Introduction to MARISTEM - Stem cells of marine/aquatic invertebrates: from basic research to innovative applications

Marine/aquatic invertebrates constitute the largest biodiversity and the widest phylogenetic radiation on Earth, from morphologically simple organisms (e.g., sponges, cnidarians), to the more complex mollusks, crustaceans, echinoderms, and protochordates. Today, adult marine/aquatic invertebrate stem cell (MISC) biology is of prime research and medical interest. However, studies on stem cells from organisms outside the classical vertebrate (e.g., human, mouse, and zebrafish) and invertebrate (e.g., Drosophila, Caenorhabditis) models have not been pursued vigorously. These organisms contain a variety of MISC types that allow the production of a large number of novel bioactive-molecules, many of which are of significant potential interest for human health. MISCs further participate in aging and regeneration phenomena, including whole-body regeneration. For years, the European MISC-community has been highly fragmented and has established scarce ties with biomedical industries in an attempt to harness MISCs for human welfare. Thus, it is important to (i) consolidate the European community of researchers working on MISCs; (ii) promote and coordinate European research on MISC biology; (iii) stimulate young researchers to embark on research in MISCbiology; (iv) develop, validate, and share novel MISC tools and methodologies; (v) establish the MISC discipline as a forefront interest of biomedical disciplines, including nanobiomedicine; and (vi) establish collaborations with industries to exploit MISCs as sources of bioactive molecules. In order to fill the recognized gaps, the EC-COST Action 16203 \u201cMARISTEM\u201d has recently been launched. At its initial stage, the consortium unites scientists from 24 EC countries, Cooperating countries, and Near Neighbor Countries

Introduction to MARISTEM - stem cells of marine/aquatic invertebrates: from basic research to innovative applications

Marine/aquatic invertebrates constitute the largest biodiversity and the widest phylogenetic radiation on Earth, from morphologically simple organisms (e.g., sponges, cnidarians), to the more complex mollusks, crustaceans, echinoderms, and protochordates. Today, adult marine/aquatic invertebrate stem cell (MISC) biology is of prime research and medical interest. However, studies on stem cells from organisms outside the classical vertebrate (e.g., human, mouse, and zebrafish) and invertebrate (e.g., Drosophila, Caenorhabditis) models have not been pursued vigorously. These organisms contain a variety of MISC-types that allow the production of a large number of novel bioactive-molecules, many of which are of significant potential interest for human health. MISCs further participate in aging and regeneration phenomena, including whole-body regeneration. For years, the European MISC-community has been highly fragmented and has established scarce ties with biomedical industries in an attempt to harness MISCs for human welfare. Thus, it is important to (i) consolidate the European community of researchers working on MISCs; (ii) promote and coordinate European research on MISC biology; (iii) stimulate young researchers to embark on research in MISC-biology; (iv) develop, validate, and share novel MISC tools and methodologies; (v) establish the MISC discipline as a forefront interest of biomedical disciplines, including nanobiomedicine; and (vi) establish collaborations with industries to exploit MISCs as sources of bioactive molecules. In order to fill the recognized gaps, the EC-COST Action 16203 \u201cMARISTEM\u201d has recently been launched. At its initial stage, the consortium unites scientists from 24 EC countries, Cooperating countries, and Near Neighbor Countries

The complement system of Botryllus schlosseri

Among the various effector mechanisms involved in immune responses, the complement system is one of the most ancient, deeply-rooted and important for its ability to orchestrate different cells and factors of both innate and adaptive immunity. The comprehension of its roots in the evolution is useful to understand how the main complement-related proteins had changed in order to adapt to new environmental conditions and life-cycles or, in the case of vertebrates, to interact with the adaptive immunity. In this context, data from organisms evolutionary close to vertebrates, such as tunicates, are of primary importance for a better understanding of the changes in immune responses associated with the invertebratevertebrate transition. In our model tunicate Botryllus schlosseri we have described a lectin and alternative pathway of complement system activation very similar to those of Vertebrates. All the complement-related genes such as c3, bf, ficolin, mbl and masp are transcribed by morula cells, the immunocytes in immunomodulation and cytotoxic responses. Functional data suggest a complement-related cross-talk between morula cells and phagocytes immunocyte during the immune response. When B. schlosseri hemocytes are incubated with yeast (Saccharomyces cerevisiae) cells, there is an overexpression of C3 by morula cell that led to increase of phagocytosis that is prevented in the presence of the C3 inhibitor, compstatin. In the next future, we will focus our efforts on the regulation of complement system in tunicates to shed new light on the complement system function in a pre-adaptive immunity scenario

Stress granules in Ciona robusta: molecular expression of tiar and ttp and early evidence of their gene expression under stress conditions induced by metals

Stress granules are non-membranous cytoplasmic foci composed of messengers (not translated), ribonucleoproteins, translation initiation components and other additional proteins, that represent a primary mechanism by which gene expression is rapidly modulated when cells are subjected to adverse environmental conditions. Very few works have been devoted to study the presence ofmolecular components of stress granules in invertebrate animals. In this work, we characterized, for the first time in the solitary ascidian Ciona robusta, the genetic sequences of two important protein components of stress granules, TIAR (TIA-1 related to proteins) and TTP (tristetraprolin), and carried out the first studies on their gene expression. The sequences characterized for tiar and ttp genes have allowed to start a study on the molecular evolution of these proteins in animals: for TIAR the obtained results are consistent with recent phylogenetic analysis that place tunicates as sister group of vertebrates, whereas the phylogenetic position of TTP remains still uncertain. The data on mRNA expression, provided by qRT-PCR analysis, are absolutely the first obtained in non-mammalian animals. As expected, the exposure to each metal (Cu, Zn and Cd) led to a generalized decrease in mRNA expression levels for both TIAR and TTP, suggesting that the metal accumulation induce acute stress and the inhibition of the transcription of tiar and ttp genes. The data presented here improved our knowledge about the molecular evolution anti-stress proteins in metazoans and emphasize the importance of the transcription of tiar and ttp genes, which represents an efficient physiological response allowing C. robusta to survive in the presence of metals in the marine environment (Supported by M.I.U.R. grant)

Complement-mediated cooperation between immunocytes in the compound ascidian Botryllus schlosseri

Two main kinds of innate immune responses are present in ascidians: phagocytosis and cytotoxicity. They are mediated by two different types of circulating immunocytes: phagocytes and cytotoxic morula cells (MCs). MCs, once activated by non-self-recognition, can stimulate phagocytosis by the release of soluble factors able to act as opsonins. BsC3, the complement C3 homologue, like mammalian C3, contains the thioester bond required to split the molecule into BsC3a and BsC3b. BsC3b likely represents the MC opsonin as it can enhances phagocytosis. The tenet is supported by the observed reduction in phagocytosing cells after exposure of hemocytes to compstatin, a drug preventing C3 activation, or after the bsc3 knockdown by iRNA injection. In addition, the transcript for BsCR1, homologous to mammalian CR1, is present in Botryllus phagocytes and the transcription is modulated during the blastogenetic cycle. MCs also release cytokines (chemokines) able to recruit immunocytes to the infection site. The activity is inhibited by antibodies raised against human TNFa. Since no genes for TNFa are present in the Botryllus genome, the observed activity is probably related to a TNF-domain containing protein, member of the Botryllus complement system. Conversely, activated phagocytes release a rhamnose-binding lectin able to interact with microbial surfaces and act as opsonin. It can also activate MCs by inducing the release of the reported cytokine and stimulate their degranulation. Overall, the results obtained so far indicate the presence of a well-defined cross-talk between the two types of immunocytes during the immune responses of B. schlosseri

Characterization of the complement system in a colonial tunicate: C3 complement receptors and opsonic role of C3

The compound ascidian Botryllus schlosseri is a reliable model organism for the study of immunobiology. As an invertebrate, it relies only on innate immunity for its defense. We already demonstrated the presence, in Botryllus, of homologues of mammalian C3, Bf, MBL and MASP1, referred to as BsC3, BsBf, BsMBL and BsMASP, respectively. All the complement components identified so far, are expressed by morula cells, the most abundant circulating hemocytes. In mammals, once the complement system is activated, a cascade of reactions occurs resulting in the cleavage of the third complement component (C3) to C3a and C3b, the former exerting a chemotactic activity, the latter acting as opsonin and, ultimately, activating the lytic pathway. The best-known receptor for C3a in mammals is C3aR, whereas CR1 is the receptor able to recognize and bind C3b on the microbial surfaces. Here, we describe, in B. schlosseri, new genes showing homology with vertebrate C3aR and CR1, respectively, and studied their transcription in the course of the colonial blastogenetic cycle. In addition, we continued our analysis of the role of C3 in Botryllus immunity by studying the modulation of BsC3 transcription during the colonial blastogenetic cycle and the effect of bsc3 knockdown on immune responses. Results indicate that only morula cells, and no other immunocytes type, are labelled by the antisense probe for BsC3aR, whereas phagocytes and young, undifferentiated cells, known as hemoblasts, are the cells stained by the probe for BsCR1. Both the bsc3ar and bscr1 genes are constitutively transcribed. However, a modulation in the extent of transcription occurs during the colonial blastogenetic cycle as the amount of BsC3aR mRNA abruptly decreased at TO, whereas no differences were observed when EC and MC were compared. This is probably related to the renewing of circulating cells at TO, that are replaced by new, differentiating cells entering the circulation in the same period

BsTLR: 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 defense. As regards TLRs, two transcripts have been described and characterized in the solitary species Ciona robusta, referred to as CiTLR1 and CiTLR2. Using the Ciona TLR nucleotide sequences, we examined the available transcriptomes of 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 BsTLR, has a TIR and a transmembrane domain, four LLR and two LRR-CT domains. In addition, we analised bstlr transcription in vivo and in vitro, under various experimental conditions and in different phases of the Botryllus blastogenetic cycle. Our data show that, in different phases, there is a change in gene transcription and mRNA location, according to the blastogenetic phase

Characterization of the complement system in a colonial tunicate: C3 complement receptors and opsonic role of C3

The complement system is one of the most ancient immune modulator mechanism of bilaterian metazoans. In vertebrates, three complement-activation pathways are known: the classical, the alternative and the lectin pathways: all of them converge on the cleavage of C3. The compound ascidian Botryllus schlosseri is a reliable model organism for the study of immunobiology. As an invertebrate, B. schlosseri relies only on innate immunity for its defense and immunocytes. We already demonstrated the presence, in Botryllus, of homologues of mammalian C3, Bf, MBL and MASP1, referred to as BsC3, BsBf, BsMBL and BsMASP, respectively. All the complement components identified so far, are expressed by morula cells, the most abundant circulatingemocytes. In mammals, once the complement system isactivated, a cascade of reactions that involves proteolysis and polymerization occurs resulting in the cleavage of the third complement component (C3) to C3a and C3b, the former exerting a chemotactic activity, the latter acting as opsonin and, ultimately, activating the lytic pathway. The best-known receptor for C3a in mammals is C3aR, whereas CR1 is the receptor able to recognize and bind C3b on the microbial surfaces. Here, we describe, in B. schlosseri, two newgenes showing homology with vertebrate C3aR and CR1, respectively, and studied their transcription in the course of the colonial blastogeneticcycle. In addition, we continued our analysis ofthe role of C3 in Botryllus immunity by studying the modulation of BsC3 transcription during the colonial blastogenetic cycle and the effect of bsc3 knockdown on immune responses. Results indicate that only morula cells, and no other immunocytes type, are labelled by the antisense probe for BsC3aR, whereas phagocytes and young, undifferentiated cells, known as hemoblasts, are the cells stained by the probe for BsCR1. This suggests the presence of an important cross-talk between these two immunocytes types. Both the bsc3ar and bscr1 genes are constitutively transcribed as almost all morula cells and phagocytes, respectively, resulted labelled by the antisense probe in the ISH assay, independently of their previous challenge with zymosan, a known activator of B. schlosseri hemocytes. However, a modulation in the extent of transcriptionoccurs during the colonial blastogenetic cycle as the amount of BsC3aR mRNA abruptly decreased at TO, whereas no differences were observed when EC and MC were compared. This is probably related to the renewing of circulating cells at TO, when 20-30% of hemocytes undergo cell death by apoptosis and are replaced by new, differentiating cells entering the circulation in the same period