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

Histocompatibility--the ability of an organism to distinguish its own cells and tissue from those of another--is a universal phenomenon in the Metazoa. In vertebrates, histocompatibility is a function of the immune system controlled by a highly polymorphic major histocompatibility complex (MHC), which encodes proteins that target foreign molecules for immune cell recognition. The association of the MHC and immune function suggests an evolutionary relationship between metazoan histocompatibility and the origins of vertebrate immunity. However, the MHC of vertebrates is the only functionally characterized histocompatibility system; the mechanisms underlying this process in non-vertebrates are unknown. A primitive chordate, the ascidian Botryllus schlosseri, also undergoes a histocompatibility reaction controlled by a highly polymorphic locus. Here we describe the isolation of a candidate gene encoding an immunoglobulin superfamily member that, by itself, predicts the outcome of histocompatibility reactions. This is the first non-vertebrate histocompatibility gene described, and may provide insights into the evolution of vertebrate adaptive immunity.

Summary The mechanisms that sustain stem cells are fundamental to the maintenance of tissues/organs. 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 germ-line 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 germ-line 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 allow them to escape the constraints imposed by the niche, thereby promoting their self-preservation throughout life.

Throughout its adult life, the colonial urochordate, Botryllus schlosseri, produces its entire body, every week, from endogenous stem cells. In this developmental process, all of the body organs including heart, digestive system, branchial sac, endostyle nerve cells and others are created de novo. Here we discover a stem cell niche in these animals- an organ called the endostyle, which also produces and exports hormones. In this study using a combination of in vivo cell labeling, cell engraftments, and chimeric fusion techniques, all followed by automated time lapse microscopy we identifies the endostyle as a stem cell niche that harbors and export adult stem cells to developing organs, wherein they participate in tissue regeneration.

Histocompatibility is the basis by which multicellular organisms of the same species distinguish self from non-self. Relatively little is known about the mechanisms underlying histocompatibility reactions in lower organisms. Botryllus schlosseri is a colonial urochordate, a sister group of vertebrates, that exhibits a genetically determined natural transplantation reaction, whereby self-recognition between colonies leads to formation of parabionts with a common vasculature, whereas rejection occurs between incompatible colonies. Using genetically defined lines, whole-transcriptome sequencing, and genomics, we identified a single gene that encodes self/non-self and determines “graft” outcomes in this organism. This gene is significantly upregulated in colonies poised to undergo fusion or rejection, is highly expressed in the vasculature, and is functionally linked to histocompatibility outcomes. These findings establish a platform for advancing the science of allorecognition.

Summary Hematopoiesis 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 an animal’s life 1 . While there are comprehensive studies on vertebrate HSC self-renewal, differentiation, physiological regulation and niche occupation, relatively little is known about their evolutionary origin and their niches. Here we study the hematopoietic system of Botryllus schlosseri , a colonial tunicate that has vasculature, circulating blood cells, and interesting stem cell biology and immunity characteristics 2 – 8 . Self-recognition between genetically compatible B. schlosseri colonies leads to the formation of natural parabionts with shared circulation, whereas incompatible colonies reject each other 3 , 4 , 7 . Using flow-cytometry, whole-transcriptome sequencing of defined cell populations and diverse functional assays, we identified HSCs, progenitors, immune-effector cells, and an HSC niche, and demonstrated that self-recognition inhibits allospecific cytotoxic reactions. Our study reveals that HSC and myeloid lineage immune cells emerged in a common ancestor of tunicates and vertebrates, and these results also suggest that hematopoietic bone marrow and the B. schlosseri endostyle niche evolved from a common origin.

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.

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.