The stress granule component TIAR during the non-embryonic development of the colonial ascidian Botryllusschlosseri.

TIAR, is a nucleic acid binding protein involved in the formation of cytoplasmic foci known as stress granules, in which mRNA translation is temporarily blocked in response to stressful conditions. TIAR is used as stress granules molecular marker in vertebrates, but it is not so deeply investigated in invertebrates, especially in marine organisms. In the present work, we investigated the role of TIAR in the colonial ascidian Botryllus schlosseri during its non-embryonic development, featured by the cyclical renewal of the colony. We studied the extent of transcription during the colonial blastogenetic cycle and the location of the transcripts in Botryllus tissues. Using an anti-TIAR antibody specific for ascidians, by immunocytochemistry and immunohistochemistry assays, we studied the expression of the protein in haemolymph cells and body tissues and by transmission electron microscopy we identified its subcellular localisation. The anti-TIAR antibody was also microinjected in the circulatory system of B. schlosseri to study its effect on non-embryonic development and immune responses. Results indicate a delay in the progression of the blastogenetic cycle in injected colonies. In addition, degranulation of circulating cytotoxic cells and phagocytosis by professional, circulating phagocytes, two fundamental processes of innate immunity, were also negatively affected.

New Data on the Rhamnose-Binding Lectin from the Colonial Ascidian Botryllus schlosseri: Subcellular Distribution, Secretion Mode and Effects on the Cyclical Generation Change.

Botryllus schlosseri in a cosmopolitan ascidian, considered a reliable model organism for studies on the evolution of the immune system. B. schlosseri rhamnose-binding lectin (BsRBL) is synthesised by circulating phagocytes and behaves as an opsonin by interacting with foreign cells or particles and acting as a molecular bridge between them and the phagocyte surface. Although described in previous works, many aspects and roles of this lectin in Botryllus biology remain unknown. Here, we studied the subcellular distribution of BsRBL during immune responses using light and electron microscopy. In addition, following the hints from extant data, suggesting a possible role of BsRBL in the process of cyclical generation change or takeover, we investigated the effects of interfering with this protein, by injecting a specific antibody in the colonial circulation, starting one day before the generation change. Results confirm the requirement of the lectin for a correct generation change and open new queries on the roles of this lectin in Botryllus biology.

Botryllus schlosseri as a Unique Colonial Chordate Model for the Study and Modulation of Innate Immune Activity.

Understanding the mechanisms that sustain immunological nonreactivity is essential for maintaining tissue in syngeneic and allogeneic settings, such as transplantation and pregnancy tolerance. While most transplantation rejections occur due to the adaptive immune response, the proinflammatory response of innate immunity is necessary for the activation of adaptive immunity. Botryllus schlosseri, a colonial tunicate, which is the nearest invertebrate group to the vertebrates, is devoid of T- and B-cell-based adaptive immunity. It has unique characteristics that make it a valuable model system for studying innate immunity mechanisms: (i) a natural allogeneic transplantation phenomenon that results in either fusion or rejection; (ii) whole animal regeneration and noninflammatory resorption on a weekly basis; (iii) allogeneic resorption which is comparable to human chronic rejection. Recent studies in B. schlosseri have led to the recognition of a molecular and cellular framework underlying the innate immunity loss of tolerance to allogeneic tissues. Additionally, B. schlosseri was developed as a model for studying hematopoietic stem cell (HSC) transplantation, and it provides further insights into the similarities between the HSC niches of human and B. schlosseri. In this review, we discuss why studying the molecular and cellular pathways that direct successful innate immune tolerance in B. schlosseri can provide novel insights into and potential modulations of these immune processes in humans.

De novo neurogenesis in a budding chordate: Co-option of larval anteroposterior patterning genes in a transitory neurogenic organ.

During metamorphosis of solitary ascidians, part of the larval tubular nervous system is recruited to form the adult central nervous system (CNS) through neural stem-like cells called ependymal cells. The anteroposterior (AP) gene expression patterning of the larval CNS regionalize the distribution of the ependymal cells, which contains the positional information of the neurons of the adult nervous system. In colonial ascidians, the CNS of asexually developed zooids has the same morphology of the one of the post-metamorphic zooids. However, its development follows a completely different organogenesis that lacks embryogenesis, a larval phase and metamorphosis. In order to describe neurogenesis during asexual development (blastogenesis), we followed the expression of six CNS AP patterning genes conserved in chordates and five neural-related genes to determine neural cell identity in Botryllus schlosseri. We observed that a neurogenesis occurs de novo on each blastogenic cycle starting from a neurogenic transitory structure, the dorsal tube. The dorsal tube partially co-opts the AP patterning of the larval CNS markers, and potentially combine the neurogenesis role and provider of positional clues for neuron patterning. This study shows how a larval developmental module is reused in a direct asexual development in order to generate the same structures.

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.

BsTLR1: A new member of the TLR family of recognition proteins from the colonial ascidian Botryllus schlosseri.

Toll-like receptors (TLRs) represent a well-known family of conserved pattern recognition receptors the importance of which, in non-self recognition, was demonstrated in both vertebrates and invertebrates. Tunicates represent the vertebrate sister group and, as invertebrates, they rely only on innate immunity for their defence. As regards TLRs, two transcripts have been described and characterised in the solitary species Ciona intestinalis, referred to as CiTLR1 and CiTLR2. Using the Ciona TLR nucleotide sequences, we mined our available transcriptome of the colonial ascidian Botryllus schlosseri looking for similar sequences. We were able to identify a sequence, with similarity to CiTLR2 and, through in silico transduction and subsequent sequence analysis, we studied the domain content of the putative protein. The sequence, called BsTLR1, has a TIR and a transmembrane domain, four LLR and two LRR-CT domains. It is actively transcribed by both phagocytes and morula cells, the two circulating immunocyte types. In addition, we analysed bstlr1 transcription in vivo and in vitro, in different phases of the Botryllus blastogenetic cycle and under various experimental conditions. Our data show that there is a change in gene expression and mRNA location, according to the blastogenetic phase. Furthermore, we used a commercial antibody raised against the ectodomain of hTLR5 to study the possible functional role of Botryllus TLR(s). We observed that anti-hTLR5 significantly decreased in vitro phagocytosis and morula cell degranulation, two typical responses to the recognition of nonself. Collectively, our data add new information on the mechanisms of nonself recognition in a colonial ascidian.

Phylogenetic analysis and screening of antimicrobial and cytotoxic activities of culturable bacteria associated with the ascidian Botryllus schlosseri.

AIMS: Isolating culturable bacteria associated with ascidian (Botryllus schlosseri) and investigating their bioactivities to discover new marine microbial resources with potential to produce novel bioactive natural products. METHODS AND RESULTS: A total of 357 bacteria were isolated from the ascidian B. schlosseri from the coast of Weihai in the north Yellow Sea, China. Of these, 203 isolates were identified by 16S rRNA gene sequencing and they belonged to 52 genera from 30 families in five phyla. The antimicrobial activities and cytotoxic activities of all isolates were determined. Of the 357 isolates, 135 isolates demonstrated antimicrobial activities, and the crude extracts of five isolates showed strong cytotoxicity against human hepatocellular carcinoma Bel 7402 or human cervical carcinoma HeLa cells. CONCLUSIONS: Our study revealed the diversity of bacteria associated with the ascidian B. schlosseri and reported a broad spectrum of antimicrobial and cytotoxic activities displayed by these isolates. SIGNIFICANCE AND IMPACT OF THE STUDY: Our results suggest that the culturable bacteria associated with the ascidian B. schlosseri may be a potential source for novel bioactive compounds.

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.

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.

Stem cells are units of natural selection for tissue formation, for germline development, and in cancer development.

It is obvious that natural selection operates at the level of individuals and collections of individuals. Nearly two decades ago we showed that in multi-individual colonies of protochordate colonial tunicates sharing a blood circulation, there exists an exchange of somatic stem cells and germline stem cells, resulting in somatic chimeras and stem cell competitions for gonadal niches. Stem cells are unlike other cells in the body in that they alone self-renew, so that they form clones that are perpetuated for the life of the organism. Stem cell competitions have allowed the emergence of competitive somatic and germline stem cell clones. Highly successful germline stem cells usually outcompete less successful competitors both in the gonads of the genotype partner from which they arise and in the gonads of the natural parabiotic partners. Therefore, natural selection also operates at the level of germline stem cell clones. In the colonial tunicate Botryllus schlosseri the formation of natural parabionts is prevented by a single-locus highly polymorphic histocompatibility gene called Botryllus histocompatibility factor. This limits germline stem cell predation to kin, as the locus has hundreds of alleles. We show that in mice germline stem cells compete for gonad niches, and in mice and humans, blood-forming stem cells also compete for bone marrow niches. We show that the clonal progression from blood-forming stem cells to acute leukemias by successive genetic and epigenetic events in blood stem cells also involves competition and selection between clones and propose that this is a general theme in cancer.

Characterization of Ambra1 in asexual cycle of a non-vertebrate chordate, the colonial tunicate Botryllus schlosseri, and phylogenetic analysis of the protein group in Bilateria.

Ambra1 is a positive regulator of autophagy, a lysosome-mediated degradative process involved both in physiological and pathological conditions. Nowadays, Ambra1 has been characterized only in mammals and zebrafish. Through bioinformatics searches and targeted cloning, we report the identification of the complete Ambra1 transcript in a non-vertebrate chordate, the tunicate Botryllus schlosseri. Tunicata is the sister group of Vertebrata and the only chordate group possessing species that reproduce also by blastogenesis (asexual reproduction). B. schlosseri Ambra1 deduced amino acid sequence is shorter than vertebrate homologues but still contains the typical WD40 domain. qPCR analyses revealed that the level of B. schlosseri Ambra1 transcription is temporally regulated along the colonial blastogenetic cycle. By means of similarity searches we identified Wdr5 and Katnb1 as proteins evolutionarily associated to Ambra1. Phylogenetic analyses on Bilateria indicate that: (i) Wdr5 is the most related to Ambra1, so that they may derive from an ancestral gene, (ii) Ambra1 forms a group of ancient genes evolved before the radiation of the taxon, (iii) these orthologous Ambra1 share the two conserved WD40/YVTN repeat-like-containing domains, and (iv) they are characterized by ancient duplications of WD40 repeats within the N-terminal domain.

Molecular evolution and in vitro characterization of Botryllus histocompatibility factor.

Botryllus schlosseri is a colonial ascidian with a natural ability to anastomose with another colony to form a vascular and hematopoietic chimera. In order to fuse, two individuals must share at least one allele at the highly polymorphic fuhc locus. Otherwise, a blood-based inflammatory response will occur resulting in a melanin scar at the sites of interaction. The single-locus genetic control of allorecognition makes B. schlosseri an attractive model to study the underlying molecular mechanisms. Over the past decade, several candidate genes involved in allorecognition have been identified, but how they ultimately contribute to allorecognition outcome remains poorly understood. Here, we report our initial molecular characterization of a recently identified candidate allodeterminant called Botryllus histocompatibility factor (bhf). bhf, both on a DNA and protein level, is the least polymorphic protein in the fuhc locus studied so far and, unlike other known allorecognition determinants, does not appear to be under any form of balancing or directional selection. Additionally, we identified a second isoform through mRNA-Seq and an EST assembly library which is missing exon 3, resulting in a C-terminally truncated form. We report via whole-mount fluorescent in situ hybridization that a subset of cells co-express bhf and cfuhc(sec). Finally, we observed BHF's localization in HEK293T at the cytoplasmic side of the plasma membrane in addition to the nucleus via a nuclear localization signal. Given the localization data thus far, we hypothesize that BHF may function as a scaffolding protein in a complex with other Botryllus proteins, rather than functioning as an allorecognition determinant.

Botryllus schlosseri, an emerging model for the study of aging, stem cells, and mechanisms of regeneration.

The decline of tissue regenerative potential with the loss of stem cell function is a hallmark of mammalian aging. We study Botryllus schlosseri, a colonial chordate which exhibits robust stem cell-mediated regeneration capacities throughout life. Larvae, derived by sexual reproduction and chordate development, metamorphose to clonal founders that undergo weekly formation of new individuals by budding from stem cells. Individuals are transient structures which die through massive apoptosis, and successive buds mature to replicate an entire new body. As a result, their stem cells, which are the only self-renewing cells in a tissue, are the only cells which remain through the entire life of the genotype and retain the effects of time. During aging, a significant decrease in the colonies' regenerative potential is observed and both sexual and asexual reproductions will eventually halt. When a parent colony is experimentally separated into a number of clonal replicates, they frequently undergo senescence simultaneously, suggesting a heritable factor that determines lifespan in these colonies. The availability of the recently published B. schlosseri genome coupled with its unique life cycle features promotes the use of this model organism for the study of the evolution of aging, stem cells, and mechanisms of regeneration.

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.

Genotype-specific expression of uncle fester suggests a role in allorecognition education in a basal chordate.

Histocompatibility is the ability to discriminate between self and non-self tissues, and has been described in species throughout the metazoa. Despite its universal presence, histocompatibility genes utilized by different phyla are unique- those found in sponges, cnidarians, ascidians and vertebrates are not orthologous. Thus, the origins of these sophisticated recognition systems, and any potential functional commonalities between them are not understood. A well-studied histocompatibility system exists in the botryllid ascidians, members of the chordate subphylum, Tunicata, and provides an opportunity to do so. Histocompatibility in the botryllids occurs at the tips of an extracorporeal vasculature that come into contact when two individuals grow into proximity. If compatible, the vessels will fuse, forming a parabiosis between the two individuals. If incompatible, the two vessels will reject- an inflammatory reaction that results in melanin scar formation at the point of contact, blocking anastomosis. Compatibility is determined by a single, highly polymorphic locus called the fuhc with the following rules: individuals that share one or both fuhc alleles will fuse, while those who share neither will reject. The fuhc locus encodes multiple proteins with roles in allorecognition, including one called uncle fester, which is necessary and sufficient to initiate the rejection response. Here we report the existence of genotype-specific expression levels of uncle fester, differing by up to 8-fold at the mRNA-level, and that these expression levels are constant and maintained for the lifetime of an individual. We also found that these differences had functional consequences: the expression level of uncle fester correlated with the speed and severity of the rejection response. These findings support previous conclusions that uncle fester levels modulate the rejection response, and may be responsible for controlling the variation observed in the timing and intensity of the reaction. The maintenance of genotype specific expression of uncle fester is also evidence of an education process reminiscent of that which occurs in mammalian Natural Killer (NK) cells. In turn, this suggests that while histocompatibility receptors and ligands evolve via convergent evolution, they may utilize conserved intracellular machinery to interpret binding events at the cell surface.

Improved Media Formulations for Primary Cell Cultures Derived from a Colonial Urochordate.

The cultivation of marine invertebrate cells in vitro has garnered significant attention due to the availability of diverse cell types and cellular potentialities in comparison to vertebrates and particularly in response to the demand for a multitude of applications. While cells in the colonial urochordate Botryllus schlosseri have a very high potential for omnipotent differentiation, no proliferating cell line has been established in Botryllus, with results indicating that cell divisions cease 24-72 h post initiation. This research assessed how various Botryllus blood cell types respond to in vitro conditions by utilizing five different refinements of cell culture media (TGM1-TGM5). During the initial week of culture, there was a noticeable medium-dependent increase in the proliferation and viability of distinct blood cell types. Within less than one month from initiation, we developed medium-specific primary cultures, a discovery that supports larger efforts to develop cell type-specific cultures. Specific cell types were easily distinguished and classified based on their natural fluorescence properties using confocal microscopy. These results are in agreement with recent advances in marine invertebrate cell cultures, demonstrating the significance of optimized nutrient media for cell culture development and for cell selection.

Yamanaka Factors in the Budding Tunicate Botryllus schlosseri Show a Shared Spatio-Temporal Expression Pattern in Chordates.

In vertebrates, the four transcription factors Sox2, c-Myc, Pou5f1 and Klf4 are involved in the differentiation of several tissues during vertebrate embryogenesis; moreover, they are normally co-expressed in embryonic stem cells and play roles in pluripotency, self-renewal, and maintenance of the undifferentiated state in adult cells. The in vitro forced co-expression of these factors, named Yamanaka factors (YFs), induces pluripotency in human or mouse fibroblasts. Botryllus schlosseri is a colonial tunicate undergoing continuous stem cell-mediated asexual development, providing a valuable model system for the study of pluripotency in the closest living relatives of vertebrates. In this study, we identified B. schlosseri orthologs of human Sox2 and c-Myc genes, as well as the closest homologs of the vertebrate-specific Pou5f1 gene, through an in-depth evolutionary analysis of the YF gene families in tunicates and other deuterostomes. Then, we studied the expression of these genes during the asexual cycle of B. schlosseri using in situ hybridization in order to investigate their possible involvement in tissue differentiation and in pluripotency maintenance. Our results show a shared spatio-temporal expression pattern consistent with the reported functions of these genes in invertebrate and vertebrate embryogenesis. Moreover, Myc, SoxB1 and Pou3 were expressed in candidate stem cells residing in their niches, while Pou2 was found expressed exclusively in the immature previtellogenic oocytes, both in gonads and circulating in the colonial vascular system. Our data suggest that Myc, SoxB1 and Pou3 may be individually involved in the differentiation of the same territories seen in other chordates, and that, together, they may play a role in stemness even in this colonial ascidian.

Characterisation and functional role of a novel C1qDC protein from a colonial ascidian.

As an invertebrate, the compound ascidian Botryllus schlosseri faces nonself only with innate immunity. In this species, we already identified the key components of the lectin and alternative complement activation pathways. In the present work, by mining the transcriptome, we identified a single transcript codifying for a protein, member of the C1q-domain-containing protein family, with a signal peptide followed by two globular C1q (gC1q) domains. It shares a similar domain organisation with C1q/TNF-related proteins 4, the only vertebrate protein family with two gC1q domains. Our gC1q domain-containing protein, called BsC1qDC, is actively transcribed by immunocytes. The transcription is modulated during the Botryllus blastogenetic cycle and is upregulated following the injection of Bacillus clausii cells in the circulation. Furthermore, the injection of bsc1qdc iRNA in the vasculature results in decreased transcription of the gene and a significant impairment of phagocytosis and degranulation, suggesting the involvement of this molecule in immune responses.

Sexual and asexual development: two distinct programs producing the same tunicate.

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.