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Background:
We performed an analysis of the transcriptome during the blastogenesis of the chordate Botryllus schlosseri, focusing in particular on genes involved in cell death by apoptosis. The tunicate B. schlosseri is an ascidian forming colonies characterized by the coexistence of three blastogenetic generations: filter-feeding adults, buds on...
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... adult; b: bud; ba: blood ampulla; bl: budlet; bv: blood vessel; e: endostyle; g: gut; rz: regressing zooid; t: tunic the nt database (Partially non-redundant nucleotide se- quences) using the TBLASTN program [26]. All mapped transcripts were classified according to the taxonomy inherited by similar and significant alignments (Fig. 4) that is also reported in Table 1. ...
Citations
... Over the last decades, a scientific community has taken shape and several research groups have made significant efforts to improve breeding conditions (17)(18)(19) and to develop and adapt imaging and molecular biology techniques (7,8,20). Several anatomical descriptions and staging methods have been proposed (4,21) and various stage-specific and tissue-specific transcriptomic databases have been produced (7,(22)(23)(24)(25)(26). In 2013, a draft genome of B. schlosseri become available (27). ...
Botryllus schlosseri (Tunicata) is a colonial chordate that has long been studied for its multiple developmental pathways and regenerative abilities and its genetically determined allorecognition system based on a polymorphic locus that controls chimerism and cell parasitism. We present the first chromosome-level genome assembly from an isogenic colony of B. schlosseri clade A1 using a mix of long and short reads scaffolded using Hi-C. The haploid genome spans 533 Mb, of which 96% is scaffolded into 16 chromosomal pseudomolecules. With a BUSCO completeness of 91.2%, this complete and contiguous B. schlosseri genome assembly provides a valuable genomic resource for the scientific community and lays the foundation for future investigations into the molecular mechanisms underlying coloniality, regeneration, histocompatibility, and the immune system in tunicates.
... Over the last decades, a scientific community has taken shape and several research groups have made significant efforts to improve breeding conditions (17)(18)(19) and to develop and adapt imaging and molecular biology techniques (7,8,20). Several anatomical descriptions and staging methods have been proposed (4,21) and various stage-specific and tissue-specific transcriptomic databases have been produced (7,(22)(23)(24)(25)(26). In 2013, a draft genome of B. schlosseri become available (27). ...
Botryllus schlosseri (Tunicata) is a colonial chordate that has long been studied for its multiple developmental pathways and regenerative abilities and its genetically determined allorecognition system based on a polymorphic locus that controls chimerism and cell parasitism. We present the first chromosome-level genome assembly from an isogenic colony of B. schlosseri clade A1 using a mix of long and short reads scaffolded using Hi-C. This haploid assembly spans 533 Mb, of which 96% are found in 16 chromosome-scale scaffolds. With a BUSCO completeness of 91.2%, this complete and contiguous B. schlosseri genome assembly provides a valuable genomic resource for the scientific community and lays the foundation for future investigations into the molecular mechanisms underlying coloniality, regeneration, histocompatibility, and the immune system in tunicates.
... Indeed, in humans and mice, the loss of SIX1 promotes apoptosis through the inactivation of pro-apoptotic proteins such as caspases [53,79]. Moreover, a previous study on B. schlosseri showed their high expression in the entire colony during takeover compared with a colony in mid-cycle [80] and a greater amount of cell death during takeover [9,15], confirming the key role of API5 and SIX1 during takeover. ...
... Between the 25 BIRC genes that B. schlosseri has in its genome [82], we found that BIRC3, a gene involved both in the programmed cell death and in the necrosis pathways has an antiapoptotic role [83], is more expressed in filter-feeding adults as compared to adults during takeover, and more expressed in adults during takeover as compared to bud ( Figure 7B). These results support previous studies on the whole colony transcriptome [80,82] that show diminished expression of BIRC during takeover. The reduced expression of this gene in the bud could indicate the absence of a need to suppress apoptosis. ...
Human neuronal loss occurs through different cellular mechanisms, mainly studied in vitro. Here, we characterized neuronal death in B. schlosseri, a marine colonial tunicate that shares substantial genomic homology with mammals and has a life history in which controlled neurodegeneration happens simultaneously in the brains of adult zooids during a cyclical phase named takeover. Using an ultrastructural and transcriptomic approach, we described neuronal death forms in adult zooids before and during the takeover phase while comparing adult zooids in takeover with their buds where brains are refining their structure. At takeover, we found in neurons clear morphologic signs of apoptosis (i.e., chromatin condensation, lobed nuclei), necrosis (swollen cytoplasm) and autophagy (autophagosomes, autolysosomes and degradative multilamellar bodies). These results were confirmed by transcriptomic analyses that highlighted the specific genes involved in these cell death pathways. Moreover, the presence of tubulovesicular structures in the brain medulla alongside the over-expression of prion disease genes in late cycle suggested a cell-to-cell, prion-like propagation recalling the conformational disorders typical of some human neurodegenerative diseases. We suggest that improved understanding of how neuronal alterations are regulated in the repeated degeneration–regeneration program of B. schlosseri may yield mechanistic insights relevant to the study of human neurodegenerative diseases.
... The ability to regenerate lost body parts is widely distributed across annelids and typically occurs through epimorphosis, which involves cell proliferation in a newly formed specialized structure called blastema that differentiates to restore the missing parts (e.g., Bely & Nyberg, 2010;de Jong & Seaver, 2018;Kostyuchenko & Kozin, 2021;Sanchez Alvarado, 2000). On the other hand, asexual reproduction or fission implies offspring production without the involvement of germ cells or gametes, and it is also a process that has evolved independently in numerous lineages throughout the tree of life (Campagna et al., 2016;Dolmatov et al., 2018;. Within the phylum Annelida, asexual reproduction can be achieved through two main ways: ...
... A member of the cadherin family (protocadherin-11 X-linked, pcdh11x) and tuberin (TSC complex subunit 2, tsc2), a tumor suppressor gene related to stem cell behavior, were found upregulated in this condition and have also been reported during the asexual cycle of the chordate Botryllus schlosseri (Campagna et al., 2016;Ricci et al., 2016). Finally, Pbx/knotted 1 homeobox 2 (pknox2) was also found upregulated, a gene whose upregulation has also been reported in fissioning individuals of the sea cucumber Cladolabes schmeltzii (Dolmatov et al., 2018). ...
... schlosseri (Campagna et al., 2016), which may indicate a positive regulation of cell death during fission zone development. Other key regulator of apoptosis also found downregulated in our analysis is pim-1 proto-oncogene, serine/threonine kinase (pim1), which has an important role in the regulation of cell proliferation during ovarian cancer (Ngoka, 2008). ...
Regeneration, the ability to replace lost body parts, is a widespread phenomenon in the animal kingdom often connected to asexual reproduction or fission, since the only difference between the two appears to be the stimulus that triggers them. Both developmental processes have largely been characterized; however, the molecular toolkit and genetic mechanisms underlying these events remain poorly unexplored. Annelids, in particular the oligochaete Pristina leidyi, provide a good model system to investigate these processes as they show diverse ways to regenerate, and can reproduce asexually through fission under laboratory conditions. Here, we used a comparative transcriptomics approach based on RNA-sequencing and differential gene expression analyses to understand the molecular mechanisms involved in anterior regeneration and asexual reproduction. We found 291 genes upregulated during anterior regeneration, including several regeneration-related genes previously reported in other annelids such as frizzled, paics, and vdra. On the other hand, during asexual reproduction, 130 genes were found upregulated, and unexpectedly, many of them were related to germline development during sexual reproduction. We also found important differences between anterior regeneration and asexual reproduction, with the latter showing a gene expression profile more similar to that of control individuals. Nevertheless, we identified 35 genes that were upregulated in both conditions, many of them related to cell pluripotency, stem cells, and cell proliferation. Overall, our results shed light on the molecular mechanisms that control anterior regeneration and asexual reproduction in annelids and reveal similarities with other animals, suggesting that the genetic machinery controlling these processes is conserved across metazoans.
... In order to validate the B. schlosseri predictions of SoxB1, Myc, Pou2 and Pou3, and to develop probes for ISH experiments, total RNA was extracted from B. schlosseri single colonies according to the protocol of Campagna et al. (2016), followed by cDNA synthesis, cloning and sequencing. Whole-mount ISHs were carried out as described in Franchi and Ballarin (2014) using at least three samples per stage. ...
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.
... Among colonial ascidians, Botryllus schlosseri is one of the reference colonial species. Several features make B. schlosseri an excellent model organism (Figure 1f,g)-namely, (i) it is abundant in shallow waters and easily cultured in the laboratory; (ii) its genome and transcriptome are available (Voskoboynik et al. 2013a(Voskoboynik et al. , 2013bCorey et al. 2016;Campagna et al. 2016;Kowarsky et al. 2021;Voskoboynik et al. 2020;Anselmi et al. 2021); (iii) asexual reproduction results in identical individuals, facilitating the ability to separate one colony (genotype) into several clonal replicates (Manni et al. 2007Kowarsky et al. 2021); (iv) it naturally forms chimeras, which allow lineage tracing by DNA fingerprints (Stoner and Weissman 1996;; (v) its transparent tissue allows in vivo tracing of labeled cells Rinkevich et al. 2013;. ...
The regeneration process assumes the presence in the body of
cells capable of self-renewal and subsequent differentiation into specialized cells. Whether these cells are stem cells or are present in circulating fluids or tissues as a pool of reserve progenitor cells, or whether they appear following dedifferentiation/transdifferentiation of specialized cells of individual tissues, are the main questions that scientists are focusing on. Understanding the origin and pathways of differentiation in coelomic fluid cells and coelomocytes of the starfish
Asterias rubens was the aim of this research. The coelomic epithelium is considered as a possible source of coelomocytes. Further effective studies of coelomocyte replenishment are difficult due to the lack of protein markers characterizing various cell morphotypes. Additional difficulties lie in the heterogeneity of analyzed cell populations. In the present study, we separated cells of the coelomic fluid and the coelomic epithelium, and a subpopulation of the coelomic epithelium enriched with poorly differentiated cells, which are proposed precursors of some types of coelomocytes, in a Percoll density gradient. Characterization of the cell morphology of different fractions and their behavior in vitro (functional characteristics) revealed
an enrichment of the gradient fractions in two of eight types of coelomocytes and three of eight morphotypes of cells of the coelomic epithelium.
... Among colonial ascidians, Botryllus schlosseri is one of the reference colonial species. Several features make B. schlosseri an excellent model organism (Figure 1f,g)-namely, (i) it is abundant in shallow waters and easily cultured in the laboratory; (ii) its genome and transcriptome are available (Voskoboynik et al. 2013a(Voskoboynik et al. , 2013bCorey et al. 2016;Campagna et al. 2016;Rosental et al. 2018;Kowarsky et al. 2021;Voskoboynik et al. 2020;Anselmi et al. 2021); (iii) asexual reproduction results in identical individuals, facilitating the ability to separate one colony (genotype) into several clonal replicates (Manni et al. 2007(Manni et al. , 2014Kowarsky et al. 2021); (iv) it naturally forms chimeras, which allow lineage tracing by DNA fingerprints (Stoner and Weissman 1996;Laird et al. 2005); (v) its transparent tissue allows in vivo tracing of labeled cells (Voskoboynik et al. 2008;Rinkevich et al. 2013;Rosental et al. 2018). ...
... Blastogenesis has been well characterized in Botryllus both anatomically and ontogenically (Manni et al. 2019;Manni et al. 2014;Sabbadin et al. 1975;Izzard 1973;Berrill 1941aBerrill , 1941b. Recent years have yielded novel insights on the molecular processes underlying blastogenesis (Franchi et al. 2017;Campagna et al. 2016;Ricci et al. 2016a ;Rodriguez et al. 2014;Rosner et al. 2014Rosner et al. , 2019Qarri et al. 2020). Transcriptomes of three major stages along the Botryllus blastogenic cycle (mid cycle, the pre-takeover and the takeover phases; Campagna et al. 2016; available at http://botryllus.cribi.unipd.it) ...
... Recent years have yielded novel insights on the molecular processes underlying blastogenesis (Franchi et al. 2017;Campagna et al. 2016;Ricci et al. 2016a ;Rodriguez et al. 2014;Rosner et al. 2014Rosner et al. , 2019Qarri et al. 2020). Transcriptomes of three major stages along the Botryllus blastogenic cycle (mid cycle, the pre-takeover and the takeover phases; Campagna et al. 2016; available at http://botryllus.cribi.unipd.it) have revealed 11,337 new genes, of which 581 transcripts were determined with complete open reading frames. ...
... have revealed 11,337 new genes, of which 581 transcripts were determined with complete open reading frames. Many sequences emerged as genes involved in apoptosis activa tion, de-activation and regulation (Campagna et al. 2016). Analyzing the differential expression for fertile vs. infertile B. schlosseri colonies, Rodriguez et al. (2014) revealed a set of genes that are differentially expressed in every blastogen esis stage analyzed. ...
Ascidians are solitary or colonial marine invertebrates that share a common ancestor with vertebrates and belong to the phylum Chordata. Colonial ascidians include more than 1,700 species and are a highly varied group in terms of sizes, shapes, colors and geographical locations. Here we discuss one of the most-studied model species, Botryllus schlosseri, as a most suitable representative of colonial ascidians. Botryllus was first described four centuries ago but has been studied extensively just in the past seven decades, revealing a promising model for diverse biological disciplines. This chapter details the history of the research, biology and geographical distributions of this cosmopolitan species. Special attention is devoted to several unique phenomena staging this species as a model system in biology, such as the continuous life and death cycles of colonial modules (blastogenesis), the capacity for whole-body regeneration and the unique self/non-self recognition (histocompatibility) resulting in either natural immunological rejection or chimerism. The description is followed by with current applications, the development of tools and the description of several leading scientific challenges. Following the wide range of phenomena developing in Botryllus, it is anticipated that this species will become a central model in biology. Working with B. schlosseri may reform and shift dogmas in biology.
... In this species, in order to better elucidate the events occurring during immune responses, we mined the available transcriptome (Campagna et al., 2016), looking for transcripts for C1qDC proteins, putatively involved in the recognition of nonself and never described before in Botryllus. This led us to identify the sequence for a novel multidomain C1qDC protein (BsC1qDC). ...
... The sequence of a transcript showing similarity to the human transcript for CTRP4, referred to as BsC1qDC, was identified through BLAST analysis of a B. schlosseri EST collection obtained in our laboratory (Campagna et al., 2016) (http://botryllus.cribi.unipd.it) and the database of the B. schlosseri genome (http://botryllus.stanford.edu/botryll ...
... The levels of transcripts in different conditions were normalised to that of BsEF1α to compensate for variations in the amounts of cDNA. Previous experiments demonstrated the stability of BsEF1α transcription during the blastogenetic cycle (Campagna et al., 2016). The subclones from MC were used as the reference samples. ...
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.
... Nevertheless, despite the abundance of studies, only few data have been provided on TLRs and their role in immune responses [21]. In order to fill this gap, in the present work, we used C. intestinalis TLR nucleotide sequences to mine our Botryllus transcriptome [22] looking for similar sequences. We identified and characterised a new member of the TLR family (called BsTLR1) and carried out new analyses to define the role of this new TLR in the context of innate immunity. ...
... In the present work, we studied the expression and the possible role of a new Botryllus TLR (BsTLR1) retrieved from in silico analysis of our transcriptome [22]. The sequence was also present in the Aniseed database under the ID g68733. ...
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.
