A broad-taxa approach as an important concept in ecotoxicological studies and pollution monitoring.

Aquatic invertebrates play a pivotal role in (eco)toxicological assessments because they offer ethical, cost-effective and repeatable testing options. Additionally, their significance in the food chain and their ability to represent diverse aquatic ecosystems make them valuable subjects for (eco)toxicological studies. To ensure consistency and comparability across studies, international (eco)toxicology guidelines have been used to establish standardised methods and protocols for data collection, analysis and interpretation. However, the current standardised protocols primarily focus on a limited number of aquatic invertebrate species, mainly from Arthropoda, Mollusca and Annelida. These protocols are suitable for basic toxicity screening, effectively assessing the immediate and severe effects of toxic substances on organisms. For more comprehensive and ecologically relevant assessments, particularly those addressing long-term effects and ecosystem-wide impacts, we recommended the use of a broader diversity of species, since the present choice of taxa exacerbates the limited scope of basic ecotoxicological studies. This review provides a comprehensive overview of (eco)toxicological studies, focusing on major aquatic invertebrate taxa and how they are used to assess the impact of chemicals in diverse aquatic environments. The present work supports the use of a broad-taxa approach in basic environmental assessments, as it better represents the natural populations inhabiting various ecosystems. Advances in omics and other biochemical and computational techniques make the broad-taxa approach more feasible, enabling mechanistic studies on non-model organisms. By combining these approaches with in vitro techniques together with the broad-taxa approach, researchers can gain insights into less-explored impacts of pollution, such as changes in population diversity, the development of tolerance and transgenerational inheritance of pollution responses, the impact on organism phenotypic plasticity, biological invasion outcomes, social behaviour changes, metabolome changes, regeneration phenomena, disease susceptibility and tissue pathologies. This review also emphasises the need for harmonised data-reporting standards and minimum annotation checklists to ensure that research results are findable, accessible, interoperable and reusable (FAIR), maximising the use and reusability of data. The ultimate goal is to encourage integrated and holistic problem-focused collaboration between diverse scientific disciplines, international standardisation organisations and decision-making bodies, with a focus on transdisciplinary knowledge co-production for the One-Health approach.

Physiological changes during torpor favor association with Endozoicomonas endosymbionts in the urochordate Botrylloides leachii.

Environmental perturbations evoke down-regulation of metabolism in some multicellular organisms, leading to dormancy, or torpor. Colonies of the urochordate Botrylloides leachii enter torpor in response to changes in seawater temperature and may survive for months as small vasculature remnants that lack feeding and reproductive organs but possess torpor-specific microbiota. Upon returning to milder conditions, the colonies rapidly restore their original morphology, cytology and functionality while harboring re-occurring microbiota, a phenomenon that has not been described in detail to date. Here we investigated the stability of B. leachii microbiome and its functionality in active and dormant colonies, using microscopy, qPCR, in situ hybridization, genomics and transcriptomics. A novel lineage of Endozoicomonas, proposed here as Candidatus Endozoicomonas endoleachii, was dominant in torpor animals (53-79% read abundance), and potentially occupied specific hemocytes found only in torpid animals. Functional analysis of the metagenome-assembled genome and genome-targeted transcriptomics revealed that Endozoicomonas can use various cellular substrates, like amino acids and sugars, potentially producing biotin and thiamine, but also expressing various features involved in autocatalytic symbiosis. Our study suggests that the microbiome can be linked to the metabolic and physiological states of the host, B. leachii, introducing a model organism for the study of symbioses during drastic physiological changes, such as torpor.

Transcriptome landscapes that signify Botrylloides leachi (Ascidiacea) torpor states.

Harsh environments enforce the expression of behavioural, morphological, physiological, and reproductive rejoinders, including torpor. Here we study the morphological, cellular, and molecular alterations in torpor architype in the colonial urochordate Botrylloides aff. leachii by employing whole organism Transmission electron (TEM) and light microscope observations, RNA sequencing, real-time polymerase chain reaction (qPCR) quantification of selected genes, and immunolocalization of WNT, SMAD and SOX2 gene expressions. On the morphological level, torpor starts with gradual regression of all zooids and buds which leaves the colony surviving as condensed vasculature remnants that may be 'aroused' to regenerate fully functional colonies upon changes in the environment. Simultaneously, we observed altered distributions of hemolymph cell types. Phagocytes doubled in number, while the number of morula cells declined by half. In addition, two new circulating cell types were observed, multi-nucleated and bacteria-bearing cells. RNA sequencing technology revealed marked differences in gene expression between different organism compartments and states: active zooids and ampullae, and between mid-torpor and naive colonies, or naive and torpid colonies. Gene Ontology term enrichment analyses further showed disparate biological processes. In torpid colonies, we observed overall 233 up regulated genes. These genes included NR4A2, EGR1, MUC5AC, HMCN2 and. Also, 27 transcription factors were upregulated in torpid colonies including ELK1, HDAC3, RBMX, MAZ, STAT1, STAT4 and STAT6. Interestingly, genes involved in developmental processes such as SPIRE1, RHOA, SOX11, WNT5A and SNX18 were also upregulated in torpid colonies. We further validated the dysregulation of 22 genes during torpor by utilizing qPCR. Immunohistochemistry of representative genes from three signaling pathways revealed high expression of these genes in circulated cells along torpor. WNT agonist administration resulted in early arousal from torpor in 80% of the torpid colonies while in active colonies WNT agonist triggered the torpor state. Abovementioned results thus connote unique transcriptome landscapes associated with Botrylloides leachii torpor.

A pan-metazoan concept for adult stem cells: the wobbling Penrose landscape.

Adult stem cells (ASCs) in vertebrates and model invertebrates (e.g. Drosophila melanogaster) are typically long-lived, lineage-restricted, clonogenic and quiescent cells with somatic descendants and tissue/organ-restricted activities. Such ASCs are mostly rare, morphologically undifferentiated, and undergo asymmetric cell division. Characterized by 'stemness' gene expression, they can regulate tissue/organ homeostasis, repair and regeneration. By contrast, analysis of other animal phyla shows that ASCs emerge at different life stages, present both differentiated and undifferentiated phenotypes, and may possess amoeboid movement. Usually pluri/totipotent, they may express germ-cell markers, but often lack germ-line sequestering, and typically do not reside in discrete niches. ASCs may constitute up to 40% of animal cells, and participate in a range of biological phenomena, from whole-body regeneration, dormancy, and agametic asexual reproduction, to indeterminate growth. They are considered legitimate units of selection. Conceptualizing this divergence, we present an alternative stemness metaphor to the Waddington landscape: the 'wobbling Penrose' landscape. Here, totipotent ASCs adopt ascending/descending courses of an 'Escherian stairwell', in a lifelong totipotency pathway. ASCs may also travel along lower stemness echelons to reach fully differentiated states. However, from any starting state, cells can change their stemness status, underscoring their dynamic cellular potencies. Thus, vertebrate ASCs may reflect just one metazoan ASC archetype.

Stem Cells and Innate Immunity in Aquatic Invertebrates: Bridging Two Seemingly Disparate Disciplines for New Discoveries in Biology.

The scopes related to the interplay between stem cells and the immune system are broad and range from the basic understanding of organism's physiology and ecology to translational studies, further contributing to (eco)toxicology, biotechnology, and medicine as well as regulatory and ethical aspects. Stem cells originate immune cells through hematopoiesis, and the interplay between the two cell types is required in processes like regeneration. In addition, stem and immune cell anomalies directly affect the organism's functions, its ability to cope with environmental changes and, indirectly, its role in ecosystem services. However, stem cells and immune cells continue to be considered parts of two branches of biological research with few interconnections between them. This review aims to bridge these two seemingly disparate disciplines towards much more integrative and transformative approaches with examples deriving mainly from aquatic invertebrates. We discuss the current understanding of cross-disciplinary collaborative and emerging issues, raising novel hypotheses and comments. We also discuss the problems and perspectives of the two disciplines and how to integrate their conceptual frameworks to address basic equations in biology in a new, innovative way.

Stem cells of aquatic invertebrates as an advanced tool for assessing ecotoxicological impacts.

Environmental stressors are assessed through methods that quantify their impacts on a wide range of metrics including species density, growth rates, reproduction, behaviour and physiology, as on host-pathogen interactions and immunocompetence. Environmental stress may induce additional sublethal effects, like mutations and epigenetic signatures affecting offspring via germline mediated transgenerational inheritance, shaping phenotypic plasticity, increasing disease susceptibility, tissue pathologies, changes in social behaviour and biological invasions. The growing diversity of pollutants released into aquatic environments requires the development of a reliable, standardised and 3R (replacement, reduction and refinement of animals in research) compliant in vitro toolbox. The tools have to be in line with REACH regulation 1907/2006/EC, aiming to improve strategies for potential ecotoxicological risks assessment and monitoring of chemicals threatening human health and aquatic environments. Aquatic invertebrates' adult stem cells (ASCs) are numerous and can be pluripotent, as illustrated by high regeneration ability documented in many of these taxa. This is of further importance as in many aquatic invertebrate taxa, ASCs are able to differentiate into germ cells. Here we propose that ASCs from key aquatic invertebrates may be harnessed for applicable and standardised new tests in ecotoxicology. As part of this approach, a battery of modern techniques and endpoints are proposed to be tested for their ability to correctly identify environmental stresses posed by emerging contaminants in aquatic environments. Consequently, we briefly describe the current status of the available toxicity testing and biota-based monitoring strategies in aquatic environmental ecotoxicology and highlight some of the associated open issues such as replicability, consistency and reliability in the outcomes, for understanding and assessing the impacts of various chemicals on organisms and on the entire aquatic environment. Following this, we describe the benefits of aquatic invertebrate ASC-based tools for better addressing ecotoxicological questions, along with the current obstacles and possible overhaul approaches.

UV-B radiation bearings on ephemeral soma in the shallow water tunicate Botryllus schlosseri.

Sedentary shallow water marine organisms acquire numerous protective mechanisms to mitigate the detrimental effects of UV radiation (UV-R). Here we investigated morphological and gene expression outcomes in colonies of the cosmopolitan ascidian Botryllus schlosseri, up to 15-days post UV-B irradiation. Astogeny in Botryllus is characterized by weekly repeating sets of asexual budding, coinciding with apoptotic elimination of functional zooids (blastogenesis). Ten UV-B doses were administered to three clusters: sublethal, enhanced-mortality, lethal (LD50 = 6.048 kJ/m2) which differed in mortality rates, yet reflected similar distorted morphotypes, and arrested blastogenesis, all intensified in the enhanced-mortality/lethal clusters. Even the sub-lethal doses inflicted expression modifications in 8 stress proteins (HSP 90/70 families and NIMA) as well as morphological blastogenesis. The morphological/gene-expression impacts in surviving colonies lasted for 15 days post irradiation (two blastogenic-cycles), where all damaged and arrested zooids/buds were absorbed, after which the colonies returned to their normal blastogenic-cycles and gene expression profiles, and initiated new buds. The above reflects a novel colonial maintenance strategy associated with the disposable-soma tenet, where the ephemeral soma in Botryllus is eliminated without engaging with the costs of repair, whereas other colonial components, primarily the pool of totipotent stem cells, are sustained under yet unknown colonial-level regulatory cues.

IAP genes partake weighty roles in the astogeny and whole body regeneration in the colonial urochordate Botryllus schlosseri.

Inhibitors of Apoptosis Protein (IAP) genes participate in processes like apoptosis, proliferation, innate immunity, inflammation, cell motility, differentiation and in malignancies. Here we reveal 25 IAP genes in the tunicate Botryllus schlosseri's genome and their functions in two developmental biology phenomena, a new mode of whole body regeneration (WBR) induced by budectomy, and blastogenesis, the four-staged cycles of botryllid ascidian astogeny. IAP genes that were specifically upregulated during these developmental phenomena were identified, and protein expression patterns of one of these genes, IAP28, were followed. Most of the IAP genes upregulation recorded at blastogenetic stages C/D was in concert with the upregulation at 100 µM H2O2 apoptotic-induced treatment and in parallel to expressions of AIF1, Bax, Mcl1, caspase 2 and two orthologues of caspase 7. Wnt agonist altered the takeover duration along with reduced IAP expressions, and displacement of IAP28+ phagocytes. WBR was initiated solely at blastogenetic stage D, where zooidal absorption was attenuated and regeneration centers were formed either from remains of partially absorbed zooids or from deformed ampullae. Subsequently, bud-bearing zooids developed, in concert with a massive IAP28-dependent phagocytic wave that eliminated the old zooids, then proceeded with the establishment of morphologically normal-looking colonies. IAP4, IAP14 and IAP28 were also involved in WBR, in conjunction with the expression of the pro-survival PI3K-Akt pathway. IAPs function deregulation by Smac mimetics resulted in severe morphological damages, attenuation in bud growth and differentiation, and in destabilization of colonial coordination. Longtime knockdown of IAP functions prior to the budectomy, resulted in colonial death.

Coupling astogenic aging in the colonial tunicate Botryllus schlosseri with the stress protein mortalin.

Botryllus schlosseri, a colonial marine invertebrate, exhibits three generations of short-lived astogenic modules that continuously grow and die throughout the colony's entire lifespan, within week-long repeating budding cycles (blastogenesis), each consisting of four stages (A-D). At stage D, aging is followed by the complete absorption of adult modules (zooids) via a massive apoptotic process. Here we studied in Botryllus the protein mortalin (HSP70s member), a molecule largely known for its association with aging and proliferation. In-situ hybridization and qPCR assays reveal that mortalin follows the cyclic pattern of blastogenesis. Colonies at blastogenic stage D display the highest mortalin levels, and young modules exhibit elevated mortalin levels compared to old modules. Manipulations of mortalin with the specific allosteric inhibitor MKT-077 has led to a decrease in the modules' growth rate and the development of abnormal somatic/germinal morphologies (primarily in vasculature and in organs such as the endostyle, the stomach and gonads). We therefore propose that mortalin plays a significant role in the astogeny and aging of colonial modules in B. schlosseri, by direct involvement in the regulation of blastogenesis.

The involvement of three signal transduction pathways in botryllid ascidian astogeny, as revealed by expression patterns of representative genes.

The patterning of the modular body plan in colonial organisms is termed astogeny, as distinct from ontogeny, the development of an individual organism from embryo to adult. Evolutionarily conserved signaling pathways suggest shared roots and common uses for both ontogeny and astogeny. Botryllid ascidians, a widely dispersed group of colonial tunicates, exhibit an intricate modular life form, in which astogeny develops as weekly, highly synchronized growth/death cycles termed blastogenesis, abiding by a strictly regulated plan. In these organisms both astogeny and ontogeny form similar body structures. Working on Botryllus schlosseri, and choosing a representative gene from each of three key Signal Transduction Pathways (STPs: Wnt/ß-catenin; TGF-ß, MAPK/ERK), we explored and compared gene expression at different stages of ontogeny and blastogenesis. Protein expression was studied via immunohistochemistry, ELISA and Western blotting. Five specific inhibitors and an activator for the selected pathways were used and followed to assess their impact during the blastogenic cycle and the development of distinctive phenotypes. Outcomes show that STPs are activated and function (while not necessarily co-localized) during both ontogeny and astogeny. Cellular patterns in blastogenesis, such as colony architecture, are shaped by these STPs. These results are further supported by administering Wnt agonist and anatagonist, TGF-ß receptor antagonists and inhibitors of Mek1/Mek2. Independent of their expression during ontogeny, some of the spatiotemporal patterns of STPs developed within short blastogenic windows. The results support the notion that while the same molecular machinery is functioning in Botryllus schlosseri astogeny and ontogeny, astogenic development is not an ontogenic replicate.

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 ß-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.

Repeated, long-term cycling of putative stem cells between niches in a basal chordate.

The mechanisms that sustain stem cells are fundamental to tissue maintenance. Here, we identify "cell islands" (CIs) as a niche for putative germ and somatic stem cells in Botryllus schlosseri, a colonial chordate that undergoes weekly cycles of death and regeneration. Cells within CIs express markers associated with germ and somatic stem cells and gene products that implicate CIs as signaling centers for stem cells. Transplantation of CIs induced long-term germline and somatic chimerism, demonstrating self-renewal and pluripotency of CI cells. Cell labeling and in vivo time-lapse imaging of CI cells reveal waves of migrations from degrading CIs into developing buds, contributing to soma and germline development. Knockdown of cadherin, which is highly expressed within CIs, elicited the migration of CI cells to circulation. Piwi knockdown resulted in regeneration arrest. We suggest that repeated trafficking of stem cells allows them to escape constraints imposed by the niche, enabling self-preservation throughout life.

Marine invertebrates cross phyla comparisons reveal highly conserved immune machinery.

Naturally occurring histocompatibility responses, following tissue-to-tissue allogeneic contacts, are common among numerous colonial marine invertebrate taxa, including sponges, cnidarians, bryozoans and ascidians. These responses, often culminating in either tissue fusions or rejections, activate a wide array of innate immune components. By comparing two allorejection EST libraries, developed from alloincompatible challenged colonies of the stony coral Stylophora pistillata and the ascidian Botryllus schlosseri, we revealed a common basis for innate immunity in these two evolutionary distant species. Two prominent genes within this common basis were the immunophilins, Cyclophilin A (CypA) and FK506-binding protein (FKBP). In situ hybridizations revealed that mRNA expression of the coral and ascidian immunophilins was restricted to specific allorecognition effector cell populations (nematoblasts and nematocytes in the coral and morula cells in the ascidian). The expressions were limited to only some of the effector cells within a population, disclosing disparities in numbers and location between naïve colonies and their immune challenged counterparts. Administration of the immunosuppression drug Cyclosporine-A during ascidian's allogeneic assays inhibited both fusion and rejection reactions, probably through the inhibition of ascidian's immunocytes (morula cells) movement and activation. Our results, together with previous published data, depict an immunophilins-based immune mechanism, which is similarly activated in allogeneic responses of distantly related animals from sponges to humans.

Piwi positive cells that line the vasculature epithelium, underlie whole body regeneration in a basal chordate.

The colonial tunicate Botrylloides leachi can regenerate functional adults from minute vasculature fragments, in a poorly understood phenomenon termed Whole Body Regeneration (WBR). Using Piwi expression (Bl-Piwi), blood cell labeling and electron microscopy, we show that WBR develops through activation, mobilization and expansion of 'dormant' cells which normally line the internal vasculature epithelium of blood vessels. Following a mechanical insult, these cells express Bl-Piwi de novo, change morphology and invade niches of the vasculature lumen, where they proliferate and differentiate, regenerating a functional organism. Mitomycin C treatments and siRNA knockdown of Bl-Piwi result in deficient cells incapable of expanding or differentiating and to subsequent regeneration arrest. Last, we find similar transient mobilization of Piwi(+) cells recurring every week, as part of normal colony development, and also during acute environmental stress. This recurrent activation of Piwi(+) cells in response to developmental, physiological and environmental insults may have enabled the adaptation of colonial tunicates to the imposed varied conditions in the marine, shallow water environment.

'Rejected' vs. 'rejecting' transcriptomes in allogeneic challenged colonial urochordates.

In botryllid ascidians, allogeneic contacts between histoincompatible colonies lead to inflammatory rejection responses, which eventually separate the interacting colonies. In order to elucidate the molecular background of allogeneic rejection in the colonial ascidian Botryllus schlosseri, we performed microarray assays verified by qPCR, and employed bioinformatic analyses of the results, revealing disparate transcription profiles of the rejecting partners. While only minor expression changes were documented during rejection when both interacting genotypes were pooled together, analyses performed on each genotype separately portrayed disparate transcriptome responses. Allogeneic interacting genotypes that developed the morphological markers of rejection (points of rejection; PORs), termed 'rejected' genotypes, showed transcription inhibition of key functional gene groups, including protein biosynthesis, cell structure and motility and stress response genes. In contrast, the allogeneic partners that did not show PORs, termed 'rejecting' genotypes, showed minor expression changes that were different from those of the 'rejected' genotypes. This data demonstrates that the observed morphological changes in the 'rejected' genotypes are not due to active transcriptional response to the immune challenge but reflect transcription inhibition of response elements. Based on the morphological and molecular outcomes we suggest that the 'rejected' colony activates an injurious self-destructive mechanism in order to disconnect itself from its histoincompatible neighboring colony.

Vasa and the germ line lineage in a colonial urochordate.

Germ cell sequestering in Animalia is enlightened by either, launching true germ line along epigenetic or preformistic modes of development, or by somatic embryogenesis, where no true germ line is set aside. The research on germ line-somatic tissue segregation is of special relevancy to colonial organisms like botryllid ascidians that reconstruct, on a weekly basis, completely new sets of male and female gonads in newly formed somatic tissues. By sequencing and evaluating expression patterns of BS-Vasa, the Botryllus schlosseri orthologue of Vasa, in sexually mature and asexual colonies during blastogenesis, we have demonstrated that the BS-Vasa mRNA and protein are not expressed exclusively in germ cell lineages, but appeared in cells repeatedly emerging de novo in the colony, independently of its sexual state. In addition, we recorded an immediate Vasa response to cellular stress (UV irradiation) indicating additional functions to its germ line assignments. To confirm germ lineage exclusivity, we examined the expression of three more stem cell markers (BS-Pl10, Bl-piwi and Oct4). Vasa co-expression with Pl10 and Oct4 was detected in germ line derivatives and with Bl-piwi in somatic tissues. Presumptive primordial germ cells (PGC-like cells), that are Vasa(+)/Pl10(+)/Oct4(+) and 6-12 microm in diameter, were first detected in wrapped-tail embryos, in oozooids, in sexual/asexual colonies, within a newly identified PGC niche termed as 'budlet niche', and in circulating blood borne cells, indicating epigenetic embryogenesis. Alternatively, BS-Vasa co-expression with piwi orthologue, an omnipresent bona fide stemness flag, in non germ line cell populations, may indicate germ cell neogenesis (somatic embryogenesis) in B. schlosseri. Both alternatives are not necessarily mutually exclusive.

Identification of the endostyle as a stem cell niche in a colonial chordate.

Stem cell populations exist in "niches" that hold them and regulate their fate decisions. Identification and characterization of these niches is essential for understanding stem cell maintenance and tissue regeneration. Here we report on the identification of a novel stem cell niche in Botryllus schlosseri, a colonial urochordate with high stem cell-mediated developmental activities. Using in vivo cell labeling, engraftment, confocal microscopy, and time-lapse imaging, we have identified cells with stemness capabilities in the anterior ventral region of the Botryllus' endostyle. These cells proliferate and migrate to regenerating organs in developing buds and buds of chimeric partners but do not contribute to the germ line. When cells are transplanted from the endostyle region, they contribute to tissue development and induce long-term chimerism in allogeneic tissues. In contrast, cells from other Botryllus' regions do not show comparable stemness capabilities. Cumulatively, these results define the Botryllus' endostyle region as an adult somatic stem cell niche.

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.

Divergent roles of the DEAD-box protein BS-PL10, the urochordate homologue of human DDX3 and DDX3Y proteins, in colony astogeny and ontogeny.

Proteins of the highly conserved PL-10 (Ded1P) subfamily of DEAD-box family, participate in a wide variety of biological functions. However, the entire spectrum of their functions in both vertebrates and invertebrates is still unknown. Here, we isolated the Botryllus schlosseri (Urochordata) homologue, BS-PL10, revealing its distributions and functions in ontogeny and colony astogeny. In botryllid ascidians, the colony grows by increasing the number of modular units (each called a zooid) through a whole colony synchronized and weekly cyclical astogenic budding process (blastogenesis). At the level of the colony, both BS-PL10 mRNA and its protein (78 kDa) fluctuate in a weekly pattern that corresponds with the animal's blastogenic cycle, increasing from blastogenic stage A to blastogenic stage D. At the organ/module level, a sharp decline is revealed. Primary and secondary developing buds express high levels of BS-PL10 mRNA and protein at all blastogeneic stages. These levels are reduced four to nine times in the new set of functional zooids. This portrait of colony astogeny differed from its ontogeny. Oocytes and sperm cells express high levels of BS-PL10 protein only at early stages of development. Young embryos reveal background levels with increased expressions in some organs at more developed stages. Results reveal that higher levels of BS-PL10 mRNA and protein are characteristic to multipotent soma and germ cells, but patterns deviate between two populations of differentiating stem cells, the stem cells involved in weekly blastogenesis and stem cells involved in embryogenesis. Two types of experimental manipulations, zooidectomy and siRNA assays, have confirmed the importance of BS-PL10 for cell differentiation and organogenesis. BS-PL10 (phylogenetically matching the animal's position in the evolutionary tree), is the only member of this subfamily in B. schlosseri, featuring a wide range of biological activities, some of which represent pivotal roles. The surprising weekly cyclical expression and the participation in cell differentiation posit this molecule as a model system for studying PL10 protein subfamily.