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Nervous system differentiation in the colonial ascidian Botryllus schlosseri: molecular and cellular aspects and evolutive implications
Full text linkIn the last years, several studies are addressed to the investigation of mechanisms that permitted the appearance and evolution of those structures considered as extremely important in the vertebrates radiation. The rise of vertebrates was accompanied by the acquisition of a great complexity in the structural plan of the organisms that is related to the evolution of features associated with the nervous system, such as neural crests, cranial placodes and an elaborated brain.
The aim of the doctoral project is inserted in this line of research. Particularly, the attention is addressed to those characters, which in the non- vertebrate chordates can be interpreted as crucial for the subsequent evolution of the vertebrate body-plan. The starting point is represented by previous morphological studies that evidenced the presence, in the tunicate embryo, of transitory ectodermal and multipotential territories located at the neural plate border. Our research, carried out using different approaches, is focused on the characterisation and description of structures that differentiate from these domains. In this regard, it is investigated the organisation of the larval papillae and their formation from the rostral placode. These structures play a pivotal role in triggering the mechanisms and changes that characterise the metamorphosis, which in ascidians constitute the lost of the chordate body-plan of the larva and the begin of the sessile post-embryonic phase.
The analysis of the sensory components in ascidians, finalised to the identification of homologies with the structures that derive form the placodes in vertebrates, is also extended to the coronal organ. The coronal organ is recently discovered and possesses morphological, positional and ultrastructural features that, together with the presence of hair cells, are comparable to the lateral line and inner ear of vertebrates, which components derive from the acoustic-lateral placodes.
A substantial part of the work is dedicated to the investigation, with a molecular approach, of the presence of structures comparable to the neural placodes in ascidians. The attention is focused on the colonial ascidian Botryllus schlosseri that permits a comparative study on the mechanisms and genic networks involved in both embryogenetic and blastogenetic development. We characterised orthologues of placodal genes and constructed probes for in situ hybridisation experiments. During the stages of bud differentiation, we localised territories interested by expression of genes normally involved in the placodal induction and specification in vertebrates. These regions, for their position and differentiative potentialities, are comparable to other embryonic domains of B. schlosseri that are considered as homologues to the neural placodes.
The larva of ascidians possesses a striated symmetrical musculature sharing some features with that of vertebrates and it flanks the dorsal tube and the notochord, representing a peculiar propriety of chordates. The acquisition of this new locomotory system probably required the parallel appearance of a sophisticated control of the coordination. The nervous system established new interactions and differentiated sensory structures that permitted the rapid perception of the environment by the mobile organism. At metamorphosis, the larval musculature is reabsorbed, while the cardiac and unstriated muscle fibres differentiate de novo from circulating mesenchymal cells. This plasticity constitutes the source of the evolutive potentiality of ascidians and thus we investigate its molecular and morphological bases. We isolated and characterised transcripts coding for muscle-specific genes, analysing the expression during the blastogenetic cycle of Botryllus, from the bud appearance to the adult regression. The use of different methods allowed the description of the organisation and differentiation of the unstriated muscle, confirming its unique proprieties. Taking together, our results contribute to the understanding of the origin and development of structures that represent an important starting point in the evolution and radiation of vertebrates.Negli ultimi anni numerosi studi si sono rivolti all’approfondimento di quei meccanismi che hanno permesso la comparsa ed evoluzione di strutture ritenute di estrema importanza nella radiazione dei vertebrati. La comparsa dei vertebrati è stata accompagnata da un enorme balzo nella complessità del piano strutturale degli organismi, largamente ascrivibile all’evoluzione di strutture associate al sistema nervoso come le creste neurali, i placodi craniali ed un cervello elaborato.
La tematica trattata durante lo svolgimento del progetto di dottorato si inserisce in questa attuale linea di ricerca. In particolare, l’attenzione è stata rivolta a quei caratteri che nei cordati non-vertebrati possono essere letti come cruciali per la successiva evoluzione del piano strutturale dei vertebrati. Il punto di partenza è rappresentato da precedenti studi morfologici che hanno evidenziato la presenza, nell’embrione dei tunicati, di territori ectodermici transitori e multipotenti localizzati al confine con la piastra neurale. Il nostro studio, svolto mediante l'utilizzo di vari approcci metodologici, si è rivolto alla caratterizzazione e descrizione delle strutture che queste aree sono in grado di differenziare.
A questo proposito, è stata analizzata l’organizzazione delle papille larvali e la loro formazione a partire dal placode rostrale. Queste strutture giocano un ruolo primario nell’innescare i meccanismi e i cambiamenti che caratterizzano la metamorfosi, ovvero quel processo che nelle ascidie segna la perdita del piano corporeo da cordato della larva e il passaggio alla fase post-embrionale sessile. L’analisi riguardante strutture sensoriali presenti nelle ascidie, allo scopo di identificare eventuali omologie con le corrispondenti strutture derivanti dai placodi nei vertebrati, è stata poi estesa all’organo coronale. L’organo coronale è stato scoperto solo recentemente e presenta caratteristiche morfologiche generali, posizionali e ultrastrutturali tali, come la presenza di cellule capellute, che lo rendono comparabile alla linea laterale ed all’orecchio interno dei vertebrati, i cui componenti derivano dai placodi acustico-laterali.
Una parte consistente del lavoro è stata dedicata all’indagine, da un punto di vista molecolare, della presenza di strutture accomunabile ai placodi neurali nelle ascidie. L’attenzione è stata rivolta all’ascidia coloniale Botryllus schlosseri, che permette di svolgere uno studio comparativo sui meccanismi e reti geniche che intervengono sia durante lo sviluppo embriogenetico che blastogenetico. Abbiamo caratterizzato specifici geni e prodotto sonde utilizzate in esperimenti di ibridazione in situ. Durante le fasi di differenziamento della gemma sono stati individuati specifici territori caratterizzati da espressione di alcuni geni normalmente coinvolti nell’induzione e specificazione placodale nei vertebrati. Grazie alla loro posizione e potenzialità differenziativa, queste stesse regioni sono apparse confrontabili con altri territori embrionali di B. schlosseri e di altre ascidie considerati omologhi a placodi neurali dei vertebrati.
La larva delle ascidie presenta una muscolatura simmetrica striata, con caratteri comuni a quella dei vertebrati, la quale fiancheggia il tubo dorsale e la notocorda e che rappresenta una proprietà peculiare dei cordati. L’acquisizione di questo nuovo sistema locomotorio ha verosimilmente richiesto la comparsa parallela di un sofisticato sistema di controllo della coordinazione. Il sistema nervoso ha stabilito nuove interazioni e differenziato strutture sensoriali che hanno permesso all’organismo mobile la rapida percezione dell’ambiente circostante. Alla metamorfosi, la muscolatura larvale viene completamente riassorbita, mentre le fibre muscolari non striate della parete del corpo e quelle cardiache si differenziano de novo da cellule mesenchimali circolanti. Questa plasticità sta alla base della potenzialità evolutiva delle ascidie e quindi ne abbiamo indagato le basi molecolari e morfologiche. Abbiamo quindi isolato e caratterizzato trascritti e geni muscolo-specifici, studiandone l’espressione durante il ciclo blastogenetico di Botryllus, dalla comparsa della gemma alla regressione dell’adulto. L ’utilizzo di vari approcci ha permesso la descrizione dell’organizzazione e differenziamento della muscolatura non striata, confermandone le caratteristiche uniche. Nel complesso i diversi risultati rappresentano contributi significativi per la conoscenza delle origini e sviluppo quelle strutture che hanno rappresentato un punto di partenza importante nell’evoluzione e radiazione dei vertebrati
The adult musculature in Botryllus schlosseri: differentiation and gene expression
Full text linkTypically, ascidians have three types of muscles: skeletal in the larva, cardiac and smooth in the post-metamorphic sessile organism. The larval and cardiac muscles are striated, with vertebrate-like arrangement of myofilaments. Instead, the smooth body-wall musculature has intermediate characters between smooth and striated muscle of vertebrates. We studied the musculature in the blastozooids of Botryllus schlosseri analysing its organisation, differentiation and gene expression. We isolated and characterised two transctipts resulted homologous to muscle genes of other adult ascidians: a muscle-type actin (BsMA2) and troponin T (BsTnT-a); moreover, we obtained also the genomic sequence coding for BsMA2.
Phylogenetic analyses showed a close relationship between urochordates and vertebrates muscle genes. The BsMA2 genomic sequence was compared in the exon-intron organization with other muscle and cytoplasmic–type actin genes of both invertebrates and vertebrates. Our analysis showed that intron positions are conserved in ascidian and in the other deuterostomes.
We detected the expression of the two genes by in situ hybridization (ISH), in order to follow the muscle development throughout the blastogenetic cycle of B. schlosseri. The ISH, in parallel with phalloidin staining experiments, showed that the first diffuse signal of muscle differentiation appears in the intersiphonal epidermis of young buds. Then, the muscle fibers differentiate in the body-wall, while an intense expression of BsMA2 marks the heart myocardium just when it begins contractions. Moreover, the ultrastructure of smooth muscle cells was investigated during differentiation from mesenchymal cells in the bud, in the adult, and in the fibers contraction during regression of zooids
Characterization of a MuSashI-like transcript in a colonial chordate, phylogenetic analysis of the protein group and differential expression patterns in sexual versus asexual development.
No full textTunicates are the unique chordates to possess species reproducing sexually and asexually. Between them, Botryllus schlosseri forms similar organisms (oozooids and blastozooids) trough embryogenetic and blastogenetic pathways respectively. We here illustrate the characterization and expression pattern, during both pathways, of a transcript for a gene that we named BsMSI.We demonstrate that BsMSI falls into MuSashI-like (MSI-like) group, formed by MSI1, MSI2 and DAZAP1 genes. They encode for RNA-binding proteins phylogenetically related to other protein groups: the TARDBP and several hnRNPs. These proteins share same modular domain structure: a 2xRRM motif. Analyses show that MSIs-like and 2xRRM-hnRNPs had a common origin from a TARDBP type, and that 2xRRM-hnRNPs form a monophyletic group. A unique MSI-like, orthologous to DAZAP1, was recognized in tunicates. We hypothesize that, as MSIs are widely present in metazoans, tunicates have lost them during their evolution.In vertebrates MSI1 is considered a stem cells marker, but also MSI2 and DAZAP1 are involved in differentiative processes. BsMSI is expressed in all the tissues of developing buds. This ubiquitous blastogenetic expression differs from the embryogenesis one, which varies spatio-temporally according to the differentiation of embryonic, larval or oozooid tissues. Interestingly, BsMSI expression ends in differentiated or regressing structures (adult blastozooids, differentiated larval structures).Because the BsMSI expression patterndoesn’t overlap between blastogenesis and embryogenesis, we speculate it operates substitutive functions of the vertebrates orthologues DAZAP, MSI1 and MSI2, and that the function, related to differentiation or tissue maintaining, varies during the B. schlosseri life cycle
Gene expression during body muscle differentiation in ascidians (Tunicata) and the evolution of muscle in chordates
No full textAscidians present three types of muscles in their life: striated in the larval tail and heart, and smooth in the post-metamorphic sessile phase. The larval and cardiac muscles have an arrangement of myofilaments like the striated muscle of vertebrates. Instead, the smooth body-wall musculature has intermediate characters between smooth and striated muscle of vertebrates. A great number of ascidians form colonies constituted of clonal individuals (blastozooids) originated by asexual reproduction. We studied the musculature in the colonial ascidian Botryllus schlosseri (Fig. 1) analysing its organization, differentiation and gene expression during development of blastozooids, beginning from the early bud stage to adult and regression stage. We isolated and characterised two transcripts from colonies that resulted homologous to muscle genes of solitary ascidians: a muscletype actin (BsMA2), a cytoplasmic-type actin (BsCA1) and a troponin T (BsTnT-c). Moreover, we obtained also the genomic sequences coding for BsMA2 and BsCA1.
Phylogenetic analyses showed a close relationship between urochordates and vertebrates muscle genes. The BsMA2 and BsCA1 genomic sequences were compared in the exon-intron organization with other muscle and cytoplasmic–type actin genes of both invertebrates and vertebrates. Our data revealed that intron positions are conserved in ascidians and in the other deuterostomes.
We detected the expression of the two genes by in situ hybridization on section (ISH; Fig. 2), in order to follow the muscle development throughout the blastogenetic cycle of B. schlosseri. The ISH, in parallel with phalloidin staining experiments, showed that the first diffuse signal of BsMA2 and BsTnT-c labels differentiating muscles which appear in the intersiphonal area of young buds. Then, the muscle fibres differentiate into the body-wall, while an intense expression only of BsMA2 marks the myocardium just when it begins contractions. The organization and differentiation of striated cardiac and smooth muscle cells were also investigated at ultrastructural level (Fig. 2) during all the phases of the blastogenetic cycle from early bud to zooid regression
Gene expression during body muscle differentiation in the colonial ascidian Botryllus schlosseri.
No full textAscidians display three types of muscles during their life: striated in the larval tail and heart, and smooth in the post-metamorphic sessile phase. The larval and cardiac muscles have an arrangement of myofilaments like the striated muscle of vertebrates. Instead, the smooth body-wall musculature has intermediate characters between smooth and striated muscle of vertebrates. A great number of ascidians form colonies constituted of clonal individuals (blastozooids) originated by asexual reproduction. We studied the musculature in Botryllus schlosseri analysing its organization, differentiation with electron microscopy. Moreover, we followed gene expression of muscle actin and troponin T in the larva and during the development of blastozooids, beginning from the early bud stage to adult and regression stage. We isolated and characterised two transcripts from colonies that resulted homologous to muscle genes of solitary ascidians: a muscle-type actin (BsMA2), a cytoplasmic-type actin (BsCA1) and a troponin T (BsTnT-c). Moreover, we obtained also the genomic sequences coding for BsMA2 and BsCA1.
Phylogenetic analyses showed a close relationship between urochordates and vertebrates muscle genes. The BsMA2 and BsCA1 genomic sequences were compared in the exon-intron organization with other muscle and cytoplasmic–type actin genes of both invertebrates and vertebrates. Our data revealed that intron positions are conserved in ascidians and in the other deuterostomes.
We detected the expression of the two genes by in situ hybridization on section (ISH) throughout the blastogenetic cycle of B. schlosseri. The ISH, in parallel with phalloidin staining experiments, showed that the first diffuse signal of BsMA2 and BsTnT-c labels differentiating muscles which appear in the intersiphonal area of young buds. Then, the muscle fibres differentiate into the body-wall, while an intense expression only of BsMA2 marks the myocardium just when it begins contractions. Thus, all the phases of muscle development and regression were documented by an integrated study showing the close correspondence of in situ expression of BsMA2 and BsTnT-c, phalloidin signal and ultrastructure
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Variations on the Author
Full text link“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Appropriate Similarity Measures for Author Cocitation Analysis
Full text linkWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
Dispelling the Myths Behind First-author Citation Counts
Full text linkWe conducted a full-scale evaluative citation analysis study of scholars in the XML research field to explore just how different from each other author rankings resulting from different citation counting methods actually are, and to demonstrate the capability of emerging data and tools on the Web in supporting more realistic citation counting methods. Our results contest some common arguments for the continued
use of first-author citation counts in the evaluation of scholars, such as high correlations between author rankings by first-author citation counts and other citation
counting methods, and high costs of using more realistic citation counting methods that are not well-supported by the ISI databases. It is argued that increasingly available digital full text research papers make it possible for citation analysis studies to go beyond what the ISI databases have directly supported and to employ more
sophisticated methods
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