Reaping the benefits of liquid handlers for high-throughput gene expression profiling in a marine model invertebrate.

BACKGROUND: Modern high-throughput technologies enable the processing of a large number of samples simultaneously, while also providing rapid and accurate procedures. In recent years, automated liquid handling workstations have emerged as an established technology for reproducible sample preparation. They offer flexibility, making them suitable for an expanding range of applications. Commonly, such approaches are well-developed for experimental procedures primarily designed for cell-line processing and xenobiotics testing. Conversely, little attention is focused on the application of automated liquid handlers in the analysis of whole organisms, which often involves time-consuming laboratory procedures. RESULTS: Here, we present a fully automated workflow for all steps, from RNA extraction to real-time PCR processing, for gene expression quantification in the ascidian marine model Ciona robusta. For procedure validation, we compared the results obtained with the liquid handler with those of the classical manual procedure. The outcome revealed comparable results, demonstrating a remarkable time saving particularly in the initial steps of sample processing. CONCLUSIONS: This work expands the possible application fields of this technology to whole-body organisms, mitigating issues that can arise from manual procedures. By minimizing errors, avoiding cross-contamination, decreasing hands-on time and streamlining the procedure, it could be employed for large-scale screening investigations.

Conserved Signaling Pathways in the Ciona robusta Gut.

The urochordate Ciona robusta exhibits numerous functional and morphogenetic traits that are shared with vertebrate models. While prior investigations have identified several analogies between the gastrointestinal tract (i.e., gut) of Ciona and mice, the molecular mechanisms responsible for these similarities remain poorly understood. This study seeks to address this knowledge gap by investigating the transcriptional landscape of the adult stage gut. Through comparative genomics analyses, we identified several evolutionarily conserved components of signaling pathways of pivotal importance for gut development (such as WNT, Notch, and TGFß-BMP) and further evaluated their expression in three distinct sections of the gastrointestinal tract by RNA-seq. Despite the presence of lineage-specific gene gains, losses, and often unclear orthology relationships, the investigated pathways were characterized by well-conserved molecular machinery, with most components being expressed at significant levels throughout the entire intestinal tract of C. robusta. We also showed significant differences in the transcriptional landscape of the stomach and intestinal tract, which were much less pronounced between the proximal and distal portions of the intestine. This study confirms that C. robusta is a reliable model system for comparative studies, supporting the use of ascidians as a model to study gut physiology.

Transcriptomic and Bioinformatic Analyses Identifying a Central Mif-Cop9-Nf-kB Signaling Network in Innate Immunity Response of Ciona robusta.

The Ascidian C. robusta is a powerful model for studying innate immunity. LPS induction activates inflammatory-like reactions in the pharynx and the expression of several innate immune genes in granulocyte hemocytes such as cytokines, for instance, macrophage migration inhibitory factors (CrMifs). This leads to intracellular signaling involving the Nf-kB signaling cascade that triggers downstream pro-inflammatory gene expression. In mammals, the COP9 (Constitutive photomorphogenesis 9) signalosome (CSN) complex also results in the activation of the NF-kB pathway. It is a highly conserved complex in vertebrates, mainly engaged in proteasome degradation which is essential for maintaining processes such as cell cycle, DNA repair, and differentiation. In the present study, we used bioinformatics and in-silico analyses combined with an in-vivo LPS exposure strategy, next-generation sequencing (NGS), and qRT-PCR to elucidate molecules and the temporal dynamics of Mif cytokines, Csn signaling components, and the Nf-κB signaling pathway in C. robusta. A qRT-PCR analysis of immune genes selected from transcriptome data revealed a biphasic activation of the inflammatory response. A phylogenetic and STRING analysis indicated an evolutionarily conserved functional link between the Mif-Csn-Nf-kB axis in ascidian C. robusta during LPS-mediated inflammation response, finely regulated by non-coding molecules such as microRNAs (miRNAs).

Ciona robusta macrophage migration inhibitory factor (Mif1 and Mif2) genes are differentially regulated in the lipopolysaccharide-challenged pharynx.

The effects of lipopolysaccharide (LPS) on Mif (macrophage migration inhibitory factor) gene expression in the pharynx (haemapoetic tissue) of Ciona robusta were investigated using quantitative reverse-transcription PCR (qRT-PCR) and in situ hybridisation (ISH). To verify the induction of an inflammatory response in the pharynx, a qRT-PCR analysis was performed to evaluate the change in the expression of proinflammatory marker genes such as Mbl, Ptx-like, Tnf-α and Nf-kb, which were shown to be upregulated 1 h post LPS challenge. The change in the expression of the two Mif paralogs in the pharynx was assessed before and after stimulation, and qRT-PCR and ISH results showed that, although Mif2 and Mif2 were expressed in clusters of haemocytes in pharynx vessels, only Mif1 expression increased after LPS stimulation. This indicates that the Mif genes are differently regulated and respond to different ambient inputs that need further analysis.

A Manually Curated Gene Model Set for an Ascidian, Ciona robusta (Ciona intestinalis Type A).

Gene/transcript model sets predicted from decoded genome sequences are an important resource for a wide range of biological studies. Accuracy of gene models is therefore critical for deducing accurate conclusions. Computationally predicted models are sometimes inconsistent with experimental data from cDNA clones and RNA-sequencing. In an ascidian, Ciona robusta (Ciona intestinalis type A), a manually curated gene/transcript model set, which was constructed using an assembly in which 68% of decoded sequences were associated with chromosomes, had been used during the last decade. Recently a new genome assembly was published, in which over 95% of decoded sequences are associated with chromosomes. In the present study, we provide a high-quality version of the gene/transcript model set for the latest assembly. Because the Ciona genome has been used in a variety of studies such as developmental biological studies, evolutionary studies, and physiological studies, the current gene/transcript model set provides a fundamental biological resource.

Novel Insights on Nitric Oxide Synthase and NO Signaling in Ascidian Metamorphosis.

Nitric oxide (NO) is a pivotal signaling molecule involved in a wide range of physiological and pathological processes. We investigated NOS/NO localization patterns during the different stages of larval development in the ascidia Ciona robusta and evidenced a specific and temporally controlled pattern. NOS/NO expression starts in the most anterior sensory structures of the early larva and progressively moves towards the caudal portion as larval development and metamorphosis proceeds. We here highlight the pattern of NOS/NO expression in the central and peripheral nervous system of Ciona larvae which precisely follows the progression of neural signals of the central pattern generator necessary for the control of the movements of the larva towards the substrate. This highly dynamic localization profile perfectly matches with the central role played by NO from the first phase of settlement induction to the next control of swimming behavior, adhesion to substrate and progressive tissue resorption and reorganization of metamorphosis itself.

Evolutionary and transcriptional analyses of a pentraxin-like component family involved in the LPS inflammatory response of Ciona robusta.

Pentraxins (PTXs) are a superfamily of conserved proteins which are components of the humoral arm of innate immunity. They are considered to be functional ancestors of antibodies and are classified into short and long types. In this study, we show that a pentraxin-like component (Ptx-like) with a C-terminal PTX domain, highly homologous to the short PTX of H. sapiens CRP, and a long N-terminal domain typical of long PTXs, is involved in the inflammatory response of Ciona robusta under LPS exposure in vivo. Analyses of protein domains as well as 3D modelling and phylogenetic tree supported the close relationship of Ptx-like with mammalian CRP, suggesting that C. robusta Ptx-like shares a common ancestor in the chordate lineages. qRT-PCR analysis showed that Ptx-like was transcriptionally upregulated during the inflammatory process induced by LPS inoculation and that it is involved in the initial phase as well as the secondary phase of the inflammatory response in which matrix remodelling and the achievement of homeostasis occur. In situ hybridisation assays revealed that gene transcription was upregulated in the pharynx post-LPS challenge in vivo, and that Ptx-like was expressed by clusters of haemocytes, mainly granulocytes, inside the pharynx vessels. We also found transcript-expressing granulocytes flowing in the musculature and in the lacunae of the circulatory system. These data supported that Ptx-like is a potential molecule of the acute-phase response in C. robusta immune defence systems against bacterial infection.

The conservation and diversity of ascidian cells and molecules involved in the inflammatory reaction: The Ciona robusta model.

Ascidians are marine invertebrate chordates belonging to the earliest branch (Tunicata) in the chordate phylum, therefore, they are of interest for studying the evolution of immune systems. Due to the known genome, the non-colonial Ciona robusta, previously considered to be C. intestinalis type A, is a model species for the study of inflammatory response. The internal defense of ascidians mainly relies on hemocytes circulating in the hemolymph and pharynx. Hemocytes can be in vivo challenged by LPS injection and various granulocyte and vacuolated cell populations differentiated to produce and release inflammatory factors. Molecular biology and gene expression studies revealed complex defense mechanisms involving different inflammatory hemocytes. Furthermore, cloning procedures allowed sequence analyses and molecular studies disclose immune-related gene families including TOLL-like receptors, galectins, C-type lectins, collectins, interlectins, pentraxine-like, peroxinectins, complement factors-like, TNFα-like, IL-17-like, TGF-like, MIF-like. These genes are promptly upregulated by the inflammatory stimulus and show a time course of transcription similar to each other. Domains sequence similarity and phylogenetic relationships with the vertebrate counterparts are shedding some light on immune-related gene evolution. Selective bioassays as well as bioinformatic approaches have allowed the characterization of antimicrobial peptides and the identification of post transcriptional molecular mechanisms able of influencing dynamics of gene regulation are described. In synthesis, the purpose of this article is to further explore the topic of hemocyte and molecules related to internal defence of ascidians involved in the inflammatory reaction, as well as to discuss current and future study options through a detailed literature review.

Transcriptomic Analyses Reveal 2 and 4 Family Members of Cytochromes P450 (CYP) Involved in LPS Inflammatory Response in Pharynx of Ciona robusta.

Cytochromes P450 (CYP) are enzymes responsible for the biotransformation of most endogenous and exogenous agents. The expression of each CYP is influenced by a unique combination of mechanisms and factors including genetic polymorphisms, induction by xenobiotics, and regulation by cytokines and hormones. In recent years, Ciona robusta, one of the closest living relatives of vertebrates, has become a model in various fields of biology, in particular for studying inflammatory response. Using an in vivo LPS exposure strategy, next-generation sequencing (NGS) and qRT-PCR combined with bioinformatics and in silico analyses, compared whole pharynx transcripts from naïve and LPS-exposed C. robusta, and we provide the first view of cytochrome genes expression and miRNA regulation in the inflammatory response induced by LPS in a hematopoietic organ. In C. robusta, cytochromes belonging to 2B,2C, 2J, 2U, 4B and 4F subfamilies were deregulated and miRNA network interactions suggest that different conserved and species-specific miRNAs are involved in post-transcriptional regulation of cytochrome genes and that there could be an interplay between specific miRNAs regulating both inflammation and cytochrome molecules in the inflammatory response in C. robusta.

Identification of an LPS-Induced Chemo-Attractive Peptide from Ciona robusta.

BACKGROUND: Previously published work has demonstrated that the LPS injection of Ciona robusta leads to the overexpression of a truncated form of an immune-related mRNA (C8short) by means of Ciona robusta (CR) alternative polyadenylation (APA) (CR-APA). METHODS: The 3D structure of the C8short-derived Ciona robusta chemo-attractive peptide (CrCP) was evaluated by homology modeling. The biological activity of the CrCP was studied in vitro using a primary human dermal cell line (HuDe). Real-Time PCR was used to investigate the expression levels of genes involved in cell motility. NF-κB signaling was studied by western blotting. RESULTS: In silico modeling showed that CrCP displayed structural characteristics already reported for a short domain of the vertebrate CRK gene, suggesting its possible involvement in cell migration mechanisms. In vitro assays demonstrated that CrCP was capable of inducing the motility of HuDe cells in both wound healing and chemo-attractive experiments. qPCR demonstrated the capability of CrCP to modulate the expression of the matrix metalloproteinase-7 (MMP-7) and E-cadherin genes. Finally, western blot analysis demonstrated that treatment with CrCP induced activation of the NF-κB signaling pathway. CONCLUSION: Our results describe the characterization of the 3D structure and chemo-attractive activity of an LPS-induced CrCP peptide from Ciona robusta.

The gelatinase MMP-9like is involved in regulation of LPS inflammatory response in Ciona robusta.

Matrix metalloproteinases (MMPs) are a family of endopeptidases collectively able to degrade the components of the extracellular matrix (ECM), with important roles in many biological processes, such as embryogenesis, normal tissue remodelling, angiogenesis and wound healing. New views on the function of MMPs reveal that they regulate inflammatory response and therefore might represent an early step in the evolution of the immune system. MMPs can affect the activity of cytokines involved in inflammation including TGF-ß and TNF-α. MMPs are widely distributed in all kingdoms of life and have likely evolved from a single-domain protein which underwent successive rounds of duplications. In this study, we focused on the Ciona robusta (formerly known as Ciona intestinalis) MMP gelatinase homologue. Gene organization, phylogenetic analysis and 3D modeling supported the closest correlation of C. robusta gelatinase with the human MMP-9. Real-time PCR analysis and zymographic assay showed a prompt expression induced by LPS inoculation and an upregulation of enzymatic activity. Furthermore, we showed that before of the well-known increase of TGF-ß and TNF-α levels, a MMP-9like boost occurred, suggesting a possible involvement of MMP-9like in regulating inflammatory response in C. robusta.

Rapid microevolution during recent range expansion to harsh environments.

BACKGROUND: Adaptive evolution is one of the crucial mechanisms for organisms to survive and thrive in new environments. Recent studies suggest that adaptive evolution could rapidly occur in species to respond to novel environments or environmental challenges during range expansion. However, for environmental adaptation, many studies successfully detected phenotypic features associated with local environments, but did not provide ample genetic evidence on microevolutionary dynamics. It is therefore crucial to thoroughly investigate the genetic basis of rapid microevolution in response to environmental changes, in particular on what genes and associated variation are responsible for environmental challenges. Here, we genotyped genome-wide gene-associated microsatellites to detect genetic signatures of rapid microevolution of a marine tunicate invader, Ciona robusta, during recent range expansion to the harsh environment in the Red Sea. RESULTS: The Red Sea population was significantly differentiated from the other global populations. The genome-wide scan, as well as multiple analytical methods, successfully identified a set of adaptive genes. Interestingly, the allele frequency largely varied at several adaptive loci in the Red Sea population, and we found significant correlations between allele frequency and local environmental factors at these adaptive loci. Furthermore, a set of genes were annotated to get involved in local temperature and salinity adaptation, and the identified adaptive genes may largely contribute to the invasion success to harsh environments. CONCLUSIONS: All the evidence obtained in this study clearly showed that environment-driven selection had left detectable signatures in the genome of Ciona robusta within a few generations. Such a rapid microevolutionary process is largely responsible for the harsh environmental adaptation and therefore contributes to invasion success in different aquatic ecosystems with largely varied environmental factors.

The Interplay of TLR-NFκB Signalling Pathway and Functional Immune-Related Enzymes in the Inflammatory Response of Ciona robusta.

The close phylogenetic relationship between ascidians (Tunicata) and vertebrates makes them a powerful model for studying the innate immune system. To better understand the nature and dynamics of immune responses and the mechanisms through which bacterial infections are detected and translated into inflammation in Ciona robusta, we applied an approach combining in vivo lipopolysaccharide (LPS) stimulation, immune-labelling techniques and functional enzymatic analyses. The immunohistochemistry showed that Toll-like receptor 4 (TLR4) and nuclear factor kappa B (NFκB) were expressed during the inflammatory pharynx response 4 h post-LPS, with the formation of nodules in pharynx vessel lumen. Also, the endothelium vessels were involved in the inflammatory response. Observations of histological sections from naive and buffer-inoculated ascidians confirmed an immuno-positive response. Enzyme immune parameters-which included the activity of phenoloxidase, glutathione peroxidase, lysozyme, alkaline phosphatase and esterase-showed up-modulation 4 h after LPS injection, confirming their participation during ascidian inflammatory response. These findings provide new insights into the mechanisms underlying the LPS-induced C. robusta response and suggest that a broad innate immune mechanism, as in vertebrates, is involved in the regulation of inflammatory responses. Further findings in this direction are needed to cover knowledge gaps regarding the organized set of molecular and cellular networks involved in universal immune interactions with pathogens.

An organic extract from ascidian Ciona robusta induces cytotoxic autophagy in human malignant cell lines.

The last decades have seen an increase in the isolation and characterization of anticancer compounds derived from marine organisms, especially invertebrates, and their use in clinical trials. In this regard, ascidians, which are included in the subphylum Tunicata, represent successful examples with two drugs, Aplidine© and Yondelis© that reached the market as orphan drugs against several malignancies. Here, we report that an organic extract prepared from homogenized tissues of the Mediterranean ascidian Ciona robusta inhibited cell proliferation in HT-29, HepG2, and U2OS human cells with the former being the most sensitive to the extract (EC50 = 250 µg/mL). We demonstrated that the ascidian organic extract was not cytotoxic on HT-29 cells that were induced to differentiate with sodium butyrate, suggesting a preference for the mixture for the malignant phenotype. Finally, we report that cell death induced by the organic extract was mediated by the activation of a process of cytotoxic autophagy as a result of the increased expression of the LC3-II marker and number of autophagic vacuoles, which almost doubled in the treated HT-29 cells. In summary, although the detailed chemical composition of the Ciona robusta extract is still undetermined, our data suggest the presence of bioactive compounds possessing anticancer activity.

Laboratory automation and high-throughput biology.

BACKGROUND: Modern high-throughput technologies enable the processing of a large number of samples simultaneously, while also providing rapid and accurate procedures. In recent years, automated liquid handling workstations have emerged as an established technology for reproducible sample preparation. They offer flexibility, making them suitable for an expanding range of applications. Commonly, such approaches are well-developed for experimental procedures primarily designed for cell-line processing and xenobiotics testing. Conversely, little attention is focused on the application of automated liquid handlers in the analysis of whole organisms, which often involves time-consuming laboratory procedures. RESULTS: Here, Annona et al present a fully automated workflow for all steps, from RNA extraction to real-time PCR processing, for gene expression quantification in the ascidian marine model Ciona robusta. For procedure validation, the authors compared the results obtained with the liquid handler with those of the classical manual procedure. The outcome revealed comparable results, demonstrating a remarkable time saving particularly in the initial steps of sample processing. CONCLUSIONS: This work expands the possible application fields of this technology to whole-body organisms, mitigating issues that can arise from manual procedures. By minimizing errors, avoiding cross-contamination, decreasing hands-on time and streamlining the procedure, it could be employed for large-scale screening investigations.

Environmental stress and nanoplastics' effects on Ciona robusta: regulation of immune/stress-related genes and induction of innate memory in pharynx and gut.

In addition to circulating haemocytes, the immune system of the solitary ascidian Ciona robusta relies on two organs, the pharynx and the gut, and encompasses a wide array of immune and stress-related genes. How the pharynx and the gut of C. robusta react and adapt to environmental stress was assessed upon short or long exposure to hypoxia/starvation in the absence or in the presence of polystyrene nanoplastics. We show that the immune response to stress is very different between the two organs, suggesting an organ-specific immune adaptation to the environmental changes. Notably, the presence of nanoplastics appears to alter the gene modulation induced by hypoxia/starvation in both organs, resulting in a partial increase in gene up-regulation in the pharynx and a less evident response to stress in the gut. We have also assessed whether the hypoxia/starvation stress could induce innate memory, measured as gene expression in response to a subsequent challenge with the bacterial agent LPS. Exposure to stress one week before challenge induced a substantial change in the response to LPS, with a general decrease of gene expression in the pharynx and a strong increase in the gut. Co-exposure with nanoplastics only partially modulated the stress-induced memory response to LPS, without substantially changing the stress-dependent gene expression profile in either organ. Overall, the presence of nanoplastics in the marine environment seems able to decrease the immune response of C. robusta to stressful conditions, hypothetically implying a reduced capacity to adapt to environmental changes, but only partially affects the stress-dependent induction of innate memory and subsequent responses to infectious challenges.

Transcriptional and proteomic analysis of the innate immune response to microbial stimuli in a model invertebrate chordate.

Inflammatory response triggered by innate immunity can act to protect against microorganisms that behave as pathogens, with the aim to restore the homeostatic state between host and beneficial microbes. As a filter-feeder organism, the ascidian Ciona robusta is continuously exposed to external microbes that may be harmful under some conditions. In this work, we used transcriptional and proteomic approaches to investigate the inflammatory response induced by stimuli of bacterial (lipopolysaccharide -LPS- and diacylated lipopeptide - Pam2CSK4) and fungal (zymosan) origin, in Ciona juveniles at stage 4 of metamorphosis. We focused on receptors, co-interactors, transcription factors and cytokines belonging to the TLR and Dectin-1 pathways and on immune factors identified by homology approach (i.e. immunoglobulin (Ig) or C-type lectin domain containing molecules). While LPS did not induce a significant response in juvenile ascidians, Pam2CSK4 and zymosan exposure triggered the activation of specific inflammatory mechanisms. In particular, Pam2CSK4-induced inflammation was characterized by modulation of TLR and Dectin-1 pathway molecules, including receptors, transcription factors, and cytokines, while immune response to zymosan primarily involved C-type lectin receptors, co-interactors, Ig-containing molecules, and cytokines. A targeted proteomic analysis enabled to confirm transcriptional data, also highlighting a temporal delay between transcriptional induction and protein level changes. Finally, a protein-protein interaction network of Ciona immune molecules was rendered to provide a wide visualization and analysis platform of innate immunity. The in vivo inflammatory model described here reveals interconnections of innate immune pathways in specific responses to selected microbial stimuli. It also represents the starting point for studying ontogeny and regulation of inflammatory disorders in different physiological conditions.

Transitions of motor neuron activities during Ciona development.

Motor neurons (MNs) are one of the most important components of Central Pattern Generators (CPG) in vertebrates (Brown, Proceedings of The Royal Society B: Biological Sciences (The Royal Society), 1911, 84(572), 308-319). However, it is unclear how the neural activities of these components develop during their embryogenesis. Our previous study revealed that in Ciona robusta (Ciona intestinalis type A), a model organism with a simple neural circuit, a single pair of MNs (MN2L/MN2R) was determining the rhythm of its spontaneous early motor behavior (developmental stage St.22-24). MN2s are known to be one of the main components of Ciona CPG, though the neural activities of MN2s in the later larval period (St.25-) were not yet investigated. In this study, we investigated the neural activities of MN2s during their later stages and how they are related to Ciona's swimming CPG. Long-term simultaneous Ca2+ imaging of both MN2s with GCaMP6s/f (St.22-34) revealed that MN2s continued to determine the rhythm of motor behavior even in their later larval stages. Their activities were classified into seven phases (I-VII) depending on the interval and the synchronicity of MN2L and MN2R Ca2+ transients. Initially, each MN2 oscillates sporadically (I). As they develop into swimming larvae, they gradually oscillate at a constant interval (II-III), then start to synchronize (IV) and fully synchronize (V). Intervals become longer (VI) and sporadic again during the tail aggression period (VII). Interestingly, 76% of the embryos started to oscillate from MN2R. In addition, independent photostimulations on left and right MN2s were conducted. This is the first report of the live imaging of neural activities in Ciona's developing swimming CPG. These findings will help to understand the development of motor neuron circuits in chordate animals.

Xbp1 and Brachyury establish an evolutionarily conserved subcircuit of the notochord gene regulatory network.

Gene regulatory networks coordinate the formation of organs and structures that compose the evolving body plans of different organisms. We are using a simple chordate model, the Ciona embryo, to investigate the essential gene regulatory network that orchestrates morphogenesis of the notochord, a structure necessary for the proper development of all chordate embryos. Although numerous transcription factors expressed in the notochord have been identified in different chordates, several of them remain to be positioned within a regulatory framework. Here, we focus on Xbp1, a transcription factor expressed during notochord formation in Ciona and other chordates. Through the identification of Xbp1-downstream notochord genes in Ciona, we found evidence of the early co-option of genes involved in the unfolded protein response to the notochord developmental program. We report the regulatory interplay between Xbp1 and Brachyury, and by extending these results to Xenopus, we show that Brachyury and Xbp1 form a cross-regulatory subcircuit of the notochord gene regulatory network that has been consolidated during chordate evolution.