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We studied the function, development and aging of the adult nervous system in the colonial tunicate Botryllus schlosseri. Adults, termed zooids, are filter-feeding individuals. Sister zooids group together to form modules, and modules, in turn, are linked by a shared vascular network to form a well-integrated colony. Zooids undergo a weekly cycle of regression and renewal during which mature zooids are replaced by developing buds. The zooid brain matures and degenerates on this 7-day cycle. We used focal extracellular recording and video imaging to explore brain activity in the context of development and degeneration and to examine the contributions of the nervous system and vascular network to behavior. Recordings from the brain revealed complex firing patterns arising both spontaneously and in response to stimulation. Neural activity increases as the brain matures and declines thereafter. Motor behavior follows the identical time course. The behavior of each zooid is guided predominantly by its individual brain, but sister zooids can also exhibit synchronous motor behavior. The vascular network also generates action potentials that are largely independent of neural activity. In addition, the entire vascular network undergoes slow rhythmic contractions that appear to arise from processes endogenous to vascular epithelial cells. We found that neurons in the brain and cells of the vascular network both express multiple genes for voltage-gated Na+ and Ca2+ ion channels homologous (based on sequence) to mammalian ion channel genes.

Colonial tunicates are marine organisms that possess multiple brains simultaneously during their colonial phase. While the cyclical processes of neurogenesis and neurodegeneration characterizing their life cycle have been documented previously, the cellular and molecular changes associated with such processes and their relationship with variation in brain morphology and individual (zooid) behavior throughout adult life remains unknown. Here, we introduce Botryllus schlosseri as an invertebrate model for neurogenesis, neural degeneration, and evolutionary neuroscience. Our analysis reveals that during the weekly colony budding (i.e., asexual reproduction), prior to programmed cell death and removal by phagocytes, decreases in the number of neurons in the adult brain are associated with reduced behavioral response and significant change in the expression of 73 mammalian homologous genes associated with neurodegenerative disease. Similarly, when comparing young colonies (1 to 2 y of age) to those reared in a laboratory for ∼20 y, we found that older colonies contained significantly fewer neurons and exhibited reduced behavioral response alongside changes in the expression of 148 such genes (35 of which were differentially expressed across both timescales). The existence of two distinct yet apparently related neurodegenerative pathways represents a novel platform to study the gene products governing the relationship between aging, neural regeneration and degeneration, and loss of nervous system function. Indeed, as a member of an evolutionary clade considered to be a sister group of vertebrates, this organism may be a fundamental resource in understanding how evolution has shaped these processes across phylogeny and obtaining mechanistic insight.

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

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.

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

Expression levels of circadian clock genes, which regulate 24-hour rhythms of behavior and physiology, have been shown to change with age. However, a study holistically linking aging and circadian gene expression is missing. Using the colonial chordate Botryllus schlosseri, we combined transcriptome sequencing and stem cell-mediated aging phenomena to test how circadian gene expression changes with age. This revealed that B. schlosseri clock and clock-controlled genes oscillate organism-wide, with daily, age-specific amplitudes and frequencies. These age-related, circadian patterns persist at the tissue level, where dramatic variations in cyclic gene expression of tissue profiles link to morphological and cellular aging phenotypes. Similar cyclical expression differences were found in hundreds of pathways associated with known hallmarks of aging, as well as pathways that were not previously linked to aging. The atlas we developed points to alterations in circadian gene expression as a key regulator of aging.One Sentence SummaryThe Ticking Clock: Systemic changes in circadian gene expression correlates with wide-ranging phenotypes of aging

Haematopoiesis is an essential process that evolved in multicellular animals. At the heart of this process are haematopoietic stem cells (HSCs), which are multipotent and self-renewing, and generate the entire repertoire of blood and immune cells throughout an animal's life1. Although there have been comprehensive studies on self-renewal, differentiation, physiological regulation and niche occupation in vertebrate HSCs, relatively little is known about the evolutionary origin and niches of these cells. Here we describe the haematopoietic system of Botryllus schlosseri, a colonial tunicate that has a vasculature and circulating blood cells, and interesting stem-cell biology and immunity characteristics2-8. Self-recognition between genetically compatible B. schlosseri colonies leads to the formation of natural parabionts with shared circulation, whereas incompatible colonies reject each other3,4,7. Using flow cytometry, whole-transcriptome sequencing of defined cell populations and diverse functional assays, we identify HSCs, progenitors, immune effector cells and an HSC niche, and demonstrate that self-recognition inhibits allospecific cytotoxic reactions. Our results show that HSC and myeloid lineage immune cells emerged in a common ancestor of tunicates and vertebrates, and also suggest that haematopoietic bone marrow and the B. schlosseri endostyle niche evolved from a common origin.

Evolution and selection have shaped diverse immune systems throughout phylogeny, the vast majority of which remain unexplored. Botryllus schlosseri is a colonial tunicate, a sister group to vertebrates, that develops as a chordate, then metamorphoses to an asexually reproductive invertebrate that every week makes the same body plan from budded stem cells. Genetically distinct B. schlosseri colonies can fuse to form a chimera, or reject each other based on allogeneic recognition. In chimeras, circulating germline and somatic stem cells participate in development; stem cells compete in all individuals in the fused colonies, with rejection preventing germline parasitism. Here we review the isolation and characterization of B. schlosseri hematopoietic stem cells (HSC) and their niches, and the role of the immune effector cells in allorecognition.

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

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

In a primitive chordate model of natural chimerism, one chimeric partner is often eliminated in a process of allogeneic resorption. Here, we identify the cellular framework underlying loss of tolerance to one partner within a natural Botryllus schlosseri chimera. We show that the principal cell type mediating chimeric partner elimination is a cytotoxic morula cell (MC). Proinflammatory, developmental cell death programs render MCs cytotoxic and, in collaboration with activated phagocytes, eliminate chimeric partners during the "takeover" phase of blastogenic development. Among these genes, the proinflammatory cytokine IL-17 enhances cytotoxicity in allorecognition assays. Cellular transfer of FACS-purified MCs from allogeneic donors into recipients shows that the resorption response can be adoptively acquired. Transfer of 1 × 10(5) allogeneic MCs eliminated 33 of 78 (42%) recipient primary buds and 20 of 76 (20.5%) adult parental adult organisms (zooids) by 14 d whereas transfer of allogeneic cell populations lacking MCs had only minimal effects on recipient colonies. Furthermore, reactivity of transferred cells coincided with the onset of developmental-regulated cell death programs and disproportionately affected developing tissues within a chimera. Among chimeric partner "losers," severe developmental defects were observed in asexually propagating tissues, reflecting a pathologic switch in gene expression in developmental programs. These studies provide evidence that elimination of one partner in a chimera is an immune cell-based rejection that operates within histocompatible pairs and that maximal allogeneic responses involve the coordination of both phagocytic programs and the "arming" of cytotoxic cells.

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.

Chimerism is defined as the coexistence of two or more genomes of separate origin within an individual. In placental mammals such as humans, natural chimerism develops during pregnancy between a mother and fetus and has an important role in the induction of fetal tolerance to maternal tissues. Natural chimerism between kin also occurs in colonial ascidians, the closest extant ancestors of chordates. In the ascidian, Botryllus schlosseri, some colonies fuse to create lifelong chimeric entities of two allogeneic genomes. The decision to fuse in B. schlosseri is governed by a polymorphic histocompatibility gene called the Botryllus histocompatibility factor (BHF). Colonies that share at least one BHF allele fuse upon contact, whereas colonies without any BHF alleles in common ultimately reject. Following vasculature fusion, stem cells from each histocompatible B. schlosseri colony compete to overtake germline or somatic lineages. Stem cell competition may lead to elimination of the other colony’s genome, or it may produce a chimeric colony with mixed genotypes. In this way, chimerism in B. schlosseri represents a nexus between stem cell competition, genome parasitism, and allorecognition. Here we review studies conducted over six decades that led to the discoveries of the nature of the cells that mediate chimerism in colonial ascidians and the gene that controls it.

SummaryHematopoiesis is an essential process that evolved in multicellular animals. At the heart of this process are hematopoietic stem cells (HSCs), which are multipotent, self-renewing and generate the entire repertoire of blood and immune cells throughout life. Here we studied the hematopoietic system of Botryllus schlosseri, a colonial tunicate that has vasculature, circulating blood cells, and interesting characteristics of stem cell biology and immunity. Self-recognition between genetically compatible B. schlosseri colonies leads to the formation of natural parabionts with shared circulation, whereas incompatible colonies reject each other. Using flow-cytometry, whole-transcriptome sequencing of defined cell populations, and diverse functional assays, we identified HSCs, progenitors, immune-effector cells, the HSC niche, and demonstrated that self-recognition inhibits cytotoxic reaction. Our study implies that the HSC and myeloid lineages emerged in a common ancestor of tunicates and vertebrates and suggests that hematopoietic bone marrow and the B. schlosseri endostyle niche evolved from the same origin.

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.

Tunicates are invertebrate members of the chordate phylum, and are considered to be the sister group of vertebrates. Tunicates are composed of ascidians, thaliaceans, and appendicularians. With the advent of inexpensive high-throughput sequencing, the number of sequenced tunicate genomes is expected to rise sharply within the coming years. To facilitate comparative genomics within the tunicates, and between tunicates and vertebrates, standardized rules for the nomenclature of tunicate genetic elements need to be established. Here we propose a set of nomenclature rules, consensual within the community, for predicted genes, pseudogenes, transcripts, operons, transcriptional cis-regulatory regions, transposable elements, and transgenic constructs. In addition, the document proposes guidelines for naming transgenic and mutant lines.

A Gene for Early Acceptance One of the fundamental properties of the immune system is the ability to distinguish self- from nonself–histocompatibility. To gain insight into the evolution and molecular basis of histocompatibility, Voskoboynik et al. (p. 384 ) sought to determine the genetic basis for a natural transplantation reaction that occurs in Botryllus schlosseri , a colonial urochordate. Compatibility allows vascular fusion among individuals, whereas incompatibility results in an inflammatory rejection response. A single gene determined the outcome of the reaction. Like histocompatibility genes in higher organisms, this gene is polymorphic and is expressed in the tissues that participate in the transplantation reaction.

SummaryThe 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.

International audience; Angiogenesis, the growth and remodeling of a vascular network, is an essential process during development, growth and disease. Here we studied the role of the vascular endothelial growth factor receptor (VEGFR) in experimentally-induced angiogenesis in the colonial ascidian Botryllus schlosseri (Tunicata, Ascidiacea). The circulatory system of B. schlosseri is composed of two distinct, but interconnected regions: a plot of sinuses and lacunae which line the body, and a transparent, macroscopic extracorporeal vascular network. The vessels of the extracorporeal vasculature are morphologically inverted in comparison to the vasculature in vertebrates: they consist of a single layer of ectodermally-derived cells with the basal lamina lining the lumen of the vessel. We found that when the peripheral circulatory system of a colony is surgically removed, it can completely regenerate within 24 to 48 h and this regeneration is dependent on proper function of the VEGF pathway: siRNA-mediated knockdown of the VEGFR blocked vascular regeneration, and interfered with vascular homeostasis. In addition, a small molecule, the VEGFR kinase inhibitor PTK787/ZK222584, phenocopied the siRNA knockdown in a reversible manner. Despite the disparate germ layer origins and morphology of the vasculature, the developmental program of branching morphogenesis during angiogenesis is controlled by similar molecular mechanisms, suggesting that the function of the VEGF pathway may be co-opted during the regeneration of an ectoderm-derived tubular structure.

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.

SummaryHistocompatibility in the primitive chordate, Botryllus schlosseri, is controlled by a single, highly polymorphic locus, the FuHC. By taking a forward genetic approach, we have identified a locus encoded near the FuHC, called fester, which is polymorphic, polygenic, and inherited in distinct haplotypes. Somatic diversification occurs through extensive alternative splicing, with each individual expressing a unique repertoire of splice forms, both membrane bound and potentially secreted, all expressed in tissues intimately associated with histocompatibility. Functional studies, via both siRNA-mediated knockdown and direct blocking by monoclonal antibodies raised against fester, were able to disrupt predicted histocompatibility outcomes. The genetic and somatic diversity, coupled to the expression and functional data, suggests that fester is a receptor involved in histocompatibility.

SUMMARY Apoptosis is an important tool for shaping developing organs and for maintaining cellular homeostasis. In the colonial urochordate Botryllus schlosseri, apoptosis is also the hallmark end point in blastogenesis, a cyclical and weekly developmental phenomenon. Then the entire old generation of zooids are eliminated (resorbed) by a process that lasts 24–36 h. Administration of the antioxidant butylated hydroxytoluene (BHT) resulted in resorption being arrested by 1–8 days on average. At high doses(2.5–15.0 mg BHT l-1) resorption was completed only after removal of BHT. Colonies that were not removed in time, died. In treated colonies, although DNA fragmentation was high, tissues and organs that would normally have died, survived, and the general oxidative levels of lipids were reduced. Blood vessels were widened, containing aggregates of blood cells with a significantly increased proportion of empty macrophage-like cells without inclusion. In colonies rescued from BHT treatment, resorption of zooids started immediately and was completed within a few days. We propose three possible mechanisms as to how BHT may affect macrophage activity: (1) by interrupting signals that further promote apoptosis; (2) through the respiratory burst initiated following a phagocytic stimulus; and (3) by reducing lipid oxidation and changing cell surface markers of target cells. Our results point, for the first time, to the role of phagocytic cells in the coordination of death and clearance signals in blastogenesis.

Two commonly accepted metabolic theories of aging interpret senescence either in terms of the rate of living, where a fixed total metabolic potential is consumed over an expected lifetime (after which the organism wears out and dies) or, in terms of accumulative oxidative damage resulting in progressive and irreversible changes in metabolic pathways. Protocols based on restricted diets, chronically administered anti-oxidants and the use of established lines of organisms resistant to free radical damage support the metabolic theories of aging by revealing, in many cases, significant extensions of life spans or dramatic anti-aging effects. To test the universality of these metabolic hypotheses of aging, we acutely treated ramets (clonal replicates) from old, long-lived colonies of the urochordate Botryllus schlosseri with lethal doses of the anti-oxidant butylated hydroxytoluene (BHT). This group of organisms has a weekly cyclical and highly synchronized developmental process (blastogenesis), during which all existing zooids are removed by massive apoptosis and phagocytosis processes. In animals treated with BHT, blastogenesis was completely arrested and colonies deteriorated to a morphologically chaotic state. Rescued ramets resorbed BHT treated zooids, regenerated entirely new sets of zooids and then revealed: (1) rejuvenescence and enhanced growth rates and in many cases, (2) up to 4.6 times extension of post-treatment life expectancy. Both metabolic theories for senescence were therefore falsified in B. schlosseri. The possible existence of an aging clock that can be set by the environment is suggested.

International audience; Ontologies provide an important resource to integrate information. For developmental biology and comparative anatomy studies, ontologies of a species are used to formalize and annotate data that are related to anatomical structures, their lineage and timing of development. Here, we have constructed the first ontology for anatomy and asexual development (blastogenesis) of a bilaterian, the colonial tunicate Botryllus schlosseri. Tunicates, like Botryllus schlosseri, are non-vertebrates and the only chordate taxon species that reproduce both sexually and asexually. Their tadpole larval stage possesses structures characteristic of all chordates, i.e. a notochord, a dorsal neural tube, and gill slits. Larvae settle and metamorphose into individuals that are either solitary or colonial. The latter reproduce both sexually and asexually and these two reproductive modes lead to essentially the same adult body plan. The Botryllus schlosseri Ontology of Development and Anatomy (BODA) will facilitate the comparison between both types of development. BODA uses the rules defined by the Open Biomedical Ontologies Foundry. It is based on studies that investigate the anatomy, blastogenesis and regeneration of this organism. BODA features allow the users to easily search and identify anatomical structures in the colony, to define the developmental stage, and to follow the morphogenetic events of a tissue and/or organ of interest throughout asexual development. We invite the scientific community to use this resource as a reference for the anatomy and developmental ontology of B. schlosseri and encourage recommendations for updates and improvements.

15 páginas, 6 figuras, 2 tablas., Ascidians are a fascinating group of filter-feedingmarine chordates characterized by rapid evolution of both sequences and structure of their nuclear andmitochondrial genomes.Moreover, they include several model organisms used to investigate complex biological processes in chordates. To study the evolutionary dynamics of ascidians at short phylogenetic distances, we sequenced 13 new mitogenomes and analyzed them, together with 15 other availablemitogenomes, using a novel approach involving detailedwholemitogenome comparisons of conspecific and congeneric pairs. The evolutionary rate was quite homogeneous at both intraspecific and congeneric level, and the lowest congeneric rateswere found in cryptic (morphologically undistinguishable) and inmorphologically very similar species pairs. Moreover, congeneric nonsynonymous rates (dN)were up to two orders ofmagnitude higher than in intraspecies pairs. Overall, a clear-cut gap sets apart conspecific from congeneric pairs. These evolutionary peculiarities allowed easily identifying an extraordinary intraspecific variability in the model ascidian Botryllus schlosseri, where most pairs show a dN value between that observed at intraspecies and congeneric level, yet consistently lower than that of the Ciona intestinalis cryptic species pair. These data suggest ongoing speciation events producing genetically distinct B. schlosseri entities. Remarkably, these ongoing speciation events were undetectable by the cox1 barcode fragment, demonstrating that, at low phylogenetic distances, the whole mitogenomehas a higher resolvingpower than cox1.Our study showsthatwhole-mitogenomecomparative analyses, performedon a suitable sample of congeneric and intraspecies pairs, may allow detecting not only cryptic species but also ongoing speciation events., This work was supported by the Ministero dell’Istruzione, dell’Universita` e della Ricerca, Italy (PRIN- 2009) to C.G., Lifewatch (Consiglio Nazionale delle Ricerche, Italy) to G.P., the National Institute of Health grants RO1GM100315 and 1R01AG037968 to A.V., and the Agence Nationale de la Recherche “Programme Blanc” (Tunicate Evo-Devo “TED”) to E.J.P.D. This is contribution No. 2014-YYY of the Institut des Sciences de l’Evolution de Montpellier (UMR 5554 – UM2 + CNRS + IRD).

Botryllus schlosseri is a colonial urochordate that follows the chordate plan of development following sexual reproduction, but invokes a stem cell-mediated budding program during subsequent rounds of asexual reproduction. As urochordates are considered to be the closest living invertebrate relatives of vertebrates, they are ideal subjects for whole genome sequence analyses. Using a novel method for high-throughput sequencing of eukaryotic genomes, we sequenced and assembled 580 Mbp of the B. schlosseri genome. The genome assembly is comprised of nearly 14,000 intron-containing predicted genes, and 13,500 intron-less predicted genes, 40% of which could be confidently parceled into 13 (of 16 haploid) chromosomes. A comparison of homologous genes between B. schlosseri and other diverse taxonomic groups revealed genomic events underlying the evolution of vertebrates and lymphoid-mediated immunity. The B. schlosseri genome is a community resource for studying alternative modes of reproduction, natural transplantation reactions, and stem cell-mediated regeneration. DOI: http://dx.doi.org/10.7554/eLife.00569.001, eLife Digest The tunicates are an evolutionary group that includes species such as sea squirts and sea tulips. Their name comes from the structure known as a ‘tunic’ that surrounds their sac-like bodies. As marine filter feeders, tunicates obtain nutrients by straining food particles from water, and they can live either alone or in colonies depending on the species. Charles Darwin suggested that tunicates may be the key to understanding the evolution of vertebrates, and indeed today they are regarded as the closest living relatives of this group. Colonial tunicates can reproduce either sexually, or asexually by budding. Compatible colonies have the ability to recognize one another and to fuse their blood vessels to form a single organism, whereas incompatible colonies reject one another and remain separate. This recognition process bears some resemblance to the rejection of foreign organ transplants in mammals. Here, Voskoboynik and co-workers present the first genome sequence of a colonial tunicate, Botryllus schlosseri. They used a novel sequencing approach that significantly increased the length of a DNA molecule that can be determined by next-generation sequencing, and allowed large DNA repeat regions to be easily resolved. In total, they sequenced 580 million base pairs of DNA, which they estimate contains roughly 27,000 genes. By comparing the B. schlosseri genome with those of a number of vertebrates, Voskoboynik et al. identified multiple B. schlosseri genes that also participate in the development and functioning of the vertebrate eye, heart, and auditory system, as well as others that may have contributed to the evolution of the immune system and of blood cells. The genome of B. schlosseri thus provides an important new tool for studying the genetic basis of the evolution of vertebrates. DOI: http://dx.doi.org/10.7554/eLife.00569.002

We are characterizing the immune system and cell populations of the tunicate model Botryllus schlosseri on functional and molecular levels. The chordate B. schlosseri belongs to a group that is considered the closest living invertebrate relative of vertebrates. Using FACS analysis we can isolate 12 populations of B. schlosseri cells by size, granularity and auto fluorescence. In order to increase the number of isolated cell populations, we used Cytof to scan large numbers of antibodies. Antibodies that differentially bind to B. schlosseri cells were validated by flow cytometry. Serum against the Botryllus Histocompatibility Factor, and lectins and fluorescent reagents activated by enzymatic activity were also used to differentiate live B. schlosseri cell types. Using these markers we isolated 34 cell populations and created RNA libraries for deep sequencing. Additionally, we developed immunological assays for cytotoxicity, phagocytosis and stem cell tracking. Using gene expression analysis we found hematopoietic stem cells and progenitors; in transplantation assays we showed their capability to induce chimerism of pigmentation and differentiation to other cell types, as well as localization to the stem cell niches. We characterized 3 different phagocytic cell types including a previously undescribed myeloid derived cell population. We describe the B. schlosseri cytotoxic cell population that originates as a large granular lymphocyte-like cell type that become morula cells upon allogeneic challenge activation. Altogether, it seems that the common ancestor of tunicates and vertebrates had a true hematopoietic myeloid lineage, while the cytotoxic cells are a convergent evolutionary system.

Chimerism is the presence of cells derived from more than one individual in a given individual. This phenomenon has been detected in a wide variety of multicellular organisms, including vertebrates. In mammals natural chimerism can develop from bidirectional trafficking of cells between multiple fetuses or between the fetus and its mother during pregnancy. Because stem cells are the only self-renewing cells in a tissue, it is likely that chimerism is sustained by stem cells. Colonial marine ascidians, like Botryllus schlosseri, may serve as an evolutionary model of chimerism. Colonies are initially formed by asexual reproduction of a founder individual, and the progeny clone members are united under a single gelatinous tunic by a network of anastomosed extracorporeal blood vessels. In these organisms, pairs of genetically distinct colonies can establish a natural chimerism upon physical contact by anastomosis of extracorporeal blood vessels between colonies. The ability to create a chimeric entity between colonies is determined by a single, highly polymorphic, fusion/histocompatibility locus (Fu/HC). Colonies that share at least one Fu/HC allele (mainly kin under in situ conditions) fuse vessels upon contact. A pair that does not share any Fu/HC allele does not, but instead has an inflammatory immune response that forms a permanent scar between colonies. Following fusion, cells transmigrate between colonies and, in some cases, replace the germline and/or the somatic tissues of the host. The replacement of host tissues by a donor genotype is pre-determined genetically and follows hierarchies of “winner strains” replacing “loser strains” tissues.

SummaryStem 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.

Embryogenesis and asexual reproduction are commonly considered to be coordinated developmental processes, which depend on accurate progression through a defined sequence of developmental stages. Here we report a peculiar developmental scenario in a simple chordate, Botryllus schlosseri, wherein a normal colony of individuals (zooids and buds) is regenerated from the vasculature (vascular budding) through a sequence of morphologically abnormal developmental stages. Vascular budding was induced by surgically removing buds and zooids from B. schlosseri colonies, leaving only the vasculature and the tunic that connects them. In vivo imaging and histological sections showed that the timing and morphology of developing structures during vascular budding deviated significantly from other asexual reproduction modes (the regular asexual reproduction mode in this organism and vascular budding in other botryllid species). Subsequent asexual reproduction cycles exhibited gradual regaining of normal developmental patterns, eventually leading to regeneration of a normal colony. The conversion into a normal body form suggests the activation of an alternative pathway of asexual reproduction, which involves gradual regaining of normal positional information. It presents a powerful model for studying the specification of the same body plan by different developmental programs.