molecular mechanisms of morphogenesis and their evolution in chordates. We review organogenesis of the ascidian neural complex comparing embryos and buds in terms of topology, developmental mechanisms and terminology. We propose a new interpretation of bud territories, and reconsider nervous system development based on recent results suggesting that ascidians have vertebrate placodal and neural-crest-like cells. Comparing embryonic and blastogenic development in Botryllus schlosseri, we propose that the bud has territories with a placodal potentiality, suggesting that chordate ancestors possessed neurogenic placodes, and that the genetic pathways regulating neurogenic placode formation were co-opted for new developmental processes, such as blastogenesis., ((c) 2006 Wiley periodicals, Inc.)

Programmed cell death (PCD) by apoptosis is a physiological mechanism by which cells are eliminated during embryonic and post-embryonic stages of animal life cycle. During asexual reproduction, the zooids of colonial ascidians originate from an assorted cell population instead of a single zygote, so that we assume that regulation of the equilibrium among proliferation, differentiation and cell death may follow different pathways in comparison to the embryonic development. Here we investigate the presence of apoptotic events throughout the blastogenetic life cycle of the colonial ascidian Botryllus schlosseri, by means of terminal deoxynucleotidyl transferase dUTP Nick End Labeling (TUNEL) coupled with histochemical and electron microscopy techniques. The occurrence of low levels of morphogenetic cell death suggests that, in contrast to what happens during sexual development (embryogenesis and metamorphosis), apoptosis does not play a pivotal role during asexual propagation in botryllid ascidian. Nevertheless, PCD emerges as a key force to regulate homeostasis in adult zooids and to shape and modulate the growth of the whole colony.

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.

Phagocytes of the compound ascidian Botryllus schlosseri are capable of constitutive macropinocytosis (MP) at sites of membrane ruffling along the leading edge. This gives rise to the formation of initially irregular vesicles which then move to the inside of the cells and acquire a more regular morphology. Both phagocyte spreading and MP are enhanced by the recognition of molecules containing the sequence Arg-Gly-Asp (RGD): this suggests that, as in mammals, integrin activation is involved in the induction of both cell spreading and endocytosis. The occurrence of MP is associated with increased oxygen consumption and a rise in the production of superoxide anion, as indicated by nitroblue tetrazolium reduction, and ATP, as indicated by increased cytochrome oxidase activity. On the whole, our results indicate the conservation of common mechanisms of MP induction throughout the Chordate phylum.

In the present report, we describe the identification of unusual free amebocytes, completely exposed to seawater, which inhabit the inner surface of the oral and atrial siphons of the compound ascidian Botryllus schlosseri (Urochordata). The origin and biological role of these cells were studied by cytochemical and ultrastructural analysis. These amebocytes are mononucleate cells, with numerous round granules, varying in content, and long filopodia, which contact the cuticle protrusions of the tunic in the siphon. Histochemical, histoenzymatic and immunohistochemical assays were carried out under light microscopy on sections and on living and fixed cultured hemocytes. Results showed that the phagocytic blood cells and the free amebocytes of the siphons shared: (i) affinity for the alpha-mannose specific agglutinin of Narcissus pseudonarcissus (NPA), (ii) occurrence of hydrolytic activities of acid phosphatase and non-specific esterases inside lysosomal vesicles and large vacuoles, (iii) membrane labeling with the lipophilic dye PKH26 specific for phagocytic cells, (iv) anti-CD39 immunocytochemical labeling specific for lysosomes of mammalian macrophages. All histochemical data support the hypothesis that these cells are 'sentinel cells' belonging to the hyaline amebocyte population of the phagocytic differentiation line of the immunocytes, since they can also recognize and phagocytize carmine experimentally administered as target particles.

The ascidian larva has a central nervous system which shares basic characteristics with craniates, such as tripartite organisation and many developmental genes. One difference, at metamorphosis, is that this chordate-like nervous system regresses and the adult's neural complex, composed of the cerebral ganglion and associated neural gland, forms. It is known that neural complex differentiation involves two ectodermal structures, the neurohypophysial duct, derived from the embryonic neural tube, and the stomodeum, i.e. the rudiment of the oral siphon; nevertheless, their precise role remains to be clarified. We have shown that in Ciona intestinalis, the neural complex primordium is the neurohypophysial duct, which in the early larva is a short tube, blind anteriorly, with its lumen in continuity with that of the central nervous system, i.e. the sensory vesicle. The tube grows forwards and fuses with the posterior wall of the stomodeum, a dorsal ectodermal invagination of the larva. The duct then loses posterior communication with the sensory vesicle and begins to grow on the roof of the vesicle itself. The neurohypophysial duct differentiates into the neural gland rudiment; its dorsal wall begins to proliferate neuroblasts, which migrate and converge to build up the cerebral ganglion. The most anterior part of the neural gland organizes into the ciliated duct and funnel, whereas the most posterior part elongates and gives rise to the dorsal strand. The hypothesis that the neurohypophysial duct/stomodeum complex possesses cell populations homologous to the craniate olfactory and adenohypophysial placodes and hypothalamus is discussed., (Copyright 2005 Wiley-Liss, Inc.)

The colonial ascidians reproduce either sexually or asexually, having evolved a rich variety of modes of propagative development. During embryogenesis, the fertilized egg develops into a swimming tadpole larva that subsequently metamorphoses into a sessile oozooid. Clonal individuals (blastozooids), resembling oozooids, are formed from few bud-forming multipotent somatic cells, following a wide range of ways that seem to characterize each family of this class. Here, we compare these two developmental processes in the compound ascidian species Botryllus schlosseri to determine whether similar gene activities are used during embryogenesis/metamorphosis and recruited in the asexual development. We analyzed expression of Pitx, a Paired-related homeobox gene. Pitx genes are key developmental genes in vertebrates, and their expression is reported to be conserved in chordate stomodea and in the establishment of left/right asymmetries. Here, we report full-length cDNA cloning of a B. schlosseri Pitx ortholog (Bs-Pitx) and expression analysis during both embryo/metamorphosis and blastogenesis. During organogenesis of both developmental sequences, Bs-Pitx was detected in identical domains: the stomodeum/neural complex and asymmetrically in the left digestive system. In striking contrast, expression patterns at early stages differ deeply. These observations provide the first evidence for a key developmental gene being deployed in essentially similar ways in two different developmental sequences that eventually give rise to similar zooids., (Copyright 2004 Wiley-Liss, Inc.)

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.

The alimentary tract of Kowalevskia tenuis and K. oceanica, the only species of the appendicularian family Kowalevskiidae, was studied both at the light and electron microscope levels and compared with species belonging to the other two families of the class. Kowalevskids show interesting specializations: 1) the pharynx opens on both sides through two opposing spiracles, modified into long ciliated fissures, and possesses an original filtering system of ciliated combs arranged in two pairs of opposing longitudinal rows; 2) the endostyle is absent, its place being taken by a ciliated groove without any glandular cell; 3) posterior to the esophagus, the globular stomach and rectum form a digestive nucleus comprising a few, large cells including two well-developed, specialized valves, cardiac and pyloric; 4) special apical junctions bearing characteristics of both gap and adherens junctions are diffuse along the gut epithelium; 5) the heart is absent. Our data suggest that Kowalevskiidae underwent a high degree of specialization for food filtering and are more closely related to Fritillariidae, with which they share several characters, rather than Oikopleuridae, the latter probably representing the most primitive family of appendicularians., (Copyright 2003 Wiley-Liss, Inc.)

Spherule cells are specific types of coelomocytes found in both the coelomic fluids and the connective tissues of many echinoderm groups and are characterised by large membrane-bound inclusions which completely fill their cytoplasm. In holothurians they are present in massive number in the coelomic fluids and are employed in brown body formation. Brown bodies are products of encapsulation and mainly consist of phagocytic amoebocytes and spherule cells: they surround foreign particles too large to be ingested by circulating phagocytes. During brown body formation, phagocytic amoebocytes flatten out over the surface of foreign particles to form unpigmented nodules which eventually aggregate into a single brown body in which many spherule cells are entrapped. Morphological modifications of spherule cells were studied in Holothuria polii following the induction of brown body formation by intracoelomic injection of sheep erythrocytes. Our ultrastructural observations provide evidence that the granules undergo typical exocytosis after previous disorganisation of their content and suggest a specific secretory activity for the spherule cells. The possible functional role of the secreted vacuolar material in brown body formation is discussed.

A new mechanoreceptor organ, the "coronal organ," located in the oral siphon, is described by light and electron microscopy in the colonial ascidians Botryllus schlosseri and Botrylloides violaceus. It is composed of a line of sensory cells (hair cells), accompanied by supporting cells, that runs continuously along the margin of the velum and tentacles of the siphon. These hair cells resemble those of the vertebrate lateral line or, in general, the acoustico-lateralis system, because they bear a single cilium, located centrally or eccentrically to a hair bundle of numerous stereovilli. In contrast to other sensory cells of ascidians, the coronal hair cells are secondary sensory cells, since they lack axonal processes directed towards the cerebral ganglion. Moreover, at their base they form synapses with nerve fibers, most of which exhibit acetylcholinesterase activity. The absence of axonal extensions was confirmed by experiments with lipophilic dyes. Different kinds of synapses were recognized: usually, each hair cell forms a few afferent synapses with dendrites of neurons located in the ganglion; efferent synapses, both axo-somatic (between an axon coming from the ganglion and the hair cell) and axo-dendritic (between an axon coming from the ganglion and an afferent fiber) were occasionally found. The presence of secondary sensory cells in ascidians is discussed in relation to the evolution of sensory cells and placodes in vertebrates. It is proposed that the coronal organ in urochordates is homologous to the vertebrate acoustico-lateralis system., (Copyright 2003 Wiley-Liss, Inc.)

In this study, we have analysed ultrastructurally the mechanism of epithelial fusion and perforation during the development of branchial fissures in the larva and bud of the colonial urochordate Botryllus schlosseri. Perforation of membranes represents an important process during embryogenesis, occurring to create communication between two separate compartments. For example, all chordate embryos share the formation of pharyngeal plates, which are constituted of apposed endodermal and ectodermal epithelia, which have the capacity to fuse and perforate. Although the process of perforation is extremely common, its cellular mechanism remains little understood in detail, because of the complexity of the structures involved. In B. schlosseri, two epithelial monolayers, the peribranchial and the branchial ones, with no interposed mesenchymal cells, participate in pharyngeal perforation. Blood flows in the interspace between the two cellular leaflets. Apico-lateral zonulae occludentes seal the cells of each epithelium, so that the blood compartment is separated from the environment of the peribranchial and branchial chambers; here, sea water will flow when the zooid siphons open. Stigmata primordia appear as contiguous thickened discs of palisading cells of branchial and peribranchial epithelia. The peribranchial component invaginates to contact the branchial one. Here, the basal laminae intermingle, compact, and are degraded, while the intercellular space between the two epithelia is reduced to achieve the same width as that found between the lateral membranes of adjacent cells. Cells involved in this fusion rapidly change their polarity: they acquire a new epithelial axis, because part of the adhering basal membrane becomes a new lateral surface, whereas the original lateral membranes become new apical surfaces. Before disassembling the old tight junctions and establishing communication between branchial and peribranchial chambers, cells of the stigmata rudiments form new tight junctions organised as distinct entities, so that the structural continuum of the epithelial layers is maintained throughout the time of fusion and perforation., (Copyright 2002 Wiley-Liss, Inc.)

We have initiated a project aimed at documenting molecular and cellular changes underlying the emergence of the hypothalamo-hypophyseal axis in Chordates. Considering the phylogenetic position of Tunicates and the 'pan-hypophyseal' expression pattern of Pitx genes in Vertebrate pituitary, we searched for a Pitx-related homeobox gene in the ascidian Ciona intestinalis, and identified Ci-Pitx (ona intestinalis uitary homeobo gene). We also isolated Cs-Pitx and Bs-Pitx, the Ci-Pitx respective counterparts of Ciona savignyi and Botryllus schlosseri, two other Tunicate species. Ci-Pitx mRNA encodes a putative protein exhibiting the diagnostic K50-Paired-class homeodomain and a conserved C-terminal Aristaless domain. Embryonic expression pattern of Ci-Pitx revealed a conserved expression domain in the anterior neural ridge and subsequently in the pharyngeal primordium, defined in Vertebrates as the stomodeal ectomere, which encompasses the presumptive pituitary territory. This shows that expression at early steps of pituitary development is a feature of Pitx-related genes that was already present in the last common ancestor of Chordates.

The motor nervous system of adult ascidians consists of neurons forming the cerebral ganglion from which axons run out directly to the effectors, i.e., muscular and ciliary cells. In this study, we analyzed the development of the motor fibers, correlating this with organ differentiation during asexual reproduction in Botryllus schlosseri. We used a staining method for acetylcholinesterase, whose reaction product is visible with both light and electron microscopy and which labels entire nerves, including their thin terminals, making them identifiable between tissues. While the cerebral ganglion is forming, the axons elongate and follow stereotypical pathways to reach the smooth muscle cells of the body, the striated muscle of the heart, and the ciliated cells of the branchial stigmata and the gut. A strict temporal relation links the development of the local neural network with its target organ, which is approached by nerves before the effector cells are fully differentiated. This process occurs for oral and cloacal siphons, branchial basket, gut, and heart. Axons grow through the extracellular matrix and arrive at their targets from different directions. In some cases, the blood sinuses constitute the favorite roads for growing axons, which seem to be guided by a mechanism involving contact guidance or stereotropism. The pattern of innervation undergoes dynamic rearrangements and a marked process of elimination of axons, when the last stages of blastogenesis occur. The final pattern of motor innervation seems to be regulated by axon withdrawal, rather than apoptosis of motor neurons., (Copyright 2002 Wiley-Liss, Inc.)

Ascidian and vertebrate nervous systems share basic characteristics, such as their origin from a neural plate, a tripartite regionalization of the brain, and the expression of similar genes during development. In ascidians, the larval chordate-like nervous system regresses during metamorphosis, and the adult's neural complex, composed of the cerebral ganglion and the associated neural gland is formed. Classically, the homology of the neural gland with the vertebrate hypophysis has long been debated. We show that in the colonial ascidian Botryllus schlosseri, the primordium of the neural complex consists of the ectodermal neurohypophysial duct, which forms from the left side of the anterior end of the embryonal neural tube. The duct contacts and fuses with the ciliated duct rudiment, a pharyngeal dorsal evagination whose cells exhibit ectodermic markers being covered by a tunic. The neurohypophysial duct then differentiates into the neural gland rudiment whereas its ventral wall begins to proliferate pioneer nerve cells which migrate and converge to make up the cerebral ganglion. The most posterior part of the neural gland differentiates into the dorsal organ, homologous to the dorsal strand. Neurogenetic mechanisms in embryogenesis and vegetative reproduction of B. schlosseri are compared, and the possible homology of the neurohypophysial duct with the olfactory/adenohypophysial/hypothalamic placodes of vertebrates is discussed. In particular, the evidence that neurohypophysial duct cells are able to delaminate and migrate as neuronal cells suggests that the common ancestor of all chordates possessed the precursor of vertebrate neural crest/placode cells., (Copyright 1999 Wiley-Liss, Inc.)

In adult ascidians, the neural complex consists of a cerebral ganglion (the brain) and the associated neural gland. We have studied the development of the neural complex during the vegetative reproduction of the colonial ascidian Botryllus schlosseri, the buds of which arise from the atrial mantle of the parental zooid. Each bud develops into a new organism within which a neural complex becomes differentiated. We found that the presumptive (pioneer) nerve cells that ultimately form the cerebral ganglion of the adult arise as migratory cells from a primordial cluster of rudimentary gland cells. Hence, the neural gland appears to be neurogenic in that it serves as the cellular source of components that differentiate into conventional nerve cells. In the adult, these cells take on the form of a typical invertebrate ganglion with an outer cortex of nerve cell bodies and an internal medulla. This medulla consists of a neuropile of neuronal processes making classical synaptic contacts. The adult neural gland differentiates into a structure with a ciliated duct that opens into the branchial chamber, the body of the gland, and the dorsal organ, which is quite distinct from the dorsal strand of other ascidians. The rudimentary neural gland cells, therefore, differentiate into one of two distinct pathways: the first, glandular, is possibly involved in the evaluation of environmental signals, and the other, nervous, leads to brain formation. This compares with the vertebrate situation in which the olfactory-pituitary placodes are thought to originate from a common cellular source. Thus, these data support the earlier contention of a homology between the tunicate neural gland and the vertebrate adenohypophysis.

The effects of tributyltin chloride (TBT) on cytoskeletal components, as possible cell targets of toxicity, were examined on cultured hemocytes of the colonial ascidian Botryllus schlosseri by means of indirect immunofluorescence. The immunotoxic effect of 10 microM TBT (sublethal concentration) consists of (1) inhibition of yeast phagocytosis, Ca2+ ATPase activity, and respiratory burst; (2) increase in intracellular Ca2+ concentration; and (3) alterations in cell morphology. After 60 min, TBT-exposed amebocytes become spherical, withdrawing their long pseudopodia, and lose motility. Their microfilaments assemble in clusters around the peripheric cytoplasm, indicating massive disassembly, with the exception of unaltered adhesion plaques. Analogously, their microtubules reveal extensive disaggregation, being scattered in the cytoplasm and not recognizable as single filaments, whereas the microtubule organizing center (MTOC) is still visible. Treatment together with 20 micrograms/ml calmodulin (CaM) can partially restore the cytoskeleton architecture. These results suggest a relationship between TBT and Ca2+ homeostasis in ascidian hemocytes. By interfering with Ca2+ ATPase activity through CaM inhibition, either directly or indirectly, TBT induces an excess of intracellular Ca2+ accumulation, which first causes internal disorganization of cytoskeletal proteins and consequently inhibition of phagocytosis, beginning from chemotaxis and particle adhesion.

In order to clarify the interaction mechanism between organotin compounds and organisms, the effects of these compounds on the development of a benthonic filter-feeding invertebrate were studied. Embryos of the ascidian Styela plicata were obtained in laboratory by cross-fertilization and their development was followed in vivo after incubation with 0.1, 1, and 10 microM organotin compounds for various exposure times. Moreover, embryos selected at opportune stages after incubation with 10 microM tributyltin (TBT) or triphenyltin (TPT) for 1 hr were observed at the electron microscope to recognize cell alterations. Results indicate that organotins significantly affect all stages of ascidian development in a dose- and time-dependent manner and the most sensitive stages are gastrula and neurula. These compounds are able to block development, giving rise to anomalous embryos with irreversible effects. The order of inhibition appears to be strongly dependent on the organotin liposolubility: TBT > dibutyltin (DBT) > monobutyltin (MBT) and TPT > tricyclohexyltin (TCHT). The mitosis block of blastomeres in the early stages may be related to an inhibition of the microtubule polymerization. Observations with light and electron microscopes reveal globeshaped blastomeres with large intercellular spaces in the morula and gastrula stages, suggesting a toxic damage with alteration of the cytoskeleton. Moreover, the occurrence of electron-dense precipitates of organotins in the inner membrane of mitochondria and morphological changes of their cristae suggest an inhibitory effect on oxidative phosphorylation which is conspicuous in the gastrula stage. In this stage, the size of the electron-dense aggregates grow from 50-70 to 110-170 nm, while at the same time the alteration of the cristae increases.

The cells of the intestinal tract and the stigmatal cells of the branchial basket have been studied in a range of tunicates including phlebobranch, aplousobranch and stolidobranch ascidians, as well as the doliolid and pyrosomatid thaliaceans. The intercellular gap junctions between gut cells appear conventional in thin section as do those found in the lower part of adjacent stigmatal cells. However, save for the stolidobranchs, the stigmatal cells also have a second kind of gap junction which exhibit an unusual fibrous density in association with their junctional cytoplasmic surfaces; these are found in the apical region of the cells. The fibrous density is particularly well demonstrated in specimens treated with tannic acid during fixation, and subsequent en bloc uranyl acetate staining. In the branchial basket the position of these apical gap junctions is at regular intervals between adhaering junctions, which have a more substantial paramembranous fibrous mat; these two kinds of junctions alternate along deeply undulating membrane appositions. With freeze-fracture, after chemical or cryo-fixation, the gap junctions of the gut and those of the lower part of the stigmatal cells appear typical, with P-face connexons, while in the apical part of cells of the branchial basket the two faces of the gap junctions are very difficult to cleave apart. Frequently the P- and E-faces are found to adhere together in replicas, so that in these apical gap junctional regions, plaques of E-face with pits overlie the PF particles. In addition, regions of cytoplasm, into which the dense fibres project, often cleave over these adhaering E-faces of the apical gap junctions. The presence of these unusual gap junctional features in the apical region of the stigmata in the vicinity of cilia is discussed as regards their functional role.

During vitellogenesis, oocytes of Botryllus schlosseri always exhibit an unusual system scattered in the cytoplasm. It consists of an association between a single fenestrated endoplasmic reticulum cisterna and one or a few smooth vesicles (cisterna vesicle association: CVA) containing a dense core facing the cisterna itself. The latter is smooth and perforated by numerous small pores (about 25 nm in diameter) in the area of association; towards the periphery, it extends into several branches with ribosomes bound to their membranes. In the vesicles, fibrillar material radiates from the dense core and is sometimes organized into a long, dense lamina. The membranes of both cisterna and vesicles appear to be coupled, but are in fact separated by a constant narrow space occupied by short densities. The presence in B. schlosseri of this unusual fenestrated membrane system contrasts with the absence of a typical porous cytoplasmic organelle, the annulate lamellae (ALs), which is widely distributed in female gametes. However, as in other animals, B. schlosseri oocytes possess intranuclear annulate lamellae (IALs) and vesicles. Comparative observations extended to the oocytes of the ascidian Ciona intestinalis have shown that the latter species exhibits typical ALs and IALs, but not the CVA. The morphology of the CVA is analysed here in detail, and similarities and differences with ALs are pointed out. Hypotheses regarding CVA function are discussed in terms of possible relations with ALs.

The protein kinase inhibitor H-7 prevents the assembly of tight junctions in cultured Madin Darby Canine Kidney (MDCK) epithelial cells (Balda et al. (1991) J. Membr. Biol. 122, 193-202; Nigam et al. (1991) Biochem. Biophys. Res. Commun. 181, 548-553); however, its mechanism of action is unknown. To understand the basis of the activity of H-7 and other inhibitors we compared the effect of H-7 on the localization of proteins belonging to tight junctions and adherens-type junctions (zonula adhaerens and desmosome), and on the organization of actin microfilaments. Junction assembly was induced in MDCK cells either by the 'Ca2+ switch' procedure or by incubating trypsinized cells at normal extracellular Ca2+, and the cells were then immunofluorescently labeled with antibodies against cingulin, ZO-1, E-cadherin and desmoplakin, and with FITC-phalloidin. Here we show by measuring the transepithelial resistance that, in addition to H-7, H-8 and staurosporine can also significantly block the assembly of tight junctions, whereas HA1004 is poorly active. H-7 inhibited the accumulation of cingulin and ZO-1 in junctional areas most effectively when added during assembly at normal extracellular Ca2+. On the other hand, H-7 did not have major effects on the accumulation of E-cadherin and desmoplakin in the regions of cell-cell contact using either assembly protocol. Electron microscopy confirmed that H-7 does not abolish the formation of adherens-type junctions, suggesting that phosphorylation plays a different role in the assembly of tight junctions versus adherens-type junctions. Finally, in both protocols of junction assembly H-7 caused a major disorganization of actin microfilaments, suggesting that H-7 may prevent TJ assembly through its effect on the cytoskeleton.

Tissues from the pharynx of five representative species of the protochordates (subphylum Tunicata, the three classes Ascidiacea, Thaliacea and Appendicularia, and subphylum Cephalochordata) were examined in both thin sections and freeze-fracture replicas. In all species, the stigmatal cilia of the branchial chamber are neatly arranged and move continuously to propel sea-water in a fixed direction for respiration and feeding of the organism. A number of specializations are found in the basal region of these cilia and are represented by: a) bridges connecting axonemal doublets numbers 5 and 6; b) dense fibrous material linking the doublet microtubules of the axoneme to the ciliary membrane, sometimes in the shape of longitudinal strands or as clusters of filaments; c) intramembrane particles (IMPs) associated with the P-face of the membrane, often arranged in clusters evenly aligned along the ciliary shaft in relation to the underlying axonemal doublets. Ciliary specializations are distributed along the plane of the effective stroke of the beat in both the ascidian Botryllus schlosseri and in the thaliacean Pyrosoma atlanticum and the amphioxus Branchiostoma lanceolatum, whereas in the thaliacean Doliolum nationalis and the appendicularian Oikopleura dioica a more uniform distribution of these specializations all around the basal portion of the cilia is observed. Whatever the disposition of the ciliary specializations in all the examined species, they are always present at the base of the water-propelling cilia. Some morphological evidence suggests that these specializations play a mechanical function in tethering the ciliary membrane to the axoneme. We propose that they help maintain the orientation of the cilia during beating, enhance their stiffness and improve their efficiency.

The following five cell types have been recognized and defined on the basis of their fine structure in the gastric epithelium of B. schlosseri: vacuolated and zymogenic cells (described in a previous paper); ciliated mucous, endocrine and plicated cells. The ciliated mucous cells are distributed at the apex and the bottom of the gastric folds and along the dorsal groove. The mucus droplets appear to form from the Golgi complex as secretory granules of variable density and texture, which are released from the cell after fusion of their membranes with the apical plasma membrane. Holocrine or apocrine secretion has not been observed. The endocrine cells are scattered and are characterized by electron dense granules, especially numerous in the basal region of the cell. Finally, the plicated cells, present in the pyloric caecum, show rod-like microvilli, a well developed Golgi complex and abundant, deep infoldings of the basal plasma membrane, which are associated with numerous mitochondria. The possible role of the gastric cell types is discussed taking into account information concerning morphologically similar cells in other animals, as well as previously reported data on the biochemistry and physiology of digestion and excretion in ascidians.

The stigmatal cells in the branchial basket of ascidians from a number of genera have been examined as to the nature and distribution of their intercellular junctions. The branchial wall consists of ciliated and parietal cells; the ciliated cells are arranged in seven rows and are associated by junctions with other cells in the same row as well as with those in adjacent rows. They are also associated by junctions with peripheral parietal cells. Junctions between adjacent ciliated cells in all cases exhibit tight junctions or zonulae occludentes. However, these cell borders also possess fasciae or zonulae adhaerentes if they are in the same row and the ciliary rootlets insert-into these junctions. If the cells are in adjacent rows they exhibit adhaerentes junctions only in species belonging to the orders Phlebobranchiata and Aplousobranchiata. In contrast, if the cells in adjacent rows belong to the order Stolidobranchiata. they never exhibit any adhaerentes junctions and the ciliary rootlets of the basal bodies from the cilia insert instead into the tight junctions and the non-junctional membrane below them. At the homologous junctional borders between adjacent parietal cells and also at heterologous junctional borders between parietal and ciliated cells, tight junctions alone occur, with no co-existing adhaerentes junctions along their lateral borders. Again, fibrils from ciliary rootlets insert into zonulae occludentes. This shows that tight junctions are capable both of forming permeability barriers, in that they can be seen to prevent the entry of exogenous tracers such as lanthanum, and of acting as adhesive devices.

The intestinal tracts from seven different species of tunicates, some solitary, some colonial, were studied fine-structurally by freeze-fracture. These urochordates occupy an intermediate position phylogenetically between the vertebrates and the invertebrates. The various regions of their gut were isolated for examination and the junctional characteristics of each part investigated. All the species examined exhibited unequivocal vertebrate-like belts of tight-junctional networks at the luminal border of their intestinal cells. No septate junctions were observed. The tight junctions varied in the number of their component strands and the depth to which they extended basally, some becoming loose and fragmented towards that border. The junctions consisted of ridges or rows of intramembranous particles (IMPs) on the P face, with complementary, but offset, E face grooves into which IMPs sometimes fractured. Tracer studies show that punctate appositions, the thin-section correlate of these ridge/groove systems, are sites beyond which exogenous molecules do not penetrate. These junctions are therefore likely to represent permeability barriers as in the gut tract of higher chordates. Associated with these occluding zonular junctions are intermediate junctions, which exhibit no identifiable freeze-fracture profile, and macular gap junctions, characterized by a reduced intercellular cleft in thin section and by clustered arrays of P face particles in freeze-fractured replicas; these display complementary aggregates of E face pits. The diameters of these maculae are rarely very large, but in certain species (for example, Ciona), they are unusually small. In some tissues, notably those of Diplosoma and Botryllus, they are all of rather similar size, but very numerous. In yet others, such as Molgula, they are polygonal with angular outlines, as might be indicative of the uncoupled state. In many attributes, these various junctions are more similar to those found in the tissues of vertebrates, than to those in the invertebrates, which the adult zooid forms of these lowly chordates resemble anatomically.