In vitro culture of blood cells from the colonial protochordateBotryllus schlosseri

Summary Primary cultures of circulatory blood cells from the colonial tunicateBotryllus schlosseri were cultivated in 96-well plates for up to 3 mo. in a medium based on Dulbecco’s modified Eagle’s medium, supplemented with salts to the botryllid ascidian hemolymph osmolarity, HEPES buffer,l-glutamin, fetal bovine serum, and antibiotics. Intercellular bridges between granular pigment cells were established within 24 h. The viability of these cells decreased slowly, and most died within 1 mo. without any sign of cell proliferation. Other cell types remained in an arrested state and were subjected to a weekly medium exchange. Spontaneous cell proliferation was randomly recorded in 6 to 10% of the wells from 2 wk to 1 mo. This proliferation was followed by the formation of masses of cell clumps, from which uniform hemocytes (5 μm, lymphocytelike cells) migrated peripherally. Stress conditions, which included longer intervals between medium exchanges and partial medium replacement, increased the probability of cell proliferation. From each proliferating primary culture, we successfully performed up to 10 plating cycles over a period of 15 wk, during which the cells differentiate in size but are uniformly structured. This produced the firstBotryllus lymphocytelike cell line. From this stage, cell numbers remained constant for up to 6 mo. without increase in cell number. Several mitogenic factors were employed on primary cultures.Botryllus and sea cucumber hemolymphs and mixed interleukins were found to augment significantly proliferation of at least one specific cell size, whereas cells were not markedly responsive to lectins (Concavalin A, wheat germ agglutinin,Ulex europaeus agglutinin), insulin, and retinoic acid. The results are discussed with respect to future efforts in the development of tunicate blood cell cultures.     

Polar Constituents of the Tunicate Botryllus schlosseri

Eighteen compounds were identified by GC–MS of their trimethylsilyl derivatives in n-butanolic extract from the biomass of Botryllus schlosseri. Three of them, 5-oxoproline, 5-hydroxyhydantoin, and kinurenic acid, were found in marine invertebrates for the first time. In addition to cellulose, the biomass was also shown to contain complex water-soluble sulfated polysaccharides. These were extracted and fractionated, and sulfate content and monosaccharide composition were determined in the fractions; fucose, xylose, galactose, mannose, glucose, glucosamine, galactosamine, and uronic acids were found. Unlike several other tunicate species, Botryllus schlosseri does not seem to contain any simple galactan sulfate.     

BS-cadherin in the colonial urochordate Botryllus schlosseri: one protein, many functions

Botryllus schlosseri is a colonial urochordate composed of coexisting modules of three asexually derived generations, the zooids and two cohorts of buds, each at disparate developmental stage. Functional zooids are replaced weekly by the older generation of buds through a highly synchronized developmental cycle called blastogenesis (which is, in turn, divided into four major stages, A to D). In this study, we examined the mode of expression of BS-cadherin, a 130-kDa transmembrane protein isolated from this species, during blastogenesis. BS-Cadherin is expressed extensively in internal organs of developing buds, embryos, ampullae and, briefly, in the digestive system of zooids at early blastogenic stage D (in contrast to low mRNA expression at this stage). In vitro trypsin assays on single-cell suspensions prepared from blastogenic stage D zooids, confirmed that BS-cadherin protein is expressed on cell surfaces and is, therefore, functional. BS-Cadherin expression is also upregulated in response to various stress conditions, such as oxidative stress, injury and allorecognition. It plays an important role in colony morphogenesis, because siRNA knockdown during D/A blastogenic transition causes chaotic colonial structures and disrupts oocytes homing onto their bud niches. These results reveal that BS-cadherin protein functions are exerted through a specific spatiotemporal pattern and fluctuating expression levels, in both development/regular homeostasis and in response to various stress conditions.     

Initial characterization of a protochordate histocompatibility locus

The colonial protochordate, Botryllus schlosseri, undergoes a natural transplantation reaction which is controlled by a single, highly polymorphic locus called the Fu/HC. We are using map-based cloning to identify Fu/HC gene(s), and have currently delineated their location to an approximately 1-cM region of the B. schlosseri genome. The Fu/HC physical map currently consists of 85 sequence-tagged sites mapped on a minimum tiling path of 800 kb which consists of five contigs, with four gaps remaining to be crossed, and is estimated to be 75% completed. Approximately half this region has been sequenced throughout the locus, allowing the first analysis of a metazoan histocompatibility locus outside of vertebrates. This has resulted in the identification of 18 predicted genes, a number of which have been found to be expressed. Several of these genes are well conserved among the chordates; however, none of the predicted or expressed genes are linked within the genome of any organism in the databases. In addition, the Fu/HC is one of the most polymorphic loci ever described, and physical mapping has revealed that the locus is quite dynamic, and includes features such as hotspots of recombination.     

Involution of the caudal musculature during metamorphosis in the ascidian,Botryllus schlosseri

Summary The caudal musculature of the free-swimming tadpole of the ascidian, B. schlosseri consists of cylindrical mononucleated cells connected in longitudinal rows flanking the axial notochord. During resorption of the larval tail, which is apparently induced by the contraction of the epidermis, muscle cells are dissociated and pushed into the body cavity where most of them are rapidly engulfed by phagocytes. In the initial stages of tail withdrawal muscle cells display surface alterations due to the disruption of intercellular junctions and disarrangement of myofibrils. Extensive degenerative changes, with shrinkage of mitochondria and disintegration of the contractile material are subsequently observed. Lysosomes and autophagic vacuoles are rarely seen and appear to play a secondary role in the degradation of the muscle cells, which occurs predominantly within the phagocytes. Myofilaments and myofibrils have never been observed within autophagic vacuoles. Clumps of muscle fragments and degenerated phagocytes undergo eventual dissolution in the blood lacunae, concomitantly with the differentiation of the young oozooid.This investigation was supported in part by a grant from the Muscular Dystrophy Associations of America and by CNR contract No. 7100396/04115542 from the Istituto di Biologia del Mare, Venice. We gratefully acknowledge the skillful assistance of Mr. G. Gallian, Mr. M. Fabbri and Mr. G. Tognon. We also thank the staff of the Stazione Idrobiologica at Chioggia for collecting the colonies.     

Germ lineage properties in the urochordate Botryllus schlosseri – From markers to temporal niches

The primordial germ cells (PGCs) in the colonial urochordate Botryllus schlosseri are sequestered in late embryonic stage. PGC-like populations, located at any blastogenic stage in specific niches, inside modules with curtailed lifespan, survive throughout the life of the colony by repeated weekly migration to newly formed buds. This cyclical migration and the lack of specific markers for PGC-like populations are obstacles to the study on PGCs. For that purpose, we isolated the Botryllus DDX1 (BS-DDX1) and characterized it by normal expression patterns and by specific siRNA knockdown experiments. Expression of BS-DDX1 concurrent with BS-Vasa, γ-H2AX, BS-cadherin and phospho-Smad1/5/8, demarcate PGC cells from soma cells and from more differentiated germ cells lineages, which enabled the detection of additional putative transient niches in zooids. Employing BS-cadherin siRNA knockdown, retinoic acid (RA) administration or β-estradiol administration affirmed the BS-Vasa⁺BS-DDX1⁺BS-cadherin⁺γ-H2AX⁺phospho-Smad1/5/8⁺ population as the B. schlosseri PGC-like cells. By striving to understand the PGC-like cells trafficking between transient niches along blastogenic cycles, CM-DiI-stained PGC-like enriched populations from late blastogenic stage D zooids were injected into genetically matched colonial ramets at blastogenic stages A or C and their fates were observed for 9 days. Based on the accumulated data, we conceived a novel network of several transient and short lived ‘germ line niches’ that preserve PGCs homeostasis, protecting these cells from the weekly astogenic senescence processes, thus enabling the survival of the PGCs throughout the organism's life.     

On the development and reproduction of Botryllus schlossen (Tunicata) colonies from the eastern Mediterranean Sea: plasticity of life history traits

Summary

Analysis of developmental patterns of Botryllus schlosseri colonies from the eastern Mediterranean coast has been performed on 143 genets under different temperature regimens (15,20,27 °C) for up to 22 weeks. While the average maximal size as the length of the blastogenic cycle (BC) varied in respect to water temperature, ontogeny at all temperatures was characterized by 4 developmental stages: 1. the lag phase (the first 1–3 BCs, 1 bud/BC), 2. the exponential growth phase (5.5–7.8 BCs, up to 3 buds/BC), 3. the plateau phase, (7.4–8.2 BCs, 1 bud/BC), 4. the degenerative or the variable phase. Many (40.6%) colonies were not sexually reproductive, the others were male only (30.1%) or hermaphrodites. Colonies at the peak of reproduction develop 1.2 oocytes/zooid, and up to 57.7% produced >4 clutches. Analysing onset of reproduction with maximal colony size revealed 4 patterns, two for “male only” colonies and two for hermaphrodites that varied at different sea water temperatures. In two patterns, the onset of reproduction precedes colony maximal size, and in the others it starts at the peak size or thereafter. Zooids at the colony's periphery developed almost twice as many buds as did zooids at the center, but produced significantly lower numbers of eggs. Fragmentation was recorded in large colonies and was temperature dependent. Results are compared with the data available on populations from other localities indicating for dramatically different developmental modes characteristic to this cosmopolitan species.     

A soluble extract from human spermatozoa activates ascidian oocytes

A soluble extract from human spermatozoa induced calcium oscillations and extrusion of the first polar body when injected into oocytes of the ascidian Ciona intestinalis . The properties of calcium oscillations and time of polar body extrusion precisely mimic oocyte activation induced by C. intestinalis sperm or sperm extracts. The data suggest that human sperm extracts can activate oocytes of different phyla by the same mechanism as homologous spermatozoa. Injection of inositol 1,4,5-trisphosphate (IP₃) into C. intestinalis oocytes mimicked to some extent the initial stages of oocyte activation, but the results demonstrate that ascidian oocyte activation by human sperm extract cannot be explained solely in terms of IP₃-induced calcium release. Injection of other calcium releasing second messengers, cyclic adenosine diphosphate ribose, or calcium ions, does not lead to oocyte activation or release intracellular calcium in ascidian oocytes. It was concluded that human spermatozoa contain one or more molecules that can trigger intracellular calcium release in oocytes from different phyla.     

The pink-blue spot syndrome in Acropora eurystoma (Eilat, Red Sea): a possible marker of stress?

The appearance of pink-blue spots (termed here as pink-blue spot syndrome - PBSS) in the branching coral Acropora eurystoma from the Gulf of Eilat, Red Sea, is described. We monitored 18 transects (10 x 1 m2 each) in front of the H. Steinitz Marine Laboratory (Eilat), at 3, 6 and 9 m depth, during March and August in 2001 and 2002. Transect measurements revealed high frequencies of colonies with PBSS (up to 100% of colonies) between 3 and 9 m depth. Ten PBSS-affected colonies of A. eurystoma were labelled and monitored for the development of spots. From March to August 2001, the number of spots per colony increased and remained constantly high at both sampling dates in 2002. Spot size ranged between 7 and 149 mm2. Spots were primarily recorded in areas where coral tissues contacted foreign biological matter, either around regenerative wounds or when surrounded by encrusting organisms, in fast-growing areas and in allogeneic interactions. A preliminary biochemical examination suggested that the pink-blue pigment in A. eurystoma is part of a family of compounds (pocilloporin) responsible for the pink-blue colours in pocilloporid and acroporid corals. Pink-blue colour could be experimentally induced in A. eurystoma by tissue-to-tissue contacts between distressed and non-distressed allogeneic branches. PBSS was also induced in healthy coral tissue by contact with inert objects, e.g., by bandaging a branch with plastic strips. Any specific pink-blue colour spots faded within 1-3 months from onset. These results suggest that PBSS in A. eurystoma may not be considered a regular coral disease, but rather a locally induced syndrome caused by restricted environmental and/or biological stress conditions.     

The importance of life-history stage and individual variation in the allorecognition system of a marine sponge

In marine invertebrates with complex life cycles, it may often be the case that trade-offs and behaviors differ between adult and larval stages. In this study, I examined the effects of life-history stage on allorecognition system function in the sponge, Haliclona sp. For sedentary marine invertebrates, allorecognition systems allow individuals to distinguish between genetically similar and distinct tissue they may encounter and are thought to reduce costly tissue fusion with individuals other than self or kin. Although it was found that sessile adults fused preferentially with self-tissue and exhibited a functioning allorecognition system, free-swimming larvae fused equally with sibling and non-sibling larvae resulting in swimming chimeras capable of successful metamorphosis, suggesting a stage-activated allorecognition system. In addition, adult sponges differed significantly in the propensity of their larvae to fuse suggesting variation in parental strategies. Analysis of larval swimming behavior indicated that larvae aggregate and are capable of increasing their encounters with other larvae and perhaps their probability of fusing in nature. The pursuit of fusion at this motile stage, along with evidence of a functioning adult allorecognition system, suggests that larvae may not express a recognition system, or that factors other than relatedness such as benefits to larval or adult chimeras, are involved in larval fusion and a stage-activated allorecognition system. In addition, this study demonstrates the presence of variation among individuals in the allorecognition system's ontogeny in the sponge Haliclona sp.     

Genetic and Genomic Toolbox of the Chordate Ciona intestinalis

The experimental malleability and unique phylogenetic position of the sea squirt Ciona intestinalis as part of the sister group to the vertebrates have helped establish these marine chordates as model organisms for the study of developmental genetics and evolution. Here we summarize the tools, techniques, and resources available to the Ciona geneticist, citing examples of studies that employed such strategies in the elucidation of gene function in Ciona. Genetic screens, germline transgenesis, electroporation of plasmid DNA, and microinjection of morpholinos are all routinely employed, and in the near future we expect these to be complemented by targeted mutagenesis, homologous recombination, and RNAi. The genomic resources available will continue to support the design and interpretation of genetic experiments and allow for increasingly sophisticated approaches on a high-throughput, whole-genome scale.