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Nicola FRANCHI

Ricercatore t.d. art. 24 c. 3 lett. B
Dipartimento di Scienze della Vita sede ex-Biologia


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Pubblicazioni

in corso di stampa - Origin and functions of Tunicate hemocytes. [Capitolo/Saggio]
Cima, F; Franchi, N; Ballarin, L
abstract


2021 - Characterisation and functional role of a novel C1qDC protein from a colonial ascidian [Articolo su rivista]
Peronato, A; Minervini, G; Tabarelli, M; Ballarin, L; Franchi, N
abstract

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.


2021 - New data on c1qdc from the colonial ascidian botryllus schlosseri [Articolo su rivista]
Peronato, A.; Minervini, G.; Franchi, N.; Ballarin, L.
abstract

In the compound ascidian Botryllus schlosseri, we recently identified a novel C1q-domain-containing (C1qDC) protein expressed by circulating immunocytes, called BsC1qDC. It has two globular C1q domains and a signal peptide and can act either as an opsonin and facilitate the phagocytosis of nonself particles or as a cytokine and stimulate the degranulation of cytotoxic cells. In the present work, we used a commercial antibody raised against human CTRP4 (hCTRP4) to provide additional evidences of the involvement of this molecule in immune responses. The antibody was validated in immunoblot analysis and recognizes a band corresponding to the expected molecular weight inferred from the analysis of the amino acid sequence of BsC1qDC. The presence of the antibody in the culture medium in phagocytosis and degranulation assays significantly reduced the two responses. In addition, the relationships between complement C3 activation and bsc1qdc transcription was studied using the injection of C3aR agonist in the colonial vasculature.


2021 - Stress granules in Ciona robusta: First evidences of TIA-1-related nucleolysin and tristetraprolin gene expression under metal exposure [Articolo su rivista]
Drago, Laura; Peronato, Anna; Franchi, Nicola; Ballarin, Loriano; Bakiu, Rigers; Santovito, Gianfranco
abstract

Stress granules are non-membranous cytoplasmic foci, composed of non-translating messenger ribonucleoproteins, translational initiation factors and other additional proteins. They represent a primary mechanism to rapidly modulate gene expression when cells are subjected to adverse environmental conditions. Very few works have been devoted to study the presence of the molecular components of stress granules in invertebrates. In this work, we characterized the transcript sequences for two important protein components of stress granules, TIA-1-related nucleolysin (TIAR) and tristetraprolin (TTP), in the solitary ascidian Ciona robusta, an invertebrate chordate, and carried out the first studies on their gene expression under stress conditions induced by metals (Cu, Zn and Cd). Data on mRNA expression levels, provided by qRT-PCR analyses, show a generalized decrease at the second day of metal-exposure for both tiar and ttp, suggesting that metal accumulation induces acute stress and the inhibition of the transcription for the two studied proteins. In-situ hybridization analyses demonstrate that TIAR and TTP antisense riboprobes recognize circulating granular amoebocytes in the hemolymph, in both blood lacunae and tunic. The results obtained in this work increase our knowledge on the evolution of anti-stress proteins in metazoans and emphasize the importance of the transcription of tiar and ttp, which represents an efficient physiological response allowing organisms to survive in the environment under stress conditions.


2020 - BsTLR1: A new member of the TLR family of recognition proteins from the colonial ascidian Botryllus schlosseri [Articolo su rivista]
Peronato, Anna; Franchi, Nicola; Loriano, Ballarin
abstract

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.


2020 - Complement system and phagocytosis in a colonial protochordate [Articolo su rivista]
Peronato, A.; Drago, L.; Rothbächer, Ute; Macor, P.; Ballarin, L.; Franchi, N.
abstract

In the present work, we investigated, in the colonial ascidian Botryllus schlosseri, the role of complement C3 (BsC3) in phagocytosis. We studied the modulation of BsC3 transcription in the course of the colonial blastogenetic cycle, with particular reference to the takeover, when apoptotic cells in the tissues of old zooids are cleared by circulating phagocytes. In situ hybridisation with BsC3 riboprobes labelled only morula cells, the most abundant haemocytes. Anti-hC3 antibody recognised morula cells and also phagocytes when haemocytes were previously incubated with zymosan. The inhibition of C3 activation prevented the labelling of phagocytes. In phagocytosis assays with haemocytes from colonies injected with anti-hC3 antibody or bsc3 iRNA, the capability to ingest target cells was significantly (p < 0.001) reduced. Therefore, our results strongly support a key role of BsC3 in phagocytosis and open to new investigations on the nature of the receptors of the products of BsC3 activation.


2020 - Insights into the Complement System of Tunicates: C3a/C5aR of the Colonial Ascidian Botryllus schlosseri [Articolo su rivista]
Peronato, Anna; Franchi, Nicola; Ballarin, Loriano
abstract

As an evolutionary ancient component of the metazoan immune defense toolkit, the complement system can modulate cells and humoral responses of both innate and (in jawed vertebrates) adaptive immunity. All the three known complement-activation pathways converge on the cleavage of C3 to C3a and C3b. The anaphylatoxin C3a behaves like a chemokine in inflammatory responses, whereas C3b exerts an opsonic role and, ultimately, can activate the lytic pathway. C3aR, one of the mammalian receptor for C3a, is a member of the G-protein-coupled receptor family sharing seven transmembrane alpha helixes. C3aR can act as a chemokine and recruit neutrophils, triggering degranulation and respiratory burst, which initiates an inflammatory reaction. Mining the transcriptome of the colonial ascidian Botryllus schlosseri, we identified a transcript showing homology with mammalian c3ar. The gene (bsc3/c5ar) is actively transcribed in morula cells, the circulating immunocyte triggering the inflammatory reactions in response to the recognition of nonself. Its transcription is modulated during the recurrent cycles of asexual reproduction known as blastogenetic cycles. Moreover, the treatment of hemocytes with C3aR agonist, induces a significant increase in the transcription of BsC3, revealing the presence of an autocrine feedback system able to modulate the expression of C3 in order to obtain a rapid clearance of potentially dangerous nonself cells or particles. The obtained results support the previously proposed role of complement as one of the main humoral components of immune response in tunicates and stress the importance of morula cells in botryllid ascidians innate immunity.


2019 - Characterisation of the complement system of a colonial tunicate: study of the expression of C3, CR1, C3aR and role of C3 in nonself recognition [Abstract in Atti di Convegno]
Peronato, A.; Franchi, N.; Drago, L.; Ballarin, L.
abstract

The complement system is one of the most ancient immune modulation mechanism of bilaterian metazoans. Three complement-activation pathways are known in vertebrates: 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. It relies only on innate immunity for its defense and immunocytes. Recently, in the same species, we demonstrated of the lectin and alternative pathways. 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, C3 is cleaved to C3a and C3b, the former exerting a chemokine–like 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 phagocyte surfaces. In the present work, we describe, in B. schlosseri,one genes showing similarity with vertebrate C3aR and three genes with similarity to CR1 (two soluble forms and one transmembrane), and studied their transcription in the course of the colonial blastogenetic cycle. Results indicate that their mRNAs are located in different immunocytes suggesting the presence of a cross-talk between phagocytes and morula cells. 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. Only morula cells, and no other immunocytes type, were labelled by the antisense probe for BsC3aR and the soluble CR1s, whereas phagocytes and young, undifferentiated cells known as hemoblasts were the cells stained by the probe for the membrane-linked BsCR1. Both the bsc3ar and bscr1 genes are constitutively transcribed; however, a modulation of transcription occurs during the colonial blastogenetic cycle as the amount of BsC3aR mRNA abruptly decreased at take-over, whereas no differences were observed when early-cycle and mid-cycle 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.


2019 - Characterization of the complement system in a colonial protochordate: C3 complement receptors and opsonic role of C3 [Abstract in Rivista]
Peronato, A.; Franchi, N.; Schiavon, L.; Ballarin, L.
abstract

The complement system is one of the most ancient immune modulator mechanism of bilaterian metazoans, able to influence ancient cells and factors of both innate and adaptive immunity. Three complement-activation pathways are known in vertebrates: 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. Recently, in the same species, we demonstrated the presence 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 that involves proteolysis and polymerization occurs resulting in the cleavage of the third complement component (C3) to C3a and C3b, the former exerting a chemokine–like 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. In the present work, we described, in B. schlosseri, two new genes showing homology with vertebrate C3aR and CR1, respectively, and studied their transcription in the course of the colonial blastogenetic cycle. Results indicate that their mRNAs are located in different immunocyte types suggesting the presence of an important cross-talk between phagocytes and morula cells. 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. Only morula cells, and no other immunocytes type, were labelled by the antisense probe for BsC3aR, whereas phagocytes and young, undifferentiated cells known as hemoblasts were the cells stained by the probe for BsCR1. 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 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, when 20-30% of hemocytes undergo cell death by apoptosis and are replaced by new, differentiating cells entering the circulation in the same period.


2019 - Characterization of the complement system in a colonial tunicate: C3 complement receptors and opsonic role of C3 [Abstract in Atti di Convegno]
Peronato, A.; Franchi, N.; Schiavon, L.; Ballarin, L.
abstract

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.


2019 - Characterization of the complement system in a colonial tunicate: C3 complement receptors and opsonic role of C3 [Abstract in Atti di Convegno]
Peronato, A.; Franchi, N.; Drago, L.; Ballarin, L.
abstract

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.


2019 - Complement-mediated cooperation between immunocytes in the compound ascidian Botryllus schlosseri [Abstract in Atti di Convegno]
Peronato, A.; Franchi, N.; Ballarin, L.
abstract

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.


2019 - Functional amyloidogenesis in immunocytes from the colonial ascidian Botryllus schlosseri: Evolutionary perspective [Articolo su rivista]
Franchi, Nicola; Ballarin, Loriano; Peronato, Anna; Cima, Francesca; Grimaldi, Annalisa; Girardello, Rossana; de Eguileor, Magda
abstract

Cytotoxic morula cells (MCs) and phagocytes are the circulating immunocytes of the colonial ascidian Botryllus schlosseri: Both these cells can synthesise amyloid fibrils, supporting the idea that physiological amyloidogenesis is involved in inflammation and modulation of immune responses. Intriguingly, amyloid of B. schlosseri immunocytes is made of two different proteins. MCs, the first cells to sense non-self and involved in the allorejection reaction between contacting genetically incompatible colonies, use melanin encapsulation as the principal method to fight non-self. They release amyloid fibrils formed by p102 protein that allow the packaging and deposit of melanin and other toxic molecules nearby the invader or in the contact region of incompatible colonies. Phagocytes release amyloid-based extracellular traps when challenged with microbes: their amyloid fibrils harbour BsAPP, an orthologue of the vertebrate amyloidogeneic protein APP. This strategy of immune response, present also in human neutrophils, allows phagocytes to block and engulf bacteria and fungi.


2019 - Identification and expression studies of putative stem/progenitor cell markers in the urochordate Botryllus schlosseri [Abstract in Rivista]
Peronato, A.; Franchi, N.; Ballarin, L.
abstract

In the colonial ascidian Botryllus schlosseri, a cyclical generation change guarantees the recurrent (weekly at 20°C) renewal of the zooids. During the blastogenetic cycle (i.e., the interval of time between a generation change and the following one), buds progressively grow to the adult size before replacing the old zooids. With the aim of better elucidating the process stem cell differentiation, with particular reference to the genesis of haemocytes during the of the colonial ascidian, we screened the B. schlosseri genome and transcriptome, looking for transcripts/genes showing similarity to vertebrate molecular markers of haematopoietic stem/progenitor cells. On these sequences, after an in silico translation, we performed the phylogenetic reconstruction that, always, returned us the tunicate relevant position, within the protochordates cluster, of vertebrate sister group. The four mammalian orthologous genes, used as markers for the recognition of haematopoietic stem/progenitor cells, identified in B. schlosseri, are bsabcg2, bscd133, bsgata1/2/3 and bsgata4/5/6. The ISH assay, performed by antisense specific riboprobes, on haemocyte monolayers and colony sections, resulted in a labelling of the sub-endostylar haemolymph lacunae. This results matches previously morphological data that identified the endostyle as a stem cell niche, strengthening our idea to use bsabcg2, bscd133, bsgata1/2/3 and bsgata4/5/6 genes for the identification of haematopoietic stem/progenitor cells in B. schlosseri. Quantitative real time PCR (qRT-PCR) highlighted the over-expression of the considered genes in the mid-cycle phase of the blastogenetic cycle. During this phase, there is the formation of new secondary buds emerging from the primary buds. The higher transcription levels of bsabcg2, bscd133, bsgata1/2/3 and bsgata4/5/6 in the mid-cycle phase reflect the presence of undifferentiated cells involved in proliferative and differentiation events required for the formation of the new blastogenetic generation.


2018 - Amyloid and immune responses in the colonial ascidian Botryllus schlosseri [Abstract in Rivista]
Ballarin, L.; Franchi, N.; Peronato, A.; Grimaldi, A.; Girardello, R.; de Eguileor, M
abstract

Increasing evidences indicate that functional, non-toxic amyloid is widely distributed among living organisms. In invertebrates and vertebrates, functional amyloid is involved in inflammatory reactions and modulation of immune responses. In the present study, we investigated the occurrence of functional amyloid in tunicates, the closest living relatives of vertebrates. Previous studies indicate that orthologous genes for the amyloid precursor protein (APP), involved in amyloid synthesis in vertebrates, are present in solitary ascidians of the genus Ciona. In addition, colonies of compound ascidians of the genus Botryllus, respond to contacting genetically incompatible colonies with an allorecognition reaction that leads to the formation of necrotic, melanic spots along the contact border. Our data provide evidence that functional amyloid is involved in immune responses of Botryllus schlosseri and indicate that, both the circulating immunocyte types, i.e., the cytotoxic morula cells and phagocytes, can produce amyloid using two different proteins: Bsp102 and BsAPP. Bsp102 forms the amyloid functioning as scaffold to store MC granular content and, once released upon MC degranulation, the support where phenoloxidase and melanin are deposited, thus limiting the diffusion of cytotoxicity. BsAPP is released by phagocytes and contribute to the formation of extracellular nets that entrap microbes and prevent their diffusion within the organism. To the best of our knowledge, this is the first report of functional amyloidogenesis in protochordate immunity.


2018 - BsTLR: a new member of the TLR family of recognition proteins from the colonial ascidian Botryllus schlosseri [Abstract in Atti di Convegno]
Peronato, A.; Franchi, N.; Ballarin, L.
abstract

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.


2018 - Echinodermata: The complex immune system in echinoderms [Capitolo/Saggio]
Courtney Smith, L.; Arizza, V.; Barela Hudgell, M. A.; Barone, G.; Bodnar, A. G.; Buckley, K. M.; Cunsolo, V.; Dheilly, N. M.; Franchi, N.; Fugmann, S. D.; Furukawa, R.; Garcia-Arraras, J.; Henson, J. H.; Hibino, T.; Irons, Z. H.; Li, C.; Lun, C. M.; Majeske, A. J.; Oren, M.; Pagliara, P.; Pinsino, A.; Raftos, D. A.; Rast, J. P.; Samasa, B.; Schillaci, D.; Schrankel, C. S.; Stabili, L.; Stensvag, K.; Sutton, E.
abstract

The Echinodermata are an ancient phylum of benthic marine invertebrates with a dispersal-stage planktonic larva. These animals have innate immune systems characterized initially by clearance of foreign particles, including microbes, from the body cavity of both larvae and adults, and allograft tissue rejection in adults. Immune responsiveness is mediated by a variety of adult coelomocytes and larval mesenchyme cells. Echinoderm diseases from a range of pathogens can lead to mass die-offs and impact aquaculture, but some individuals can recover. Genome sequences of several echinoderms have identified genes with immune function, including expanded families of Toll-like receptors, NOD-like receptors, and scavenger receptors with cysteine-rich domains, plus signaling pathways and cytokines. The set of transcription factors that regulate proliferation and differentiation of the cellular immune system are conserved and indicate the ancestral origins of hematopoiesis. Both larval and adult echinoderms are in constant contact with potential pathogens in seawater, and they respond to infection by phagocytosis and encapsulation, and employ proteins that function in immune detection and response. Antipathogen responses include activation of the SpTransformer genes, a complement system, and the production of many types of antimicrobial peptides. Echinoderms have homologues of the recombinase activating genes plus all associated genes that function in vertebrates for immunoglobulin gene family rearrangement, although their gene targets are unknown. The echinoderm immune system has been characterized as unexpectedly complex, robust, and flexible. Many echinoderms have very long life-spans that correlate with an excellent capacity for cell damage repair. In many marine ecosystems, echinoderms are keystone predators and herbivores, and therefore are species that can serve as optimal sentinels of environmental health. Coelomocytes can be employed in sensor systems to test for the presence of marine pollutants. When Elie Metchnikoff inserted a rose prickle into a larval sea star and observed chemotaxis, phagocytosis, and encapsulation by the mesenchyme cells, he initiated not only the field of immunology but also that of comparative immunology, of which the echinoderms have been an important part.


2018 - Expression study of molecular markers involved in staminality and differentiation in the colonial ascidians Botryllus schlosseri [Abstract in Rivista]
Ballin, F.; Franchi, N.; Peronato, A.; Ballarin, L.
abstract

Ascidians are invertebrate chordates, members of the subphylum Tunicata that represents the sister group of vertebrates. They offer the opportunity to investigate and compare the behaviour of both embryonic and adult stem cells. Morphological data suggest the presence of undifferentiated haemocytes (haemoblasts) able to proliferate and give rise to terminally differentiated cells. Relevant studies were also carried out in the neural lineage, in which neural progenitor cells regenerate the brain after extirpation. In B. schlosseri, during the cyclical generation change, bud primordial cells, probably deriving from a pool of long-living stem cells, are able to give rise to the neural complex. We screened the B. schlosseri genome and transcriptome, looking for transcripts/genes showing similarity to vertebrate molecular markers of haematopoietic and neural stem cells. Four sequences, orthologous to mammalian transcripts considered markers of haematopoietic progenitor cells, were identified in B. schlosseri. They are: bsabcg2, bscd133, bsgata1/2/3 and bsgata4/5/6. In situ hybridization on haemocyte monolayers and colony sections, resulted in labelling of cells in the sub-endostylar haemolymph lacunae. This results matches previously morphological data that identified the endostyle as a stem cell niche. Quantitative real time PCR (qRT-PCR) highlighted the over-expression of the considered genes in the mid-cycle phase of the blastogenetic cycle. During this phase, there is the formation of new secondary buds emerging from the primary buds. The high expression levels of bsabcg2, bscd133, bsgata1/2/3 and bsgata4/5/6 genes in the mid-cycle phase reflect the presence of undifferentiated cells involved in proliferative and differentiation events required for giving rise to the new blastogenetic generation. For the neural lineage, we identified and characterised two transcripts orthologues of vertebrate neural stem cell markers (BsSox2 and BsMsi2). We also studied the expression, during the blastogenetic cycle, of a panel of genes already known to be involved in ascidian larvae neurogenesis, i.e., orthologues of Pax2/5/8, Hox1 and Hox3. ISH with riboprobes for BsSox2, BsMsi2, BsPax2/5/8, BsHox1 and BsHox3 revealed a common labelling in the endostyle niche. The presence of bssox2, bsmsi2, bspax2/5/8, bshox1 and bshox3 transcripts in the cells of the region known to be a stem cell niche, led us to conclude, not only that our probes identified undifferentiated cells but even that in B. schlosseri are probably present a single population of pluripotent stem cells that could differentiate into haematopoietic or neural cells. The qRT-PCR, showed an high expression level in the mid-cycle phase of all the putative neural markers considered. In this phase new secondary buds are produced from primary buds. Each new bud needs its own neural complex and this requires the proliferation of undifferentiated cells to originate neural gland rudiment and cerebral ganglion. Bssox2, bsmsi2, bspax2/5/8, bshox1 and bshox3 increased their expression associated with these neurogenesis events and this support their involvement in neural stem cell differentiation.


2018 - Molecular characterization and metal induced gene expression of the novel glutathione peroxidase 7 from the chordate invertebrate Ciona robusta [Articolo su rivista]
Ferro, D.; Franchi, N.; Bakiu, R.; Ballarin L., ⁎; Santovito, G.
abstract

In the present study, we describe the identified and characterized the gene and the transcript of a novel glutathione peroxidase-7 (GPx7) from the solitary ascidian Ciona robusta, an invertebrate chordate widely distributed in temperate shallow seawater. The putative nucleotide and amino acid sequences were compared with those of GPx7 from other metazoans and phylogenetic analysis suggests the presence of a high evolutionary pressure in the contest of neutral evolution. The mRNA of CrGPx7 is located in hemocytes and ovarian follicular cells, as revealed by in situ hybridization. The time course of CrGPx7 mRNA levels in the presence of Cd, Cu and Zn, showed upregulation in the final stages of the experiments, suggesting a role of GPx7 in late protection from oxidative stress. Our in silico analyses of the crgpx7 promoter region revealed putative consensus sequences similar to mammalian metal-responsive elements (MRE) and xenobiotic-responsive elements (XRE), suggesting that the transcription of these genes directly depends on metals. Cell-free extract from C. robusta tissues show the presence of selenium-independent GPx activity that is inhibited by the presence of metals.


2017 - Allorecognition in the ascidian Botryllus schlosseri: the importance of amyloid [Abstract in Rivista]
Franchi, N.; De Eguileor, M.; Grimaldi, A.; Girardello, R.; Ballarin, L.
abstract

In the compound ascidian Botryllus schlosseri, allorecognition manifests primarily as colony specificity, controlled by a highly polymorphic Fu/HC locus, so that contacting colonies sharing at least one allele at the Fu/HC locus can fuse into a chimeric colony; if no alleles are shared, a typical inflammation reaction occurs, with the recruitment of a specific hemocyte type, the cytotoxic morula cells (MCs), inside the tips of the ampullae (the blind termini of the tunic vasculature) extending towards the alien colony, their extravasation in the tunic and their final degranulation with the consequent release of factors inducing autocrine and paracrine cell and the formation of necrotic, melanic spots (points of rejection; PORs) along the contact border. MCs are the first cells to sense nonself and are the source of cytokines that induce the recruitment of immunocytes at the inflammatory sites and the activation of phagocytes required for the clearance of foreign material. MCs store quinones, polyphenols and the enzyme phenoloxidase (PO) inside their granules, representing their cytotoxic potential. Although PO, quinones and polyphenols are soluble factors, it is remarkable that the deposition of melanin and the cell death is confined to the immediate outside of the ampullar tips, suggesting that the diffusion of the enzyme and the products of its activity is, in some way, prevented in order to limit cytotoxicity to the immediate neighbourhood of degranulating MCs. With this idea in mind, we looked for factors released by MCs that could limit the spreading of cytotoxicity and melanisation. We found that MCs contain amyloid inside their granules and that amyloid fibrils are released in the surrounding medium upon MC degranulation forming a net-like scaffold entrapping PO and melanin, thus limiting their diffusion. In addition, the search for genes and factor controlling both melanogenesis and amyloidogenesis, revealed an evolutionary conserved machinery involved in the processes and an unexpected cross talk between the two Botryllus immunocyte types, i.e., phagocytes and MCs. This work represents the first demonstration of a physiological role of amyloid in protochordata immunity.


2017 - Amyloid and allorecognition in the colonial ascidian Botryllus schlosseri [Abstract in Atti di Convegno]
Franchi, N.; Girardello, R.; Grimaldi, A.; De Eguileor, M.; Ballarin, L.
abstract

Allorecognition, i.e., the ability of intraspecific nonself recognition is widely distributed among colonial, sessile marine organisms in the form of colony specificity. In the cosmopolitan compound ascidian Botryllus schlosseri, colony specificity is controlled by a highly polymorphic Fu/HC locus: two colonies sharing at least one alleleat the Fu/HC locus can fuse into a chimeric colony; if no alleles are shared, a typical inflammatory reaction occurs, with the recruitment of a specific hemocyte type, the cytotoxic morula cells (MCs), inside the tips of the ampullae (the blind termini ofthe tunic vasculature) extending towards the alien colony, their extravasation in the tunic and their final degranulation. As a consequence of allorecognition, necrotic, melanic spots (points of rejection; PORs) form along the contact border, due to the release, by MCs, of their granular content, mainly represented by quinones, polyphenols and the enzyme phenoloxidase (PO), upon the perception of the allogeneic humoral factors diffusing from the alien colony through the partially fused tunics. It is remarkable that the deposition of melanin and the cell death is confined to the immediate outside of the ampullar tips, suggesting that the diffusion of PO and the products of its activity are, in some way, prevented in order to limit cytotoxicity to the immediate neighbourhood of the contact region. In this context, we looked for factors released by MCs that could limit the spreading of cytotoxicity and melanisation. We found that MCs share with vertebrate melanocytes similar packaging of melanin precursors, entrapped in a 3Dscaffold of amyloid fibrils. They contribute to form the electron dense content of MC granules that, after stimulation, flake off and is released in the surrounding medium. Released amyloid fibrils limit the diffusion of the produced melanin. The search for genes and factor controlling both melanogenesis and amyloidogenesis, revealed an evolutionary conserved machinery involved in the processes and an unexpected cross talk between the two Botryllus immunocyte types, i.e., phagocytes and MCs. Furthermore, this work confirms the physiological role of amyloid in tunicate immunity.


2017 - Expression of genes involved in oxidative stress response in colonies of the ascidian Botryllus schlosseri exposed to various environmental conditions [Articolo su rivista]
Tasselli, Stefano; Ballin, Francesca; Franchi, Nicola; Fabbri, Elena; Ballarin, Loriano
abstract

Environmental stress conditions are ultimately related to the induction of oxidative stress in organisms, as a consequence of an increased production of reactive oxygen species (ROS). This could be exploited to study sub-lethal effects induced by the environment in the organisms. In the present work, we evaluate the possibility to use the colonial ascidian Botryllus schlosseri as a bioindicator, to assess the environmental quality in the Lagoon of Venice. Three colony batches were immersed, for 22 days, at two sites (1 and 2) with different grades of hydrodynamics and anthropogenic impact and physico-chemical features of seawater; a control batch was kept in a large tank with continuous seawater flow at the Marine Station of the Department of Biology, University of Padova, in Chioggia (site 3). Seawater at site 2 had higher pH and temperature than site 1. Colonies were then retrieved, their mRNA was extracted and the level of transcription of genes involved in oxidative stress response (glutathione synthase, g-glutamyl-cysteine ligase, modulatory subunit, two isoforms of glutathione peroxidases and Cu/Zn superoxide dismutase) was evaluated. In colonies from sites 1 and 2, most genes showed significantly increased transcriptional levels with respect to control values. Spectrophotometric analyses of colony homogenates revealed that the enzymatic activity of superoxide dismutase and catalase was higher in colonies from site 2 as compared to site 1, allowing us to speculate that colonies in site 2 were under higher stress level than those in site 1. Overall, we can conclude that B. schlosseri seems a good indicator of the ecological status of the Lagoon environment, within a range of pH and temperature in which colonies are used to live.


2017 - First evidences of a complement system lytic pathway in invertebrates: data from the compound ascidian Botryllus schlosseri [Abstract in Rivista]
Peronato, A.; Franchi, N.; Ballarin, L.
abstract

The complement system is well studied in mammals, where more than 30 proteins have been described, involved in the activation and regulation of this important humoral effector. However, the evolutionary history of the complement system is not yet fully elucidated and, in recent years, it has been widely demonstrated that complement system is more than just a defender against intruders. For instance, it is important for the clearance of apoptotic cells and corpses. Botryllus schlosseri is a cosmopolitan ascidian, belonging to the phylum Chordata, considered a model organism for the studies of the evolution of the immune system. Studying the complexity of the complement system in Botryllus, we identified a transcript, in our EST collection, coding a protein containing the MACPF (membrane attack complex/perforin) domain, shared by both most of the proteins involved in the lytic pathway and perforins. Invertebrates, we know that perforins are produced by T-lymphocytes and natural killer cells, whereas the activity of the proteins, with the MACPF domain is regulated by the complement component C3/C5.Comparing the domain topology of vertebrateC9 and our protein, called Botryllus C9-like protein (BsC9), a high level of similarity results. To demonstrate that our C9-like protein can be considered a part of the complement system in B. schlosseri we evaluated the expression of BsC9 with respect to C3 activity. Our previous data demonstrates that B. schlosseri C3 is activated by zymosan. We combined the microinjection of zymosan with and without a validated anti-C3 antibody (against human C3) to block the activity of C3. With this approach we studied in, time course, the expression of both BsC3 and BsC9 demonstrating that the anti-C3 antibody is able to inhibit the expression of BsC9. These results are confirmed in both ISH and ICC using the same antibody, and in vitro with the C3 inhibitor compstatin. In addition, a significant (p < 0.05) decrement of labeling with BsC9 antisense riboprobe and validated antibody anti hsC9 is observed when hemocytes are incubated with zymosan and compstatin. Collectively, these results argue in favor of the presence of components of the lytic pathway in our model organism.


2017 - Immunity in Protochordates: The Tunicate Perspective [Articolo su rivista]
Franchi, Nicola; Ballarin, Loriano
abstract

Tunicates are the closest relatives of vertebrates, and their peculiar phylogenetic position explains the increasing interest toward tunicate immunobiology. They are filter-feeding organisms, and this greatly influences their defense strategies. The majority of the studies on tunicate immunity were carried out in ascidians. The tunic acts as a first barrier against pathogens and parasites. In addition, the oral siphon and the pharynx represent two major, highly vascularized, immune organs, where circulating hemocytes can sense non-self material and trigger immune responses that, usually, lead to inflammation and phagocytosis. Inflammation involves the recruitment of circulating cytotoxic, phenoloxidase (PO)-containing cells in the infected area, where they degranulate as a consequence of non-self recognition and release cytokines, complement factors, and the enzyme PO. The latter, acting on polyphenol substrata, produces cytotoxic quinones, which polymerize to melanin, and reactive oxygen species, which induce oxidative stress. Both the alternative and the lectin pathways of complement activation converge to activate C3: C3a and C3b are involved in the recruitment of hemocytes and in the opsonization of foreign materials, respectively. The interaction of circulating professional phagocytes with potentially pathogenic foreign material can be direct or mediated by opsonins, either complement dependent or complement independent. Together with cytotoxic cells, phagocytes are active in the encapsulation of large materials. Cells involved in immune responses, collectively called immunocytes, represent a large fraction of hemocytes, and the presence of a cross talk between cytotoxic cells and phagocytes, mediated by secreted humoral factors, was reported. Lectins play a pivotal role as pattern-recognition receptors and opsonizing agents. In addition, variable region-containing chitin-binding proteins, identified in the solitary ascidian Ciona intestinalis, control the settlement and colonization of bacteria in the gut.


2017 - Morula cells as key hemocytes of the lectin pathway of complement activation in the colonial tunicate Botryllus schlosseri [Articolo su rivista]
Franchi, Nicola; Ballarin, Loriano
abstract

The complement system is deeply rooted in the evolution of humoral mechanism of innate immunity. In addition to the alternative pathway of complement activation, lectins and associated serine proteases exert important roles in the recognition of non-self and activation of the effectors. In the colonial tunicate Botryllus schlosseri, we identified, characterized and studied the expression of three orthologues of genes involved in the lectin pathway of complement activation of vertebrates, i.e., genes for a mannose-binding lectin (MBL), a ficolin and a mannose-associated serine protease 1 (MASP1). All the genes are transcribed by hemocytes, and specifically by morula cells, the same immunocytes responsible for the transcription of C3 and Bf orthologues. The transcription levels of MASP1 and ficolin orthologues are not affected by zymosan challenge, indicating a constitutive expression of complement system associated serine proteases, whereas the MBL orthologue is up-regulated after 15 min of zymosan exposure. Collectively, our data suggest the presence of a complete lectin activation pathway in Botryllus.


2017 - Protection from Oxidative Stress in Immunocytes of the Colonial Ascidian Botryllus schlosseri: Transcript Characterization and Expression Studies [Articolo su rivista]
Franchi, Nicola; Ballin, Francesca; Ballarin, Loriano
abstract

Botryllus schlosseri is a cosmopolitan colonial ascidian that undergoes cyclical generation changes, or take-overs, during which adult zooids are resorbed and replaced by their buds. At take-over, adult tissues undergo diffuse apoptosis and effete cells are massively ingested by circulating phagocytes, with a consequent increase in oxygen consumption and in production of reactive oxygen species (ROS). The latter are responsible for the death of phagocytes involved in the clearance of apoptotic cells and corpses by phagocytosis-induced apoptosis. However, the majority of phagocytes and hemocytes do not die, even if they experience oxidative stress. This fact suggests the presence of detoxification mechanisms assuring their protection. To test this assumption, we searched for transcripts of genes involved in detoxification in the transcriptome of B. schlosseri. We identified and characterized transcripts for Cu/Zn superoxide dismutase (SOD), γ-glutamyl-cysteine ligase modulatory subunit (GCLM), glutathione synthase (GS), and two glutathione peroxidases (i.e., GPx3 and GPx5), all involved in protection from ROS. We also carried out a phylogenetic analysis of the putative amino acid sequences, confirming their similarity to their vertebrate counterparts, and studied the location of their mRNAs by in situ hybridization on hemocyte monolayers. We also analyzed gene transcription during the colonial blastogenetic cycle, which is the interval of time between one take-over and the next, by qRT-PCR. In addition, we investigated the effects of cadmium (Cd), an inducer of oxidative stress, on gene transcription. Our results indicated that i) antioxidant gene expression is modulated in the course of the blastogenetic cycle and upon exposure to Cd, and ii) hemocytes synthesize both enzymatic and nonenzymatic antioxidants, in line with the idea that they represent a major detoxification system for ascidians.


2017 - The complement system in Botryllus schlosseri [Abstract in Atti di Convegno]
Franchi, Nicola; Peronato, Anna; Ballarin, Loriano
abstract

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 the evolution of the main complement components can provide clues to understand changes related to adaptations to new environmental conditions and life-cycles or, in the case of vertebrates, to interactions with the adaptive immunity. Data on tunicates, evolutionary close to vertebrates, are of primary importance for the elucidationof the changes associated with the invertebrate-vertebrate transition. In our model tunicate Botryllus schlosseri, we described both a lectin and an alternative pathway of complement activation, similar to those of vertebrates. All the described complement-related genes such as c3, bf, ficolin, mbl and masp are transcribed by morula cells, i.e., the immunocytes involved in cytotoxic responses and immunomodulation. Functional data suggest the presence of a complement-related immunocyte dialogue during the immune response. When B. schlosseri immunocytes are incubated with yeast (Saccharomyces cerevisiae) we observed an over-expression of C3 by morula cell that led to an increased amoebocyte-mediated phagocytosis. When a specific C3 inhibitor (compstatin) is added to the medium, this activity decreases. These new data pave the way for a better comprehension of the evolution lof complement-system behavior during immune responses. In the next future, our efforts will focus on the regulation of complement system in tunicates to shed light on the complement system function in a pre-adaptive immunity scenario.


2017 - The complement system of Botryllus schlosseri [Abstract in Atti di Convegno]
Franchi, N.; Ballarin, L.
abstract

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.


2017 - The C1q domain-containing protein from the ascidian Botryllus schlosseri manifests a cytokine-like behavior [Abstract in Rivista]
Franchi, N.; Ballarin, L.
abstract

Genes encoding complement component 3 (C3) have been extensively investigated in invertebrate genomes and traced back in evolutionary history to the early metazoan radiation. However, other components of the complement system, such as those related to the classical activation pathway, are still not much investigated. Currently, the genes encoding for proteins with a C1q domain, probably the main components of the classical pathway, have been only partially investigated from an evolutionary perspective. These genes exist in many of the sequenced genomes, from both vertebrates and invertebrates and functions have been described for some of the corresponding proteins. A C1q-like gene have been identified in the medicinal leech Hirudo medicinalis where a C1q-like peptide elicits a chemotactic behavior that could be blocked using a human antibody against the gC1q receptor. C1q-like genes have also been found in the urochordate Ciona intestinalis and the cephalocordate Branchiostoma floridae where it has been demonstrated that the globular domain is able to recognize and bind mammalian antibodies initiating the classical pathway of complement activation. The globular head C1q domain is a lectin domain present in transcriptomes of amphioxus, lamprey, and several teleost fishes. Few of these putative C1q-like proteins have been characterized; however, they can bind to a variety of carbohydrates. In Botryllus schlosseri we have found, in our EST collection, a single transcript with C1q characteristics (BsC1q-like). The deduced protein contains two globular head C1q domains, a feature unknown in invertebrates. As regard Vertebrates, we can find a similar architecture only in mammals, in the so called C1q/TNF-related Protein 4 (CTRP4). This protein is very poorly studied and seems to be expressed in the hypothalamus and contribute to the modulation of food intake and body weight. Our data, from the colonial ascidian, suggest a role for the BsC1q-like protein as mediator of the activation and degranulation of the cytotoxic hemocytes. Both ISH and ICC demonstrate that both cytotoxic morula cells and phagocytes express the BsC1q-like mRNA and protein; functional analyses demonstrate that the human antibody against globular head C1q is able to inhibit morula cell degranulation after bacterial challenge. It is not yet clear if it is possible to considered this molecule as member of the complement system in Botryllus but future analyses will be directed to the study of the functional relationships between BsC3 and BsC1q-like as well as of the binding capabilities of the latter.


2016 - Cytotoxic Cells of Compound Ascidians [Capitolo/Saggio]
Franchi, Nicola; Ballarin, Loriano
abstract


2016 - Data on four apoptosis-related genes in the colonial tunicate Botryllus schlosseri [Articolo su rivista]
Franchi, Nicola; Ballin, Francesca; Manni, Lucia; Schiavon, Filippo; Ballarin, Loriano
abstract

The datadescribed are related to the article entitled “Recurrent phagocytosis-induced apoptosis in the cyclical generatio nchange of the compound ascidian Botryllus schlosseri” (Franchi et al.,2016) [1]. Four apoptosis-related genes, showing high similarity with mammalian Bax(a member of the Bcl-2 protein family), AIF1 (apoptosis-inducing factor-1), PARP1 (poly ADP ribose polymerase-1) and IAP7 (inhibitor of apoptosis-7) were identified from the analysis of the trascriptome of B. schlosseri. They were named BsBax, BsAIF1, BsPARP1 and BsIAP7. Here, their deduced amino acid sequence were compared wit hknown sequences of orthologous genes from other deuterostome species together with a study of their identity/similarity.


2016 - Histamine stimulates ciliary beat frequency via the H2 receptor in the protochordate Botryllus schlosseri [Articolo su rivista]
Cima, Francesca; Franchi, Nicola
abstract

Histamine is a biogenic molecule that plays a role in many physiological pathways via binding to a specific receptor. Histaminergic receptors belong to the large family of seven-transmembrane α-helix domain receptors classified in mammals into four distinct classes: H1, H2, H3, and H4. Despite being widely studied in vertebrates, few data are available on the invertebrate receptors, with only predicted H1 and H2 sequences for nonchordate deuterostomes. Here, we report the first characterized transcript sequence for an H2 receptor from the colonial ascidian Botryllus schlosseri, describing the localization of both transcript and protein during blastogenic development through in situ hybridization and immunohistochemistry. Its phylogenetic relationships with deuterostome orthologous proteins are reported, its role in ciliary beat frequency (CBF) in cultured stigma cells of the branchial basket is outlined, and the effects of histamine and its receptor agonists and antagonists are analyzed. In the presence of increasing concentrations of histamine in the medium, CBF increases similarly to the selective H2 receptor agonist dimaprit. In contrast, ranitidine, which is an inhibitor of the H2 receptor, causes a significant inhibition of CBF, similar to that observed after preincubation with the specific anti-BsHRH2 or the anti-human HRH2 antibody. In cells bordering the branchial basket stigmata, both antibodies colocalize in the proximal region of the ciliary plasmalemma, and histamine is present inside vesicles of the apical region, thus supporting the hypothesis of a histamine-binding H2 receptor control of the pharyngeal mucociliary transport similar to that of the upper respiratory tract and middle ear in mammals.


2016 - Origin and function of tunicate hemocytes [Capitolo/Saggio]
Cima, Francesca; Franchi, Nicola; Ballarin, Loriano
abstract

Tunicates, or urochordates, are a subphylum of the phylum Chordata, sharing with other members of the phylum: (1) a permanent or temporary notochord, in the form of a dorsal rod; (2) a central nervous system, in the form of a dorsal tube; (3) a pharynx provided with gill slits or pharyngeal pouches, and a ventral gland secreting iodoproteins (endostyle or thyroid); and (4) a muscular tail. Tunicates are considered the sister group of vertebrates,1 forming with the latter the clade Olfactoria. Recently, it has been proposed to classify them as a phylum within the superphylum Chordata.2 They are traditionally subdivided in three classes: (1) Ascidiacea (benthic and sessile), (2) Thaliacea, and (3) Larvacea or Appendicularia (pelagic). Ascidians have a free-swimming, tadpole-like larva, an adult sac-like body with two siphons that allow water flux, and a large branchial basket, provided with a ventral endostyle that secretes the mucous net required for filtration. They comprise two orders: Enterogona (including the suborders Phlebobranchia and Aplousobranchia) and Pleurogona (with the suborder Stolidobranchia).3 Thaliaceans include three orders: the colonial Pyrosomida, and the solitary/colonial Doliolida and Salpida. They have a barrel-like adult body, and, with the exception of Doliolida, are devoid of larval stages.4,5 Larvaceans or appendicularians resemble the ascidian larvae and use the tail to create the water current for filtration; filters are included in the gelatinous house secreted by the animals themselves. 4 Most of the recent authors consider larvaceans as a sister group of the other tunicates, and thaliaceans as a sister group of Enterogona5–10 (Fig. 2.1). Ascidians include about 2300 species, and most of the information on tunicate hemocytes comes from studies on this group of organisms. This review will, then, focus mainly on ascidian hemocytes and will discuss their role in immunity. Where possible, information on circulating cells of pelagic tunicates will be added.


2016 - Recurrent phagocytosis-induced apoptosis in the cyclical generation change of the compound ascidian Botryllus schlosseri [Articolo su rivista]
Franchi, Nicola; Ballin, Francesca; Manni, Lucia; Schiavon, Filippo; Basso, Giuseppe; Ballarin, Loriano
abstract

Colonies of the marine, filter-feeding ascidian Botryllus schlosseri undergo cyclical generation changes or takeovers. These events are characterised by the progressive resorption of adult zooids and their replacement by their buds that grow to adult size, open their siphons and start filtering. During the takeover, tissues of adult zooids undergo extensive apoptosis; circulating, spreading phagocytes enter the effete tissues, ingest dying cells acquiring a giant size and a round morphology. Then, phagocytes reenter the circulation where they represent a considerable fraction (more than 20%) of circulating haemocytes. In this study, we evidence that most of these circulating phagocytes show morphological and biochemical signs of apoptosis. Accordingly, these phagocytes express transcripts of orthologues of the apoptosis-related genes Bax, AIF1 and PARP1. Electron microscopy shows that giant phagocytes contain apoptotic phagocytes inside their own phagocytic vacuole. The transcript of the orthologues of the antiapoptotic gene IAP7 was detected only in spreading phagocytes, mostly abundant in phases far from the take-over. Therefore, the presented data suggest that, at take-over, phagocytes undergo phagocytosis-induced apoptosis (PIA). In mammals, PIA is assumed to be a process assuring the killing and the complete elimination of microbes, by promoting the disposal of terminally differentiated phagocytes and the resolution of infection. In B. schlosseri, PIA assumes a so far undescribed role, being required for the control of asexual development and colony homeostasis.


2016 - Transcriptome dynamics in the asexual cycle of the chordate Botryllus schlosseri [Articolo su rivista]
Campagna, Davide; Gasparini, Fabio; Franchi, Nicola; Vitulo, Nicola; Ballin, Francesca; Manni, Lucia; Valle, Giorgio; Ballarin, Loriano
abstract

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 adults, and budlets on buds. Cyclically, adult tissues undergo apoptosis and are progressively resorbed and replaced by their buds originated by asexual reproduction. This is a feature of colonial tunicates, the only known chordates that can reproduce asexually. Results: Thanks to a newly developed web-based platform (http://botryllus.cribi.unipd.it), we compared the transcriptomes of the mid-cycle, the pre-take-over, and the take-over phases of the colonial blastogenetic cycle. The platform is equipped with programs for comparative analysis and allows to select the statistical stringency. We enriched the genome annotation with 11,337 new genes; 581 transcripts were resolved as complete open reading frames, translated in silico into amino acid sequences and then aligned onto the non-redundant sequence database. Significant differentially expressed genes were classified within the gene ontology categories. Among them, we recognized genes involved in apoptosis activation, de-activation, and regulation. Conclusions: With the current work, we contributed to the improvement of the first released B. schlosseri genome assembly and offer an overview of the transcriptome changes during the blastogenetic cycle, showing up- and down-regulated genes. These results are important for the comprehension of the events underlying colony growth and regression, cell proliferation, colony homeostasis, and competition among different generations.


2015 - Complement system receptors C3aR and CR1 in Tunicates: ancient roots of immunocyte dialogue [Abstract in Atti di Convegno]
Franchi, Nicola; Ballarin, Loriano
abstract

Complement system is one of the most important humoral defense mechanism of innate immunity. Its evolutionary history is rooted in cnidarians where C3 molecules have been described. C3 is considered, in all taxa, the main protein of the complement system and the peptides C3a and C3b, derived from its proteolysis, are the effectors of all the complement-related responses towards microorganisms. These peptides have been described in many taxa, but only in Vertebrates the complement system behaviour was deeply investigated, where more than 30 proteins were described. Conversely, in invertebrates, no more than 10 proteins in few organisms have been identified, all belonging to only two pathways of complement activation: the alternative and the lectin pathway. Recently we focussed our attention on the complement system of the colonial ascidian Botryllus schlosseri. We identified C3 and the serine proteases associated with the alternative and lectin pathways. In the present work, we identified the main receptors of the activated C3: BsCR1 and BsC3aR. The former is the receptor of C3b; the latter of the anaphylotoxin C3a. The sequence and the structure of BsC3aR and BsCR1 are highly conserved. Both genes are constitutively transcribed; CR1 and C3aR are transcribed only in haemocytes, the former in circulating phagocytes and the latter in cytotoxic morula cells. . The different expression sites of CR1 and C3aR indicates a similarity with what reported in vertebrates, where C3 can contribute to the activation of phagocytes, and what happen in ascidians, where C3 is produced by morula cells which, through the complement system, can orchestrate a complex dialogue with phagocytes during the immune response.


2015 - Expression study of molecular markers involved in staminality and differentiation in the colonial ascidians Botryllus schlosseri [Abstract in Rivista]
Ballin, F.; Franchi, N.; Ballarin, L.
abstract

All multicellular organisms originate from a small set of totipotent embryonic cells that differentiate into a structured body plan during embryogenesis. The ability to generate an embryo from a single cell and the regenerative capabilities of metazoans suggest the presence of cell types with stem cell attributes. Compound ascidians, like Botryllus schlosseri, offer the opportunities to investigate the biology of both embryonic and adult stem cells thanks to the presence of sexual and asexual reproduction. In B. schlosseri new buds emerge as thickening of the peribranchial epithelium in a process call palleal budding. Sometimes, a vascular budding occurs, with the development of new buds formed by circulating multipotent or pluripotent cells. These two kinds of budding processes render B. schlosseri a good research tool for the study of staminality. In B. schlosseri, during the cyclical generation change, an increase in the number of hemoblasts occurs which will replace, after their differentiation, the circulating cells undergoing apoptotic cell death. Ascidian hematopoiesis occurs in close proximity to the pharyngeal vessels, in the so-called “hematopoietic nodules” and in the endostyle, the cells of which proliferate and migrate to regenerating organs in developing buds. Despite the morphologic suggestions that hemoblasts are the precursors of all the circulating cell types, immunocytes included, there is a general lack of biochemical and molecular data supporting this assumption. Here we report the first results on the characterisation of staminality and differentiation molecular markers such as ABCG2, CD133 and GATA2/3 considered hematopoiesis molecular marker in other deuterostomes.


2015 - Insights on cytotoxic cells of the colonial ascidian Botryllus schlosseri [Articolo su rivista]
Franchi, Nicola; Ballarin, Loriano; Cima, Francesca
abstract

Morula cells (MCs) represent the most abundant circulating hemocyte of the compound ascidian Botryllus schlosseri. They are cytotoxic cells involved in the rejection reaction between contacting, genetically incompatible colonies. Upon the recognition of foreign substances, they degranulate and release their content, which contribute to the cell death along the contact borders. A major role in MC-related cytotoxicity is exerted by the enzyme phenoloxidase (PO) that converts polyphenol substrata to quinones which, then, polymerize to form melanins. During this reaction, reactive oxygen species are formed which are the cause of MC-related cytotoxicity. Here, we carried out new analyses to investigate further the nature of MC content and its role in cytotoxicity. Results confirm that PO is located inside MC vacuoles together with arylsulfatase, iron and polyphenols/quinones, the latter probably representing ready-to-use cytotoxic molecules, deriving from the oxidation of DOPA-containing proteins. In addition, small DOPA-containing peptides, called tunichromes, are also present inside MCs. MC degranulation and PO-mediated cytotoxicity are prevented by secretion inhibitors and by H89 and calphostin C. The observation that PO activity is always detectable in MCs in the absence of protease treatment, and its inhibition by sulfites and sulfates, suggest a non-classical pathway of PO modulation in botryllid ascidians.


2015 - Life history and ecological genetics of the colonial ascidian Botryllus schlosseri [Articolo su rivista]
Cima, Francesca; Ballarin, Loriano; Caicci, Federico; Franchi, Nicola; Gasparini, Fabio; Rigon, F.; Schiavon, F.; Manni, Lucia
abstract

The colonial ascidian Botryllus schlosseri is a cosmopolitan, marine filter feeder, introduced as a laboratory research organism in the 1950s. Currently, it is widely used in many laboratories to investigate a variety of biological questions. Recently, it has become a species of concern, as it is an invasive species in many coastal environments. Here, we review studies on the geographical distribution of the species, sexual and asexual reproduction in the field, tolerance to temperature, salinity and anthropogenic activity, polychromatism, enzymatic polymorphism, and the genetic basis of pigmentation. Studying the relationship between genetic polymorphism and the adaptation of B. schlosseri to environmental stress is a challenge of future research and will improve our understanding of its evolutionary success and invasive potential.


2015 - New data on Botryllus schlosseri AMP: a transcript analysis [Abstract in Rivista]
Franchi, Nicola; Ballarin, Loriano
abstract

The innate immune system provides an immediate response against infections in animals and plants. Endogenous antimicrobial peptides (AMPs) are essential effector molecules of this first line of defence allowing the direct killing of invading micro-organisms. Moreover AMPs have attracted increasing interest because of their potential as new antibiotics. To date various natural peptides from all kinds of organisms and synthetic derivatives have been characterized for their potential use as novel therapeutics. However, the number of candidate peptides undergoing preclinical or clinical evaluation is still very low. Marine invertebrates are source of several AMPs and some of these have been isolated and characterized from different urochordate such as Styela plicata (clavanins and styelins) and Ciona intestinalis (Ci-MAM). In the colonial ascidian B. schlosseri, exploiting the transcriptome and the genome, we have been able to identify a styelin-like AMP. We also carried out some preliminary experiments to investigate the chemical properties and the expression pattern of such gene in the presence of different PAMPs.


2015 - New data on phagocytosis-induced apoptosis in the colonial ascidian Botryllus schlosseri [Abstract in Rivista]
Franchi, N.; Schiavon, F.; Ballarin, L.
abstract

Colonies of the ascidian Botryllus schlosseri undergo cyclical generation change or take-over (TO) during which diffuse apoptosis occurs in zooid tissues, as indicated by morphological, biochemical and molecular investigations. Tissues are rapidly infiltrated by circulating phagocytes, selectively recruited by dying cells, which recognize and greedily ingest them. Previous observations led us to suggest that phagocytes, once ingested apoptotic cells or corpses, undergo phagocytosis-induced apoptosis (PIA). We already demonstrated, by Western blot and immunocytochemical analyses, the release of cytochrome c by hemocytes and the expression of apoptosis-related molecules, such as BAX and caspases, in phagocytes during the TO. In order to corroborate the above assumption, we carried out new morphological analyses at the transmission electron microscope (TEM) and started a molecular analysis of apoptosis in Botryllus phagocytes looking for transcripts differentially expressed in phagocytes at TO. We identified and characterized transcript sequences for BAX, AIF and PARP (poly ADP ribose polymerase), and studied their expression and the location of the corresponding mRNA in hemocytes. The collected data clearly indicate the diffuse occurrence of PIA among phagocytes which guarantee the disposal of apoptotic cells or corpses. In addition, they extend the classical view of PIA, intended as a mechanism for the prevention of microbial diffusion within the organism, and reveal an undescribed role of the process in the control of asexual development and colonial homeostasis.


2015 - SATRAP: SOLiD assembler translation program [Articolo su rivista]
Campagna, Davide; Gasparini, Fabio; Franchi, Nicola; Manni, Lucia; Telatin, Andrea; Vitulo, Nicola; Ballarin, Loriano; Valle, Giorgio
abstract

SOLiD DNA sequences are typically analyzed using a reference genome, while they are not recommended for de novo assembly of genomes or transcriptomes. This is mainly due to the difficulty in translating the SOLiD color-space data into normal base-space sequences. In fact, the nature of color-space is such that any misinterpreted color leads to a chain of further translation errors, producing totally wrong results. Here we describe SATRAP, a computer program designed to efficiently translate de novo assembled color-space sequences into a base-space format. The program was tested and validated using simulated and real transcriptomic data; its modularity allows an easy integration into more complex pipelines, such as Oases for RNA-seq de novo assembly. SATRAP is available at http://satrap.cribi.unipd.it, either as a multi-step pipeline incorporating several tools for RNA-seq assembly or as an individual module for use with the Oases package.


2015 - Sexual and asexual reproduction in the colonial ascidian Botryllus schlosseri [Articolo su rivista]
Gasparini, F.; Manni, L.; Cima, F.; Zaniolo, G.; Burighel, P.; Caicci, F.; Franchi, N.; Schiavon, F.; Rigon, F.; Campagna, D.; Ballarin, L.
abstract

The colonial tunicate Botryllus schlosseri is a widespread filter-feeding ascidian that lives in shallow waters and is easily reared in aquaria. Its peculiar blastogenetic cycle, characterized by the presence of three blastogenetic generations (filtering adults, buds, and budlets) and by recurrent generation changes, has resulted in over 60 years of studies aimed at understanding how sexual and asexual reproduction are coordinated and regulated in the colony. The possibility of using different methodological approaches, from classical genetics to cell transplantation, contributed to the development of this species as a valuable model organism for the study of a variety of biological processes. Here, we review the main studies detailing rearing, staging methods, reproduction and colony growth of this species, emphasizing the asymmetry in sexual and asexual reproduction potential, sexual reproduction in the field and the laboratory, and self- and crossfertilization. These data, opportunely matched with recent tanscriptomic and genomic outcomes, can give a valuable help to the elucidation of some important steps in chordate evolution.


2015 - Suppression of cell-spreading and phagocytic activity on nano-pillared surface: in vitro experiment using hemocytes of the colonial ascidian Botryllus schlosseri [Articolo su rivista]
Ballarin, Loriano; Franchi, Nicola; Gasparini, Fabio; Caicci, Federico; Miyauchi, A.; Hirose, E.
abstract

Nano-scale nipple array on the body surface has been described from various invertebrates including endoparasitic and mesoparasitic copepods, but the functions of the nipple array is not well understood. Using the hydrophilized nanopillar sheets made of polystyrene as a mimetic material of the nipple arrays on the parasites’ body surface, we assayed the cell spreading and phagocytosis of the hemocytes of the colonial ascidian Botryllus schlosseri. On the pillared surface, the number of spreading amebocytes and the number of phagocytizing hemocytes per unit area were always smaller than those on the flat surface (Mann-Whitney test, p &lt; 0.05 - 0.001), probably because the effective area for the cell attachment on the pillared surface is much smaller than the area on the flat sheet. The present results supports the idea that the nipple array on the parasites' body surface reduces the innate immune reaction from the host hemocytes.


2014 - Cellular aspects of allorecognition in the compound ascidian Botrylloides simodensis [Articolo su rivista]
Franchi, N.; Hirose, E.; Ballarin, L.
abstract

When colonies of the compound ascidian Botrylloides simodensis contact each other at their cut surfaces, either fusion or rejection occurs. Contact between genetically compatible colonies leads to the complete fusion of their tunics and vasculature within 24 h. Conversely, the rejection reaction between incompatible colonies is characterized by the appearance of a melanic, necrotic band along the contact border. In the case of fusion, limited crowding of cytotoxic morula cells (MCs) was observed in the ampullae near the contact border. In rejection, limited tunic fusion occurred in the contact region and MCs were selectively recruited inside the ampullae near the cut surface: most of them leaked into the tunic where they changed their morphology and contributed to the formation of the necrotic region. Granular amebocytes, like MCs, have granules well stained by eosin and were also seen inside the ampullae involved in the rejection reaction and along the contact border between incompatible colonies. Immunohistochemical analysis using antibodies raised against Botryllus schlosseri phenoloxidase (PO) and mammalian IL-1-α and TNF-α indicate that MCs were the only cells recognized by the anti-PO antibody; they resulted immunopositive also to the anti-cytokine antibodies in both fusion and rejection, whereas granular amoebocytes were recognized by the latter antibodies only during the rejection reaction


2014 - Characterization and transcription studies of a phytochelatin synthase gene from the solitary tunicate Ciona intestinalis exposed to cadmium [Articolo su rivista]
Franchi, Nicola; Piccinni, Ester; Ferro, Diana; Basso, Giuseppe; Spolaore, Barbara; Santovito, Gianfranco; Ballarin, Loriano
abstract

The major thiol-containing molecules involved in controlling the level of intracellular ROS in eukaryotes, acting as a nonenzymatic detoxification system, are metallothioneins (MTs), glutathione (GSH) and phytochelatins (PCs). Both MTs and GSH are well-known in the animal kingdom. PC was considered a prerogative of the plant kingdom but, in 2001, a phytochelatin synthase (PCS) gene was described in the nematode Caenorhabditis elegans; additional genes encoding this enzyme were later described in the earthworm Eisenia fetida and in the parasitic nematode Schistosoma mansoni but scanty data are available, up to now, for Deuterostomes. Here, we describe the molecular characteristics and transcription pattern, in the presence of Cd, of a PCS gene from the invertebrate chordate Ciona intestinalis, a ubiquitous solitary tunicate and demonstrate the presence of PCs in tissue extracts. We also studied mRNA localization by in situ hybridization. In addition, we analyzed the behavior of hemocytes and tunic cells consequent to Cd exposure as well as the transcription pattern of the Ciona orthologous for proliferating cell nuclear antigen (PCNA), usually considered a proliferation marker, and observed that cell proliferation occurs after 96 h of Cd treatment. This matches the hypothesis of Cd-induced cell proliferation, as already suggested by previous data on the expression of a metallothionein gene in the same animal.


2014 - Evidences for antimicrobial peptides in the colonial ascidian Botryllus schlosseri [Abstract in Rivista]
Schiavon, F.; Franchi, N.; Ballarin, L.
abstract

Invertebrates have acquired many mechanisms of defence in order to overwhelm the risk of pathogen attack. In particular, the presence of various types of antimicrobial peptides (AMP) guarantees an efficient response, making them able to kill both gram positive and negative bacteria, fungi and viruses. In ascidians, AMPs have been isolated and described in different species, such as in Styela clava (Clavanins and Styelins) and in Ciona intestinalis (the recent Ci-MAM). Previous studies have revealed molecules, including phenoloxidase and rhamnose-binding lectins, with an antimicrobial effect also in Botryllus schlosseri. Here, we report that the growth of some bacteria strains is highly inhibited by extracts of haemocytes, showing alterations in their surface. Moreover, molecular analyses allowed us to identify a Botryllus sequence similar to Styelins that is abundantly transcribed in phagocytes.


2014 - In vitro effects of the nonsteroidal anti-inflammatory drug, ibuprofen, on the immune parameters of the colonial ascidian Botryllus schlosseri [Articolo su rivista]
Matozzo, Valerio; Franchi, Nicola; Ballarin, Loriano
abstract

In this study, in vitro effects of ibuprofen (IBU) on the immune parameters of the colonial ascidian Botryllus schlosseri were evaluated. Haemocytes were exposed for 1 h to 0 (control), 100 and 1000 µg IBU/L and the effects on haemocyte viability and morphology (shape factor), lysosomal membrane stability (Neutral Red retention assay), phagocytic activity, apoptosis (TUNEL reaction), hydrolytic (acid phosphatase) and oxidative (phenoloxidase and peroxidase) enzyme activities were evaluated. The exposure of haemocytes to IBU did not affect significantly their viability, but increased the percentage of cells with round shape. IBU caused a significant reduction in both phagocytic activity and lysosomal membrane stability. The percentage of haemocytes positive to TUNEL reaction (indicative of DNA fragmentation) increased significantly after IBU exposure. Significant decreases in the percentage of haemocytes positive to acid phosphatase were recorded at 1000 µg/L of IBU. Conversely, no significant variations were recorded in the percentage of haemocytes positive to phenoloxidase and peroxidase. Results obtained indicate that exposure of ascidian haemocytes to IBU induces marked alterations in cell functionality. Immunomarkers measured in this study are sensitive, rapid and reproducible. However, their responsiveness and biological relevance will need to be verified for in vivo exposure.


2014 - Lectins and immunity in compound ascidians [Abstract in Atti di Convegno]
Franchi, N.; Schiavon, F.; Ballarin, L.
abstract

Lectins are proteins able to recognize and bind specific glycoconjugates, widely distributed among plants and animals. Most of them have agglutinating activity towards vertebrate erythrocytes and other animal cells, due to the presence of multiple carbohydrate recognition domains which bind to cell surface sugars. A great number of invertebrate lectins have been described in the last two decades: they show different specificities, sizes and physico-chemical properties and are believed to be involved in various processes, such as cell-cell interaction, fertilisation, morphogenesis and defence reactions. Ascidians are invertebrate chordates phylogenetically close to vertebrates and the study of their immune responses can contribute to a better understanding of the complex immune system of vertebrates. In compound ascidians, lectins play an important role in opsonisation of foreign particles or cells having entered the organism. They can also induce cell proliferation and enhance the recruitment of immunocytes to the infection area. In the compound ascidian Botryllus schlosseri, our model organism, we recently identified a rhamnose-binding lectin (BsRBL)which can recruit phagocytes, activate their respiratory burst with the consequent production of microbicidal reactive-oxygen species, and stimulate phagocytosis of foreign target cell by opsonising them and inducing cytoskeletal changes in phagocytes. In addition, BsRBL induces the synthesis and release, by cytotoxic morula cells, of cytokines recognised by anti-IL-1 and anti-TNF antibodies, with chemotactic activity towards cytotoxic immunocytes. It also triggers the degranulation of morula cells with the consequent release of the cytotoxic enzyme phenoloxidase. Results suggest an important role of BsRBL in Botryllus immunobiology and support the existence of a cross-talk between B. schlosseri immunocytes.


2014 - New evidences of conserved pathways in complement system dynamics from the colonial ascidian Botryllus schlosseri. [Abstract in Rivista]
Franchi, N.; Ballarin, L.
abstract

In recent years, it has been widely demonstrated that complement, although often depicted as a ‘first line of defense’, is more than just a defender against microbial intruders and acts as a tightly integrated surveillance system. It is not only important against microorganisms, but also for the clearance of apoptotic cells and corpses. Botryllus schlosseri belongs, as vertebrates, to the phylum Chordata and, for this phylogenetic trait, it is unanimously considered a reliable model organism for the studies of the evolution of the immune system. Moreover it is also characterized by a peculiar life cycle with a cyclical, massive apoptosis. This two key features render B. schlosseri a good research tool for the study of the evolution of the complement system. Here we report the first results on the expression of BsC3 and BsFactorB, both components of the alternative pathway (AP) of complement activation, which form the AP C3 convertase. Since studies on mammalian models have shown that 80 % of the observed complement response is derived from AP convertase-mediated C3 amplification, even if initially induced by the classical pathway (CP), studies of complement activation in an organism that lack the adaptive immunity, as B. schlosseri, could lead to a better comprehension of the AP cascade and the behavior of C3 convertase not only in invertebrates, but also in vertebrates, mammals included. As in mammals, BsC3 is highly transcribed at basal level and over-expressed after incubation with non self (zymosan) while BsFactorB always shows limited expression. In the presence of compstatin, a 13-residue cyclic peptide able to inhibit the activation of C3 by C3 convertases, the percentage of phagocytosing hemocytes collapses. In the presence of both zymosan and compstatin, the transcription of BsC3 by hemocytes increases with respect to cells exposed only to zymosan: this suggests the presence of a conserved molecular machinery able to control and modulate B. schlosseri as well as the mammalian complement.


2014 - Phagocytosis-induced apoptosis in the compound ascidian Botryllus schlosseri [Abstract in Rivista]
Schiavon, Filippo; Franchi, Nicola; Ballarin, Loriano
abstract

Colonies of the ascidian Botryllus schlosseri contain three blastogenetic generations: functional zooids, their buds and budlets on buds. Generation change or take-over (TO) occur cyclically and assure the recurrent renewal of the colony. During these events, lasting 24-36 h, diffuse apoptosis occurs in zooid tissues, as indicated by morphological, biochemical and molecular investigations. Tissues are rapidly infiltrated by circulating phagocytes, selectively recruited by dying cells, which recognize and greedily ingest them. Using hemocytes as selected cell population for investigation, we studied the transcription rate of three recently characterized genes involved in apoptosis, bsbax, bsaif1 and bsparp1, during the TO as compared to colony developmental phases far from it. In addition, we observed that the massive ingestion leads phagocytes themselves to undergo apoptosis, probably as a consequence of the oxidative stress related to the sustained respiratory burst, as suggested by biochemical analysis. Therefore, a large fraction of circulating phagocytes needs to be replaced by new, young hemocytes, entering the circulation at the end of the generation change.


2014 - Preliminary characterization of complement in a colonial tunicate: C3, Bf and inhibition of C3 opsonic activity by compstatin [Articolo su rivista]
Franchi, Nicola; Ballarin, Loriano
abstract

The complement system is a fundamental effector mechanism of the innate immunity in both vertebrates and invertebrates. The comprehension of its roots in the evolution is a useful step 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. Data on 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 invertebrate-vertebrate transition. Here we report on the characterization of C3 and Bf transcripts from the colonial ascidian Botryllus schlosseri (BsC3 and BsBf, respectively), a reliable model organism for immunobiological research, and present a comparative analysis of amino acid sequences of C3s and Bfs suggesting that, in deuterostomes, the structure of these proteins remained largely unchanged. We also present new data on the cells responsible of the expression of BsC3 and BsBf showing that cytotoxic immunocytes are the sole cells where the relative transcripts can be found. Finally, using the C3 specific inhibitor compstatin, we demonstrate the opsonic role of BsC3 in accordance with the idea that promotion of phagocytosis is one of the main function of C3 in metazoans.


2014 - The production of amyloid requires cross-talk between immunocytes in the compound ascidian Botryllus schlosseri [Abstract in Rivista]
Ballarin, L.; Girardello, R.; Grimaldi, A.; Franchi, N.; De Eguileor, M.
abstract

Two main immunocyte types are present in the hemolymph of the compound ascidian Botryllus schlosseri, i.e. phagocytes and cytotoxic morula cells (MCs). Previous studies have demonstrated that MCs work as sentinel cells able to sense foreign molecules and, as a consequence, release cytokines which activate phagocytes leading to the synthesis and release of rhamnose-binding lectin. The latter, in turn, acts as a chemotactic factor for phagocytes and opsonizes foreign particles stimulating their clearance. In addition, upon the contact with foreign molecules, MCs can degranulate and release the content of their vacuoles, mainly phenoloxidase (PO) and its polyphenol substrata. In a recent investigation on Botryllus cytotoxic cells, we found abundance of amyloid inside MC vacuoles which likely, once released, act as a scaffold to prevent the diffusion of PO and cytotoxicity to the whole organism. In addition, the study of the molecular cascade leading to the synthesis of amyloid, is revealing a non-classical pathway in which both the phagocytes and MC are involved.


2013 - A novel platform for gene expression study [Abstract in Atti di Convegno]
Campagna, D.; Franchi, N.; Gasparini, F.; Manni, L.; Valle, G.; Ballarin, L.
abstract


2013 - A phytochelatin synthase gene in the solitary tunicate Ciona intestinalis [Abstract in Atti di Convegno]
Franchi, N.; Ferro, D.; Spolaore, B.; Santovito, G.; Ballarin, L.
abstract

The major thiol-containing molecules involved in controlling the level of intracellular ROS in eukaryotes, acting as a nonenzymatic detoxification system, are metallothioneins (MTs), glutathione (GSH) and phytochelatins (PCs). Both MTs and GSH are well-known in the animal kingdom. PC was considered a prerogative of the plant kingdom but, in 2001, a phytochelatin synthase (PCS) gene was described in the nematode Caenorhabditis elegans; additional genes encoding this enzyme were later described in the earthworm Eisenia fetida and in the parasitic nematode Schistosoma mansoni but scanty data are available, up to now, for deuterostomes. Here, we describe the molecular characterisation and transcription pattern, in the presence of Cd, of a PCS gene from the invertebrate chordate Ciona intestinalis, a ubiquitous solitary tunicate and demonstrate the presence of PCs in tissue extracts. We also studied mRNA localization by in situ hybridization. In addition, we analysed the behaviour of hemocytes and tunic cells consequent to Cd exposure as well as the transcription pattern of the Ciona orthologous for PCNA, usually considered a proliferation marker, and observed that cell proliferation occurs after 96 h of Cd treatment. This matches the hypothesis of Cd-induced cell proliferation, as already suggested by our previous data on the expression of a metallothionein gene in the same animal.


2013 - Characterization and metal-induced gene transcription of two new copper zinc superoxide dismutases in the solitary ascidian Ciona intestinalis [Articolo su rivista]
Ferro, Diana; Franchi, Nicola; Valentina, Mangano; Rigers, Bakiu; Matteo, Cammarata; Nicolò, Parrinello; Santovito, Gianfranco; Ballarin, Loriano
abstract

Antioxidant enzymes are known to protect living organisms against the oxidative stress risk, also induced by metals. In the present study, we describe the purification and molecular characterization of two Cu,Zn superoxide dismutases (SODs), referred to as Ci-SODa and Ci-SODb, from Ciona intestinalis, a basal chordate widely distributed in temperate shallow seawater. The putative amino acid sequences were compared with Cu,Zn SODs from other metazoans and phylogenetic analyses indicate that the two putative Ci-SODs are more related to invertebrate SODs than vertebrate ones. Both phylogenetic and preliminary homology modeling analyses suggest that Ci-SODa and Ci-SODb are extracellular and intracellular isoform, respectively. The mRNA of the two Cu,Zn SODs was localized in hemocytes and in ovarian follicular cells, as revealed by in situ hybridization. The time course of SOD mRNA levels in the presence of three different metals showed upregulation of ci-soda and inhibition of ci-sodb. Spectrophotometric analysis confirms the presence of SOD activity in Ciona tissues. Our in silico analyses of the ci-soda promoter region revealed putative consensus sequences similar to mammalian metal-responsive elements (MRE), suggesting that the transcription of these genes directly depends on metals. These data emphasize the importance of complex metal regulation of ci-soda and ci-sodb transcription, as components of an efficient detoxification pathway allowing the survival of C. intestinalis in continued, elevated presence of metals in the environment.


2013 - Cloning and expression of glutathione peroxidase genes in the chordate invertebrate Ciona intestinalis. [Abstract in Rivista]
Ferro, D.; Bakiu, R.; Franchi, N.; Ballarin, L.; Santovito, G.
abstract

Ascidians represent interesting models from an evolutionary and ecotoxicology point of view, because of their large distribution in temperate sea and their phylogenetic position of invertebrate chordates. Immune responses imply an increase in oxygen consumption with a consequent risk of oxidative stress. With the aim to study the components of the antioxidant defense system in the solitary ascidian Ciona intestinalis, we have characterized the genes codifying for two glutathione peroxidases (GPx), metalloenzymes that catalyze the reduction of hydrogen peroxide or organic hydroperoxides to water or corresponding alcohols, using reduced glutathione (GSH) as an electron donor. In the GeneBank database five GPx-like sequences from C. intestinalis are present, but only two of those genes demonstrated an effective transcription after cloning and sequencing analyses. The respective proteins, named Ci-GPx7 and Ci-GPxb, show a good level of sequence conservation with metazoan orthologs, especially for residues that are important for the catalytic activity. In the 3’-UTR region of Ci-GPxb cDNA we have found a typical SECIS-element, confirming that this protein may be a selenium GPx. Phylogenetic reconstruction, performed with Bayesian methods using metazoan GPxs, indicate that Ci-GPxb emerges in the tree with the tetrameric GPxs, confirming as previously hypothesized. Preliminary data, obtained by homology modeling, confirmed the tetrameric structure and show that the GPxb is similar to GPx3 from Homo sapiens, a selenium protein. Thus, we propose to name this gene ci-gpx3. As expected, Ci-GPx7 clusterized with other GPx7s. The transcription of both these genes, measured by RT-sqPCR, resulted inducible by Cd, Cu and Zn, which have different effects. The peroxidase activity decreases in the cell-free extract from specimens treated with each considered metals, probably in relation to metal-induction of GSH biosynthesis, as indicated by the presence of positive correlation by the time-dependent Cd accumulation an increase of ROS and GSH production. The data presented here improved our knowledge about the evolution of the antioxidant system in metazoans and emphasize the importance of a complex regulation for the antioxidant system, including the transcription of ci-gpx7 and ci-gpx3 genes, which can create an efficient detoxification pathway allowing C. intestinalis to survive in the presence of metals in the environment.


2013 - Evolution of the complement system: ancient molecules and new evidences from tunicates [Relazione in Atti di Convegno]
Franchi, N.; Ballarin, L.; Parrinello, N.
abstract

The complement system is a key player in innate immunity but, recently, it is becoming even more evident that complement plays also important roles in adaptive immunity. Components of the human complement system possess unique domain structures and are classified in protein families: C3, factor B (Bf), mannan-binding protein-associated serine protease (MASP), C6 and factor I (If) family. These complement families probably derive from exon shuffling, which created the unique domain structures of each family, and gene duplication and subsequent functional divergence, which increased the number of members in each family. Accumulating information on the complement system of vertebrates indicates that these gene duplications, which played a pivotal role in establishing the classical and the lytic pathway, occurred in jawed vertebrates. In contrast, information on complement genes of invertebrate chordates is limited and, so far, only the ascidians, such as Ciona intestinalis and Halocinthia roretzi, and the cephalochordate Branchiostoma floridae have an almost complete set of the complement gene families: C3-, Bf-, MASP-, and C6-like genes. In the present work we demonstrate the presence of C6-, C3-, MASP-, MBL- and Bf-like genes in the colonial ascidian Botryllus schlosseri and the enhanced transcription after zymosan infection, which indicates their involvement in the ascidian immunity. The observation of a transcribed C6 gene suggest that the presence of the lytic pathway predates the appearance of the vertebrates. We are now carrying out new investigation to demonstrate a complement-related lytic activity in B. schlosseri.


2013 - Influence of cadmium on the morphology and functionality of haemocytes in the compound ascidian Botryllus schlosseri [Articolo su rivista]
Franchi, Nicola; Ballarin, Loriano
abstract

In order to get insights into the effects of cadmium (Cd) on cell morphology and functions, we exposed haemocytes of the colonial ascidian Botryllus schlosseri to sub-lethal concentrations of CdCl2. Results indicate that Cd hampers haemocyte spreading and phagocytosis in a dose-dependent way, through the alteration of the actin cytoskeleton. In addition, the metal decreases the stability of the internal membranes, as revealed by the Neutral Red assay. The fraction of cells showing positivity for the lysosomal enzyme acid phosphatase is also reduced in the presence of Cd, whereas the number of cells responsive to the Annexin-V assay and showing chromatin condensation increases, suggesting a metal-dependent induction of apoptosis in exposed cells. As Cd is a known cause of oxidative stress, the decrease in the percentage of cells positive to the assay for superoxide anion, observed at low Cd concentrations, is indicative of the synthesis of metal-chelating molecules, such as metallothioneins, whereas, the increase at high Cd concentrations suggests a depletion of the cell reducing redox potential.


2013 - Insight on signal transduction pathways involved in phagocytosis in the colonial ascidian Botryllus schlosseri [Articolo su rivista]
Franchi, Nicola; Schiavon, Filippo; Michele, Betti; Laura, Canesi; Ballarin, Loriano
abstract

Tunicates are chordate invertebrates, closely related to vertebrates, which represent valuable organisms for the study of a variety of biological processes from an evolutionary point of view. As invertebrates, they rely on innate immunity to cope with foreign, potentially pathogenic material. Among tunicates, the compound ascidian Botryllus schlosseri is emerging as a reliable model organism for the study of innate immune responses. However, there is a general lack of knowledge on the signalling pathways activated during immune responses and, in particular, in phagocytosis. In the present work, we carried out a preliminary investigation of the signalling pathways involved in phagocytosis, with particular reference to MAPK activation. We studied in vitro zymosan phagocytosis in the presence of manumycin A, which inhibit the activation of Ras, PD98059, SP600125 and SB202190, inhibitors of Erk, JNK and p38, respectively, parthenolide, N-acetyl-L-cysteine (NAC) and pyrrolidine dithiocarbamate (PDTC), inhibiting NF-kB activation. In addition we carried out immunoblot and immunocytochemistry analysis with the use of anti-pErk1/2, anti-pp38, anti-pJNK, anti-NF-kB (p50) and antipan Ras antibodies. Results demonstrate that the recognition of foreign cells triggers a phosphorylation cascade leading to the activation of Ras-like small GTPases, MAPKs and NF-kB and argue in favour of a conservation, also in ascidians, of the main signalling pathways.


2013 - Phenoloxidase, melanin and amyloid fibrils in the compound ascidian Botryllus schlosseri [Abstract in Atti di Convegno]
Girardello, R; Tasselli, S; de Eguileor, M; Franchi, N; Ballarin, L
abstract

In invertebrates, the recognition of non-self molecules frequently leads to the release of phenoloxidase (PO) by circulating granulocytes, in the form of its precursor, pro-PO, which is quickly activated by soluble serine proteases. This response represents a powerful weapon against potentially pathogenic microorganisms and, in several taxa, independently of their phylogenetic position, the pro-PO activation system is responsible of a massive production of melanin. The latter is supported by the formation of amyloid fibrils as well as other concurrent events, such as ACTH production, NEP overexpression and α-MSH formation. In the colonial ascidian Botryllus schlosseri, allorecognition between contacting, genetically incompatible colonies, leads to the recruitment of haemocytes in the contact region and their degranulation with the consequent release of PO and the production of melanin which is deposited in the necrotic spots along the colony contacting border.


2013 - Phenoloxidases and cytotoxicity in ascidians: an overview [Abstract in Atti di Convegno]
Ballarin, L.; Franchi, N.; Schiavon, F.; Tosatto, S. C. E.
abstract

Phenoloxidases (POs) belong to a family of copper-containing proteins (including also hemocyanins) widely distributed among invertebrates. They are able to convert polyphenols to quinones and induce cytotoxicity through the production of reactive oxygen species, a fundamental event in many immune responses. In ascidians, PO activity has been described and studied in both solitary and colonial species and the enzyme is involved in inflammatory and cytotoxic reactions against foreign cells or molecules, and in the formation of the cytotoxic foci which characterize the nonfusion reaction of botryllid ascidians. Expressed genes for putative POs have been recently identified in the solitary ascidian C. intestinalis (CiPO1 and CiPO2) and the compound ascidian Botryllus schlosseri. Multiple sequence alignments evidenced the similarity between the sequences of ascidian POs and crustacean proPOs whereas the analysis of the three-dimensional structure reveals high similarity with arthropod haemocyanins, which share common precursors with arthropod proPOs. Botryllus and Ciona POs grouped in the same cluster, and all of them share the full conservation of the six histidines at the two copper-binding sites as well as of other motifs, also found in arthropod haemocyanin subunits, involved in the regulation of enzyme activity. In situ hybridisation indicated that the Botryllus PO is transcribed inside morula cells, a characteristic cytotoxic haemocyte type.


2013 - Putative rhamnose-binding lectin in the solitary ascidian Ciona intestinalis. [Abstract in Rivista]
Franchi, N.; Sanfratello, M. A.; Parrinello, D.; Vazzana, M.; Di Bella, M. L.; Cammarata, M.; Ballarin, L.; Parrinello, N.
abstract

Lectins are sugar-binding proteins involved in cell-cell interaction and the recognition of carbohydrate-containing molecules. They act as humoral factors in non-self recognition, are a key component of the innate immune systems of many metazoans and are involved in phagocyte activation through their opsonizing activity. In recent years, a new lectin family, the rhamnose-binding lectin (RBL) family, has been described and its members can modulate the inflammatory response in fish (Watanabe et al., 2009) as well as in various invertebrates, including the colonial tunicate Botryllus schlosseri. Recently, we succeeded in purifying, by affinity chromatography using a rhamnose column, a putative RBL, from the hemolymph of the solitary ascidian Ciona intestinalis. The molecule is Ca2+-independent and promptly (within 4h) inducible, after LPS inoculation. The eluted fractions, when examined by 15 % SDS-PAGE under reducing condition, showed four bands with apparent molecular masses of 65, 54, 30 and 19 kDa. The agglutinating activity of the isolated fraction was demonstrated using trypsinized rabbit erythrocytes and it was inhibited by glycosides such as rhamnose, galactose and lactose. Moreover, by immunocytochemical analysis using antibodies produced against B. schlosseri RBL and in situ hybridization with a riboprobe for the annotated C. intestinalis RBL, we have observed a positive signal in hyaline and granular amoebocytes and in the endothelium of the pharynx vessels.


2013 - Routes in Innate Immunity Evolution: Galectins and Rhamnose-binding Lectins in AscidiansMarine Proteins and Peptides [Capitolo/Saggio]
Ballarin, Loriano; Matteo, Cammarata; Franchi, Nicola; Nicolò, Parrinello
abstract

The term ‘lectin’ is commonly used to encompass a wide variety of carbohydrate-binding proteins, widely distributed in viruses, prokaryotes and eucaryotes (Vasta &amp; Ahmed, 2008). The first animal lectins were isolated by Noguchi in early 1900 from Limulus polyphemus and Homarus americanus; many years later, Watkins &amp; Morgan (1952) proposed a sugar-specific binding (l-fucose) property for the eel lectin. Animal lectins are grouped in various molecular families, differing in carbohydrate-recognition domain (CRD) structure and organization (Gabius, 1997; Kilpatrick, 2002; Loris, 2002; Vasta et al., 2004). They are involved in a variety of key biological processes, ranging from development (Kaltner &amp; Stierstorfer, 1998; Kilpatrick, 2002) to immune responses (Arason, 1996; Vasta et al., 1994). Protein–carbohydrate interactions are the basis of a mechanism for signaling functions, cell communication and self–non-self recognitions and are critical in the establishment and maintenance of highly specific mutualistic associations in organism–microbe complexes (Sharon &amp; Lis, 1993). In this respect, mutual benefit (symbiosis or commensalism) depends on the maintenance of a tightly regulated balance, whereas colonization of tissues beneficial to the microbe can lead to the loss of host fitness (pathogenesis), unless host-defense responses are able to eliminate the foreignness (Casadevall &amp; Pirofski, 2000). Microheterogeneity, originating from multiple lectin gene copies, allelic variation or post-translational modifications of the gene products, expands the molecular diversity and recognition capabilities. The molecular repertoire may provide a broad non-self-recognition capacity for an efficient innate immune recognition system based on recognition of carbohydrate moieties. Galectins, rhamnose-binding lectins, C-type lectins, fucolectins, P-type lectins and L-type lectins are some examples of animal lectins (Kilpatrick, 2002; L´opez et al., 2011; Shirai et al., 2009).


2012 - BOTRYLLUS SCHLOSSERI BLASTOGENETIC CYCLE: GENE EXPRESSION ANALYSIS USING SOLID SEQUENCING TECHNOLOGY [Abstract in Atti di Convegno]
Gasparini, Fabio; Campagna, Davide; Franchi, Nicola; Valle, Giorgio; Manni, Lucia; Ballarin, Loriano
abstract

BOTRYLLUS SCHLOSSERI BLASTOGENETIC CYCLE: GENE EXPRESSION ANALYSIS USING SOLID SEQUENCING TECHNOLOGY Fabio Gasparini, Davide Campagna, Nicola Franchi, Giorgio Valle, Lucia Manni, Loriano Ballarin Diparlimento di Biologia, Universita degli Studi di Padova. fabio.gasparini@unipd.it Colonies of the cosmopolitan ascidian Botryllus schlosseri are formed by zooids arranged in star-shaped systems, of 6-12 individuals around a common cloacal opening. They undergo cyclical generation change or take-over, during which tissues of adult zooids undergo massive apoptosis, are progressively resorbed and replaced by their buds which reach functional maturity and start filtering. The interest towards the Botryllus blastogenetic cycle has recently increased as it represents a reliable model for the study of natural apoptosis during the generation change and asexual reproduction by continuous budding. The recent development of high-throughput sequencing technologies allow the expanding of the range of the studied organisms and was applied to many model organisms for the discovery of splice variants, RNA editing sites and new microRNA. The production of a de novo transcriptome assembly is often the preferred method for the study of non-model organisms, since it is cheaper and easier than constructing a genome. We have sequenced several SOLID RNA-seq libraries coming from 3 stages of the colonial blastogenetic cycle: i) the phase immediately preceding the take-over, when the colony is preparing to the generation change; ii) the take-over, when adult zooids are resorbed and replaced by new ones, and iii), the mid-cycle phase, when buds and zooids coexist together and no generation change occurs. RNA-seq data has allowed the production of a de nova transcriptome assembly for B. schlosseri and all information has been included in an ad hoc database which interacts with a Web based interface. As resulting by gene expression analysis we have found several differentially expressed genes putatively responsible of the morphological and biochemical changes during blastogenesis. This result opens new research perspectives for the study of the interplay between death and life during the generation change as well as the regulation of asexual reproduction in B. schlosseri.


2012 - Looking for putative phenoloxidases of compound ascidians: haemocyanin-like proteins in Polyandrocarpa misakiensis and Botryllus schlosseri [Articolo su rivista]
Ballarin, Loriano; Franchi, Nicola; Schiavon, Filippo; Tosatto, Silvio C. E.; Micetic, Ivan; Kawamura, Kazuo
abstract

Phenoloxidases (POs) constitute a family of copper-containing enzymes widely distributed among invertebrates. They exert a pivotal role in immune defences as they induce cytotoxicity through the conversion of phenols to quinones and the production of reactive oxygen species. In ascidians, PO activity has been described and studied in both solitary and colonial species and the enzyme is involved in inflammatory and cytotoxic reactions against foreign cells or molecules, and in the formation of the cytotoxic foci which characterise the nonfusion reaction of botryllids. Expressed genes for two putative POs (CiPO1 and CiPO2) have been recently identified in C. intestinalis. In the present study, we determined the cDNA sequences of the POs from two colonial ascidians: Botryllus schlosseri from Mediterranean Sea and Polyandrocarpa misakiensis from Japan. Multiple sequence alignments evidenced the similarity between ascidian POs and crustacean proPOs whereas the analysis of the three-dimensional structure reveals high similarity with arthropod haemocyanins which share common precursors with arthropod proPOs. Ascidian POs and arthropod proPOs grouped in the same cluster and share the full conservation of the six histidines at the two copper-binding sites as well as of other motifs, also found in arthropod haemocyanin subunits, involved in the regulation of enzyme activity. Cytoenzymatic studies and in situ hybridisation (ISH) indicated that the genes are transcribed inside morula cells (MCs), a characteristic haemocyte type in ascidians, at the beginning of their differentiation. Sequence analysis allowed a better understanding of previous biochemical data and suggest some hypotheses for the regulation of enzyme activity.


2012 - Molecular studies on phenoloxidases of compound ascidians [Relazione in Atti di Convegno]
Ballarin, Loriano; Franchi, Nicola; Schiavon, Filippo; Tosatto, Silvio; Mičetić, I.; Kawamura, K.
abstract

Phenoloxidases (POs) constitute a family of copper-containing enzymes with orthodiphenoloxidase (catecholase) activity widely distributed among invertebrates. They exert a pivotal role in immune defences as they can induce cytotoxicity through the conversion of phenols to species. In ascidians, PO activity has been described and studied in both solitary and colonial species and the enzyme is involved in inflammatory and cytotoxic reactions against foreign cells or molecules as well as in the formation of the cytotoxic foci along the contacting edges of genetically incompatible colonies which characterises the nonfusion reaction of botryllids. Expressed genes for two putative POs (CiPO1 and CiPO2) have been identified in C. intestinalis (Immesberger and Burmester, 2004). In the present study, we determined the cDNA sequences of the POs from two colonial ascidians: Botryllus schlosseri from Mediterranean (Adriatic) Sea and Polyandrocarpa misakiensis from Japan. Multiple sequence alignments clearly evidenced the similarity between ascidian PO and crustacean proPOs whereas the analysis of the three-dimensional structure of compound ascidian POs reveal high similarity with arthropod haemocyanins which share common precursors with proPOs. Ascidian POs and arthropod proPOs grouped in the same cluster well separated from mollusc tyrosinases, and share the full conservation of the six histidines at the two copper-binding sites as well as of other motifs, also found in arthropod haemocyanins, involved in the regulation of enzyme activity. Cytoenzymatic studies and in situ hybridisation (ISH) indicated that the genes are transcribed inside morula cells (MCs), a characteristic haemocyte type in ascidians, at the beginning of their differentiation. Sequence analysis allowed a better understanding of previous biochemical data and suggest some hypothesis for the regulation of enzyme activity.


2012 - Preliminary studies of the complement system in Botryllus schlosseri [Relazione in Atti di Convegno]
Franchi, N.; Ballarin, L.
abstract

The complement system represents an important humoral component of the mammalian immune system. Complement components can be subdivided in 5 gene families: C3/C4/C5, Bf/C2, MASP/C1r-s, C6/C7/C8A/C8B/C9 and Factor I. Until 1884, it was generally believed that the complement system was an unique feature of vertebrates since all attempts to identify complement components in invertebrates failed. In recent years, the genomic approach revealed the presence of complement orthologue genes in invertebrate deuterostomes, mainly in sea urchins and tunicates (ascidians). Conversely, no complement genes were found in the genome of protostomes such as Drosophila melanogaster and Caenorhabditis elegans, suggesting that the complement system was established in the deuterostome lineage. Genome analyses carried out in the solitary ascidian Ciona intestinalis revealed that most complement gene families are present in urochordates. We recently carried out the assembling of EST collections from the colonial ascidian B. schlosseri, obtained in our and other laboratories: we found multiple transcripts showing high similarity with vertebrate complement components such as C3, MASP, MBL and C6. Preliminary in silico studies revealed close relationships between Botryllus C3, MASP and MBL and orthologues from other chordates. In particular, C6 seems related with the and Halocynthia roretzi and share with them the absence of the FIM domain which is responsible for the interaction with the other complement molecules in vertebrates. Future studies will be devoted to the analysis of the expression of genes for complement components of B. schlosseri.


2012 - Transcription of genes involved in glutathione biosynthesis in the solitary tunicate Ciona intestinalis exposed to metals [Articolo su rivista]
Franchi, Nicola; Ferro, Diana; Ballarin, Loriano; Santovito, Gianfranco
abstract

Exposure to metals is known to generate oxidative stress risk in living organisms, which are able to respond with the induction of antioxidant defenses, both enzymatic and non-enzymatic. Glutathione (GSH) is considered to be an important cellular component involved in protecting cells, both as metal chelating agent and oxygen radical scavenger. In this work we used molecular techniques to analyze the nucleotide and predicted amino acid sequences of genes involved in GSH biosynthesis, γ-glutamylcysteine ligase catalytic subunit (ci-gclc), γ-glutamyl-cysteine ligase modifier subunit (ci-gclm) and GSH synthase (ci-gs) in the solitary tunicate Ciona intestinalis. We also studied the transcription of the above genes after in vivo exposure to Cd, Cu and Zn by sq RT-PCR, to improve our knowledge on the relationship between metal-induced oxidative stress and GSH production and locate mRNA expression by in situ hybridization (ISH). These genes exhibit a good level of sequence conservation with metazoan homologs, especially for residues important for the activity of the enzymes. Phylogenetic analyses indicate that the three enzymes evolved in different ways, Ci-GCLC and Ci-GS being mostly correlated with invertebrate proteins, Ci-GCLM resulting as sister group of vertebrate GCLMs. Our in silico analyses of the ci-gs and ci-gclc promoter regions revealed putative consensus sequences similar to mammalian metal-responsive elements (MRE) and antioxidant response elements (ARE), indicating that the transcription of these genes may directly depend on metals and/or reactive oxygen species. Results highlighted a statistically significant increase in gene transcription, demonstrating that metal treatments have inducible effects on these genes. They can modulate gene transcription not only through MREs but also through AREs, as a consequence of metal-dependent ROS formation. The ISH location of Ci-GS and Ci-GCLC mRNAs shows that the cells most involved in glutathione biosynthesis are circulating hemocytes. The data presented here emphasize the importance of complex metal regulation of ci-gclc, cigclm and ci-gs transcription, which can create an efficient detoxification pathway allowing C. intestinalis to survive in continued elevated presence of metals in the environment.


2011 - Botryllus schlosseri blastogenetic cicle: gene expression analysis using solid sequencing technology [Relazione in Atti di Convegno]
Campagna, D.; Gasparini, F.; Franchi, N.; Valle, G.; Ballarin, L.
abstract


2011 - CiMT-1, an unusual chordate metallothionein gene in Ciona intestinalis genome: structure and expression studies [Articolo su rivista]
Franchi, Nicola; Boldrin, Francesco; Ballarin, Loriano; Piccinni, E.
abstract

The present article reports on the characterization of the urochordate metallothionein (MT) gene, CiMT-1, from the solitary ascidian Ciona intestinalis. The predicted protein is shorter than other known deuterostome MTs, having only 39 amino acids. The gene has the same tripartite structure as vertebrate MTs, with some features resembling those of echinoderm MTs. The promoter region shows the canonical cis-acting elements recognized by transcription factors that respond to metal, ROS, and cytokines. Unusual sequences, described in fish and echinoderms, are also present. In situ hybridization suggests that only a population of hemocytes involved in immune responses, i.e. granular amebocytes, express CiMT-1 mRNA. These observations support the idea that urochordates perform detoxification through hemocytes, and that MTs may play important roles in inflammatory humoral responses in tunicates. The reported data offer new clues for better understanding the evolution of these multivalent proteins from non-vertebrate to vertebrate chordates and reinforce their functions in detoxification and immunity.


2011 - Development of genotoxicity and immunotoxicity assays for the evaluation of nanoparticles in colonial ascidians and bivalve molluscs [Relazione in Atti di Convegno]
Franchi, N.; Domeneghetti, S.; Vecchio, L.; Libralato, G.; Sabbioni, E.; Ballarin, L.; Venier, P.
abstract

A variety of nanoparticles (NP) and nanomaterials are currently in use or under development in our society. Peculiar properties make them attractive for a wide range of applications; however, a large fraction of NPs still lack of adequate toxicological characterization, i.e. basic data for risk assessment. The European Center for the Sustainable Impact of Nanotechnology (ECSIN) was created to study the impact of innovative nanotechnologies on the environmental and human health, and to evaluate related ethical and social aspects. According to a problem-solving approach, and also in collaboration with University research groups, the advanced equipment and expertise present in ECSIN currently support specific nanotoxicology projects. In the frame of the Operative programme 2007-2013 (ESF, Human Capital), the Veneto Region is now supporting training and research activities concerning the toxicological characterization of NPs in two invertebrate organisms, colonial ascidians and bivalve molluscs, selected on the basis of ecological relevance and available knowledge. Hence, we have started to study the in vitro effects of metal NPs on hemocytes of Botryllus schlosseri and Mytilus galloprovincialis (1, 2). In addition to cytotoxicity measures, end-points informative of oxidative events and immune-related functions will be taken into account. This study will possibly contribute to the first-level characterization of commercial NPs, with particular attention to the induction of genetic damage and changes in the innate immune responses.


2011 - Hemocyte proliferation influences metallothionein and phytochelatin levels in Ciona intestinalis. [Relazione in Atti di Convegno]
Franchi, N.; Piccinni, E.; Ballarin, L.
abstract

We studied, through semiquantitative PCR and in-situ hybridization, the activities of the newly identified metallothionein and phytochelatin synthase genes from the solitary ascidian Ciona intestinalis (Ci-MT and Ci-PCS, respectively) in response to 10 μM CdCl2. Metallothioneins (MTs) and phytochelatins (PCs) are involved in detoxification systems of many organisms. An appreciable number of data are available for metazoan, especially for vertebrate, but very few data are available for urochordates. As the latter occupy the peculiar phylogenetic position of invertebrate chordates, the research on MTs and PCs in tunicates assumes a particular significance. Cd strongly induced Ci-MT, with a maximum at 4 days. Ci-PCS showed maximum expression at the same time. This result is probably related to a cell proliferation event, rather than an effective Ci-PCS gene activation. The hypothesis is supported by the strong induction of Proliferating Cell Nuclear Antigen (PCNA) transcript after 4 days of treatment and the colocalization in the hemocytes of the different riboprobes used. In addition, in literature is reported an expression profile similar to that of CiMT for C. intestinalis mannose binding lectins (CiMBL). Collectively, our data and data from the literature support the conclusion that hemocyte proliferation occurs in tunicate immune responses.


2011 - Immune roles of a rhamnose-bimnding lectin from the colonial ascidian Botryllus schlosseri [Relazione in Atti di Convegno]
Franchi, N.; Schiavon, F.; Ballarin, L.
abstract


2011 - Immune roles of a rhamnose-binding lectin in the colonial ascidian Botryllus schlosseri [Articolo su rivista]
Franchi, Nicola; Schiavon, Filippo; Carletto, M; Gasparini, Fabio; Bertoloni, G; Tosatto, Silvio; Ballarin, Loriano
abstract

The present paper describes the immune role played by a recently identified (Gasparini et al. 2008) member of the rhamnose-binding lectin (RBL) family from the colonial ascidian Botryllus schlosseri. B. schlosseri RBL (BsRBL) can activate phagocytes through: (i) induction of their directional movement towards the source of the molecule; (ii) modification of cytoskeleton, required for shape changes; (iii) stimulation of the respiratory burst, and consequent production of reactive oxygen species (ROS) with microbicidal activity, including superoxide anions and peroxides; and (iv) increase in the ability to phagocytose foreign particles. RBL also induces the synthesis and release, by cytotoxic morula cells (MCs), of cytokines recognised by anti-IL1_ and anti-TNF_ antibodies. At high concentrations, BsRBL induces degranulation of MCs and the consequent release of the cytotoxic enzyme phenoloxidase into the medium. Results are consistent with the existence of cross-talk between B. schlosseri immunocytes (phagocytes and MCs). In addition, a three-dimensional model for BsRBL is presented.


2011 - Immune roles of a rhamnose-binding lectin in the compound ascidian Botryllus schlosseri [Abstract in Rivista]
Franchi, N.; Schiavon, F.; Carletto, M.; Gasparini, F.; Bertoloni, G.; Tosatto, Sce.; Ballarin, L.
abstract

The present communication describes the immune role played by a recently identified member of the rhamnose-binding lectin (RBL) family from the colonial ascidian Botryllus schlosseri. B. schlosseri RBL (BsRBL) can activate phagocytes through: i) induction of their directional movement towards the source of the molecule; ii) modification of cytoskeleton, required for shape changes; iii) stimulation of the respiratory burst, and consequent production of reactive oxygen species (ROS) with microbicidal activity, including superoxide anions and peroxides; and iv) increase in the ability to phagocytose foreign particles. RBL also induces the synthesis and release, by cytotoxic morula cells (MCs), of cytokines recognized by anti-IL1α and anti-TNFα antibodies. At high concentrations, BsRBL induces degranulation of MCs and the consequent release of the cytotoxic enzyme phenoloxidase into the medium. Results are consistent with the existence of cross-talk between B. schlosseri immunocytes (phagocytes and MCs). In addition, a three dimensional model for BsRBL is presented.


2011 - Metal-induced antioxidant defense in the solitary ascidian Ciona intestinalis [Relazione in Atti di Convegno]
Franchi, N.; Ferro, D.; Piccinni, E.; Ballarin, L.; Santovito, G.
abstract

Antioxidant enzymes play important roles in antioxidant responses caused by metabolic process or pathogen invasion. In many organisms, it has been reported that antioxidant enzymes participate in their innate immune defense against immuno-stimulant challenges such as β-glucan and sulfated polysaccharide, LPS or viruses. Cu,Zn superoxide dismutase (SOD, EC 1.15.1.1) is one of these key enzymes, highly conserved from invertebrates to vertebrates, involved in scavenging superoxide radicals into molecular oxygen and hydrogen peroxide. However, little is known about the responses of antioxidant enzymes like Cu,Zn SOD in tunicates after exposure to environmental changes in heavy metal concentrations. The present research focuses on structural and functional studies of the Cu,Zn SOD gene in the solitary ascidian Ciona intestinalis. The gene sequence has been identified in GenBank and the amino acid sequence of the codified protein has been compared with those deduced from orthologous genes in other metazoans, both vertebrates and invertebrates, to determine if the amino acids important for catalytic activity were conserved and whether the enzyme of C. intestinalis acquired special features, relatively to the primary sequence, during its evolution. The in silico analysis was extended to the promoter regions for the presence of regulatory sequences such as antioxidant response elements (ARE), metal response elements (MRE) and xenobiotic response elements (XRE). The available sequences were used for cladistic studies, providing some interesting inference on the molecular evolution of the C. intestinalis protein. Using semi-quantitative RT-PCR technique, Cu,Zn SOD gene expression have been evaluated as a function of exposure to three different metals (Cd, Zn and Cu). For this purpose, specimens of C. intestinalis were divided into three experimental groups, which were exposed for 6, 24, 48, 72, 96 and 120 h at equimolar concentrations (10 μM) of single metal. Untreated specimens were used as controls. Cu,Zn SOD is mainly induced by Cu and Zn, metals that are components of the active site. Besides, it was provided an initial estimation on the localization of expression by in situ hybridization. The results indicate that the cells most involved in the expression of the considered genes are the hemocytes.


2011 - Recognition and clearance of apoptotic cells during the colonial blastogenetic cycle in the compound ascidian Botryllus schlosseri. [Relazione in Atti di Convegno]
Ballarin, L.; Schiavon, F.; Franchi, N.
abstract

Colonies of the compound ascidian Botryllus schlosseri feature cyclical generation changes, allowing continuous renewing of the colony, during which old zooids synchronously cease filtering and are resorbed in 24-26 h, while a new generation of buds reach the functional maturity, open their siphons and start filtering. It is possible to define a colonial blastogenetic cycle corresponding to the time interval of functional activity of a zooid generation (one week at 20°C), starting with the opening of the siphons of a new zooid generation and ending with the resorption or take-over (TO) of old zooids. During the TO, diffuse apoptosis occurs in zooid tissues followed by the massive recruitment of phagocytes which leave the circulation and infiltrate zooid tissues. The interaction between phagocytes and effete cells, leading to their internalisation, is fundamental for the progression of the TO as colonies do not feed during the generation change and the products of the digestion of senescent cells represent the main source of nutrients for the colony during this period. The recognition and engulfment of apoptotic cells is greatly promoted by molecules with opsonic activity, able to act as a bridge between the surface of effete cells and that of phagocytes. B. schlosseri rhamnose-binding lectin (BsRBL) is a new member of the rhamnose-binding protein family, which exerts an opsonic role towards non-self cells. During the generation change, we observed an increase in the levels of mRNA, as revealed by sqRT-PCR, and of the protein, in immunoblot analysis, during the TO with respect to phases of the blastogenetic cycle far from the TO (mid-cycle; MC). This goes in parallel with a change in surface carbohydrates in apoptotic cells and an increased number of cells recognised by anti-BsRBL antibody at TO with respect to MC. All these data are consistent with a pivotal role of BsRBL in the interaction between phagocytes and apoptotic cells and corpses, never described before, and open new perspectives on the possible role of RBLs in vertebrates.


2011 - THE BIOLOGY OF BOTRYLLUS SCHLOSSERI: A REVIEW OF SABBADIN'S IDEAS AND STUDIES [Relazione in Atti di Convegno]
Manni, L; Ballarin, L; Burighel, P; F., Cima; Caicci, F; Franchi, N; Gasparini, F; Rigon, F; Schiavon, F; Zaniolo, G
abstract

More thean 50 years ago, Armando sabbadin introduced at the University of Padova (Italy), the colonial ascidian Botryllus schlosseri, setting up conditions for the permanent culture of this species. Attracted by its fascinating life cycle, he spent about five years in studying its biology, laying the foundations for future studies and producing a series of almost unknown papers, published in Italian, containing the evidences of many statements that are now an aquired background of researchers. These papers acount the value of the staging method introduced by Berrill in 1941 for B. schlosseri and then modified by Sabbadin in 1955, evidencing the relationship between bud growing and water temperature. Moreover, they state the regulative ability of colonies, able to survive in adverse environmental conditions and to rescue development when conditions improve. The papers present also different experiments of repeated bud extirpation, which allowed Sabbadin to evidence competition between right and left buds for both sexual and asexual reproduction. All these conclusions were based on data coming form daily observations of a number of treated and control colonies, and were expressed in form of tables and graphs which prove what we take now for assured. We review these unknown data, so that they become more visible to the scientific community and discuss recent publications which exploit some Sabbadin's assertions.


2010 - An unusual chordate metallothionein gene in Ciona intestinalis genome: structure and expression studies [Relazione in Atti di Convegno]
Franchi, Nicola; Del Favero, M.; Piccinni, E.; Ballarin, Loriano
abstract

Metallothioneins (MTs) are able to bind essential and non-essential heavy metal ions, thus controlling cellular homeostasis and detossification. In addition, they act as scavengers for reactive oxygen species (ROS), thanks to their abundant thiols groups. They have a role also in the regulation of inflammatory responses through the modulation of immunomodulatory humoral components. Chordata represents the major phylum of Deuterostomes, including about 45,000 species distributed in three subphyla: Tunicata (Urochordata), Cephalochordata and Vertebrata. Invertebrate Chordata, about 3 % of the total chordate species, are collectively named Protochordata. Unfortunately, no MT genes have been annotated so far in Protochordates. In order to allow a comparison with the vertebrate MTs we undertook a search for MT genes in the genome of the solitary tunicate Ciona intestinalis. We were able to find a MT gene (CiMT1), which represents the first MT gene identified in Tunicates. Its expression is limited to hemocytes and modulated by Cd, Zn and Cu. The deduced protein is only 39 amino acids in length with no typical α and β domains. However the sequence shows that this protein shares the usual percentage (≥ 30) of Cys residues arranged in typical conserved motifs reported for vertebrates.


2010 - Metallothionein and phytochelatin levels in Ciona intestinalis are related to haemocyte proliferation [Relazione in Atti di Convegno]
Franchi, N.; Boldrin, F.; Piccinni, E.; Ballarin, L.
abstract


2010 - Metallothionein and phytochelatin levels in Ciona intestinalis are related to haemocytes proliferation [Relazione in Atti di Convegno]
Franchi, N; F., Boldrin; E., Piccinni; L., Ballarin
abstract


2010 - Metallothioneins from urochordates: something new has happened. [Relazione in Atti di Convegno]
Franchi, N.; Ballarin, L.; Piccinni, E.
abstract

Metallothioneins (MTs) are able to bind essential and non-essential heavy metal ions, thus controlling cellular homeostasis and detossification. In addition, they act as scavengers for reactive oxygen species (ROS), thanks to their abundant thiols groups. They have a role also in the regulation of inflammatory responses through the modulation of immunomodulatory humoral components. Chordata represents the major phylum of Deuterostomes, including about 45,000 species distributed in three subphyla: Tunicata (Urochordata), Cephalochordata and Vertebrata. Invertebrate Chordata, about 3% of the total chordate species, are collectively named Protochordata. No MT genes have been annotated so far in Protochordata. In order to allow a comparison with the vertebrate MTs we undertook a search for MT genes in the genome of the solitary tunicate Ciona intestinalis. We were able to find a MT gene (CiMT1), which represents the first MT gene identified in Tunicates. Its expression is limited to haemocytes and modulated by Cd, Zn and Cu. The deduced protein is only 39 amino acids in length with no typical α and β domains. However the sequence shows that this protein shares the usual percentage ≥( 30) of Cys residues arranged in typical conserved motifs reported for vertebrates. We were able to identify this gene also in Molgula manhattensis and Ascidiella aspersa, the search in other tunicates gave no results. This data, with the evidence that in some urochordates is present the gene for a phytochelatin syntase, could suggest that other mechanisms of detossification are present in ascidians.


2010 - Nanos homologue expression in haemocytes of two botryllid ascidians (Botryllus primigenus and Botryllus schlosseri) [Relazione in Atti di Convegno]
Ballarin, L.; Franchi, N.; Kawamura, K.
abstract

Nanos and nanos-related genes encode RNA-binding proteins and are expressed by stem cells of many metazoans. In the Japanese colonial ascidian Botryllus primigenus, a nanos homologue of 1.3 kb, Bpnos, has been recently identified (Sunanaga et al., 2008), containing two nanos-like CCHC zinc finger motifs. The gene in expressed in germ stem cells, immature and mature male germ cells, as well as in a fraction of circulating haemocytes. Using an anti-Bpnos monoclonal antibody, we confirmed that haemoblasts, i.e., circulating undifferentiated cells, are the only haemocytes recognised by the antibody in both B. primigenus and the Mediterranean species Botryllus schlosseri. In both species, the frequency of haemoblasts reaches the maximum value just after the take-over phase, when the generation change occurs and gradually decreases during the rest of the cycle. The number of circulating haemoblasts is much higher in the Japanese than in the Mediterranean species and this can be related to the occurrence of spontaneous vascular budding in B. primigenus. We also identified a nanos-related gene in B. schlosseri, sharing the nanos domain with two CCHC motifs, which we propose to call Bsnos. We are now trying to get the full cDNA sequence and the deduced amino acid sequence in order to get probes for Bsnos expression studies.


2010 - The Botryllus rhamnose-binding lectin as multifunctional immune molecule [Relazione in Atti di Convegno]
Franchi, N.; Carletto, M.; Schiavon, F.; Tosatto, S.; Ballarin, L.
abstract


2009 - An exceptional immunosurveillance system of the pharynx entry in a protochordate [Relazione in Atti di Convegno]
Cima, F; Franchi, N; Ballarin, L.
abstract

In the colonial ascidian Botryllus schlosseri, we have recently identified unusual free amoebocytes, completely exposed to the sea-water flow entering into the pharynx, which inhabit the tunic surface that internally covers the oral siphon (Cima et al., 2006 Dev Comp. Immunol., 30: 463-472). All histochemical data support the hypothesis that these cells named “siphonal guard cells” (SGC) belong to the hyaline amoebocyte population of the phagocytic differentiation pathway of the circulating immunocytes. After crossing the epidermis which bounds the siphonal blood sinus, they adhere to strategic points of the tunic and can recognise and phagocytise various foreign particles. After exposure of colonies to bacterial spores, the observations at both light and electronic microscope revealed that these cells are involved in a complex series of local and systemic immune events. Already after 5 min, the SGC show bacteria inside heterophagic vacuoles. After 10-15 min, as a transitory plug of colloidal material rich in heparin and histamine is formed in the siphonal lumen by exocytosis of some SGC, other ones with engulfed bacteria cross the epidermis reaching the siphonal sinus and probably transferring an alert signal; cells of the cytotoxic blood cell line (morula cells) crowd into the siphonal sinus, where most of them are labelled by anti-TNF- and anti-CD57 antibodies and degranulate. After this time, large scavenger phagocytes increase in number in blood circulation, show bacteria engulfed in large phagosomes and are continuously eliminated through the peribranchial chamber and the cloacal siphon with a mechanism which was not previously described.


2009 - Individuation of a new metallothionein from the urochordate Ciona intestinalis [Relazione in Atti di Convegno]
Franchi, N.; Ballarin, L.; Piccinni, E.
abstract

Metallothioneins (MTs) are metal-chelating proteins occurring in animals, plants and prokaryotes, involved in detosification and immunity. They do not constitute a monophyletic protein family, but rather a superfamily of heterogeneous, low molecular weight, cysteine-rich peptides. Current knowledge on MTs comes mainly from vertebrate molecules which are composed of approximately 60 amino acids, including 20 cysteine residues, and folded into two independent domains when coordinating divalent metal ions. Up to now, there are no descriptions of MTs in invertebrate Chordates althought it seems that the vertebrate structure is maintained also in other deuterostomes such as the echinoderms. In this study, we present some data on MTs of the solitary urochordate Ciona intestinalis. We have cloned the transcript and characterized the gene of a new MT, codifying for 39 amino acids, including 12 cys residues (30 % of total amino acids, in accordance with other MTs); moreover, the typical organization of cysteine residues in C-X-C motifs is conserved. The gene is composed of two introns (one inside the coding region and the other inside the 3’ UTR region) and three exons. The 5’ untrascribed region contains several cis elements similar to those found in vertebrate MT genes such as: metal responding elements (MRE), antioxidant responding elements and STAT3, which are involved in constitutive and metal-related, ROS and cytochines induction, respectively. The amino acid sequence of C. intestinalis MT shows only limited similarity with other MTs, e.g., Mytilus edulis MT (28.8 % identity), Strongylocentrotus purpuratus MT (23.4 % identity) and Sparus aurata MT (36.7 % identity). Phylogenetic analyses with various methods are in progress.


2009 - Individuation of a new metallothionein from the urochordate Ciona intestinalis [Relazione in Atti di Convegno]
Franchi, N; Ballarin, L.; Piccinni, E
abstract

Metallothioneins (MTs) are metal-chelating proteins occurring in animals, plants and prokaryotes, involved in detossification and immunity. In 2001, Canpolat and Lynes showed that exogenous MT can affect cell proliferation macrophage and cytotoxic T lymphocyte function, and humoral immunity to T-dependent antigens in mice. These evidences strengthen the hypothesis that MTs have an active role in extracellular environment as immunomodulatory proteins. Up to now, there are no descriptions of MTs in invertebrate Chordates althought it seems that the vertebrate structure is maintained also in other deuterostomes such as the echinoderms. As we are interested in studing the involvement and role of MTs ascidian immune responses, we undertook a preliminary investigation amied to identify these molecules in Ciona intestinalis, the genome of which has been fully sequenced but no MTs have been annotated. We have cloned the transcript and characterized the gene of a new MT, codifying for 39 amino acids, including 12 cys residues (30% of total amino acids, in accordance with other MTs). Moreover, the typical organization of cysteine residues in C-X-C motifs is conserved. The gene is composed of two introns (one inside the coding region and the other inside the 3’ UTR region) and three exons. The 5’ untrascribed region contains several cis elements similar to those found in vertebrate MT genes such as: metal responding elements (MRE) involved in constitutive and metal-related induction, antioxidant responding elements, important for ROS-dependent MT expression and STAT3, having a role in cytokine-related induction. The amino acid sequence of C. intestinalis MT shows only limited similarity with other known MTs: Mytilus edulis MT (28,8% identity), Strongylocentrotus purpuratus MT (23,4% identity) and Sparus aurata MT (36,7% identity).


2009 - Individuazione e caratterizzazione di una nuova metallotioneina nell’ascidia Ciona intestinalis [Relazione in Atti di Convegno]
Franchi, N.; Ballarin, L.; Piccinni, E.
abstract

Le metallotioneine (MT) sono proteine a basso peso molecolare in grado di legare metalli essenziali e non essenziali grazie alla presenza di cisteine organizzate in cluster conservati. La funzione principale di queste molecole è legata all’omeostasi dei metalli essenziali ma svolgono anche un ruolo importante nella detossifi cazione di metalli pesanti, nella protezione da ROS e nella regolazione della risposta immunitaria. MT sono state individuate in molti taxa animali, così come in vegetali e funghi ma, fi no ad ora, non erano state descritte metallotioneine negli urocordati. Questo importante gruppo, sistergroup dei vertebrati, off re l’occasione di studiare l’evoluzione molecolare grazie alla sua posizione fi logenetica di cordati invertebrati. La nostra attenzione è rivolta all’ascidia solitaria Ciona intestinalis. In tale organismo abbiamo individuato un trascritto la cui sequenza aminoacidica dedotta presenta le caratteristiche delle MT: il 30% dei residui aminoacidici sono cisteine organizzate in cluster e la regione promotrice presenta i siti di induzione da metalli, ROS e citochine tipici per questa famiglia di proteine. Mediante RT-PCR è stata dimostrata l’inducibilità di questi trascritti dopo esposizione per 5 giorni a 10 μM CdCl2 in acqua di mare artifi ciale. Tratti dell’apparato digerente di organismi trattati e non trattati sono stati utilizzati per studiare la localizzazione degli mRNA tramite ibridazione in situ. La metallotioneina di Ciona intestinalis risulta più corta (39 aminoacidi) se paragonata e quelle degli altri deuterostomi (circa 60 aminoacidi) e manca dei domini “alfa” e “beta” peculiari delle MT a Cd dei Vertebrati e degli echinodermi. Si è osservato che i campioni raccolti presentavano un’ elevata espressione di MT e pertanto sono stati eseguiti esperimenti di detossifi cazione in acqua di mare artifi ciale. L’ibridazione in situ ha evidenziato come la produzione di queste molecole sia a carico degli emociti, tra cui immunociti, suggerendo la possibilità che, anche in Ciona intestinalis, possano avere un ruolo chiave nella risposta immunitaria.


2009 - Rhamnose-binding lectins in the compound ascidian Botryllus schlosseri as multifaceted immune molecules [Relazione in Atti di Convegno]
Franchi, N; F, Schiavon; L, Ballarin
abstract


2009 - Rhamnose-binding lectins in the compound ascidian Botryllus schlosseri as multifunctional immune molecules [Relazione in Atti di Convegno]
Franchi, N; Schiavon, F; Ballarin, L.
abstract


2008 - A novel rhamnose-binding lectin from the colonial ascidian Botryllus schlosseri [Relazione in Atti di Convegno]
Franchi, N; Fabio, Gasparini; Barbara, Spolaore; Loriano, Ballarin
abstract


2008 - Effects of cadmium on the functionality of haemocytes from the compound ascidian Botryllus schlosseri [Relazione in Atti di Convegno]
Franchi, N; DI SILVESTRO, M; Ballarin, L.
abstract


2008 - MGE contribution to the colonial ascidian B. schlosseri genomic studies: insight into immunobiology and muscle development [Relazione in Atti di Convegno]
Gasparini, F; Burighel, P; Degasperi, V; Manni, L; Franchi, N; Ballarin, L
abstract


2008 - Novel rhamnose-binding lectins from the colonial ascidian Botryllus schlosseri [Articolo su rivista]
Gasparini, Fabio; Franchi, Nicola; Spolaore, Barbara; Ballarin, Loriano
abstract

In a full-length cDNA library from the compound ascidian Botryllus schlosseri, we identified, by BLAST search against UniProt database, five transcripts, each with complete coding sequence, homologous to known rhamnose-binding lectins (RBLs). Comparisons of the predicted amino acid sequences suggest that they represent different isoforms of a novel RBL, called BsRBL-1–5. Four of these isolectins were found in Botryllus homogenate after purification by affinity chromatography on acid-treated Sepharose, analysis by reverse-phase HPLC and mass spectrometry. Analysis of both molecular masses and tryptic digests of BsRBLs indicated that the N-terminal sequence of the purified proteins starts from residue 22 of the putative amino acid sequence, and residues 1–21 represent a signal peptide. Analysis by mass spectrometry of V8-protease digests confirmed the presence and alignments of the eight cysteines involved in the disulphide bridges that characterise RBLs. Functional studies proved the enhancing effect on phagocytosis of the affinity-purified material. Results are discussed in terms of phylogenetic relationships of BsRBLs with orthologous molecules from protostomes and deuterostomes.


2007 - A novel rhamnose-binding lectin from the compound ascidian Botryllus schlosseri [Relazione in Atti di Convegno]
Franchi, N; Gasparini, F; Spolaore, B; Ballarin, L
abstract


2007 - A novel rhamnose-binding lectin from the compound ascidian Botryllus schlosseri. [Relazione in Atti di Convegno]
Ballarin, L.; Franchi, N; Spolaore, B; Gasparini, F
abstract


2007 - Purification and characterisation of a novel rhamnose-binding lectin from the compound ascidian Botryllus schlosseri. [Relazione in Atti di Convegno]
Ballarin, L.; Franchi, N; Spolaore, B; Gasparini, F
abstract


2007 - The application of genomic approaches to colonial ascidian B. schlosseri. [Relazione in Atti di Convegno]
Franchi, N; Ballarin, L.; Gasparini, F
abstract


2005 - Morula cells and non-self recognition in the compound ascidian Botryllus schlosseri [Articolo su rivista]
Ballarin, Loriano; A., Menin; Franchi, Nicola; G., Bertoloni; Cima, Francesca
abstract

In the ascidian Botryllus schlosseri, we studied the effects of hemocyte incubation with foreign cells, such as bacteria, bacterial spores and yeast. In the presence of yeast and bacterial spores, morula cells, a common cell type in botryllid ascidians, changed their morphology, release phenoloxidase in the medium, thus causing an increase in cytotoxicity, and express molecole recognised by anti-IL-1-a- and anti-TNF-a-antibodies. These effects were not observed when hemocytes were incubated with both gram-positive (Staphylococcus aureus) and gram-negative (Escherichia coli) bacteria. Considering that morula cells are the main source of molecules recognised by anti-cytokine-antibodies we suggest an immunosurveillance role of these cells, which may influence immune responses such as phagocytosis.


2003 - Morula cell activation upon the recognition of bacteria, in the compound ascidian Botryllus schlosseri [Relazione in Atti di Convegno]
Ballarin, L.; Franchi, N; Cima, F
abstract