Nitric Oxide Function and Nitric Oxide Synthase Evolution in Aquatic Chordates.

Nitric oxide (NO) is a key signaling molecule in almost all organisms and is active in a variety of physiological and pathological processes. Our understanding of the peculiarities and functions of this simple gas has increased considerably by extending studies to non-mammal vertebrates and invertebrates. In this review, we report the nitric oxide synthase (Nos) genes so far characterized in chordates and provide an extensive, detailed, and comparative analysis of the function of NO in the aquatic chordates tunicates, cephalochordates, teleost fishes, and amphibians. This comprehensive set of data adds new elements to our understanding of Nos evolution, from the single gene commonly found in invertebrates to the three genes present in vertebrates.

Comparative analysis of novel and common reference genes in adult tissues of the mussel Mytilus galloprovincialis.

BACKGROUND: Real-time quantitative PCR is a widely used method for gene expression analyses in various organisms. Its accuracy mainly relies on the correct selection of reference genes. Any experimental plan involving real-time PCR needs to evaluate the characteristics of the samples to be examined and the relative stability of reference genes. Most studies in mollusks rely on reference genes commonly used in vertebrates. RESULTS: In this study, we focused on the transcriptome of the bivalve mollusk Mytilus galloprovincialis in physiological state to identify suitable reference genes in several adult tissues. Candidate genes with highly stable expression across 51 RNA-seq datasets from multiple tissues were selected through genome-wide bioinformatics analysis. This approach led to the identification of three genes (Rpl14, Rpl32 and Rpl34), whose suitability was evaluated together with 7 other reference genes commonly reported in literature (Act, Cyp-A, Ef1α, Gapdh, 18S, 28S and Rps4). The stability analyses performed with geNorm, NormFinder and Bestkeeper identified specific either single or pairs of genes suitable as references for gene expression analyses in specific tissues and revealed the Act/Cyp-A pair as the most appropriate to analyze gene expression across different tissues. CONCLUSION: Mytilus galloprovincialis is a model system increasingly used in ecotoxicology and molecular studies. Our transcriptome-wide approach represents the first comprehensive investigation aimed at the identification of suitable reference genes for expression studies in this species.

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 Adaptation of the Thyroid Hormone Signaling Toolkit in Chordates.

The specification of the endostyle in non-vertebrate chordates and of the thyroid gland in vertebrates are fundamental steps in the evolution of the thyroid hormone (TH) signaling to coordinate development and body physiology in response to a range of environmental signals. The physiology and biology of TH signaling in vertebrates have been studied in the past, but a complete understanding of such a complex system is still lacking. Non-model species from non-vertebrate chordates may greatly improve our understanding of the evolution of this complex endocrine pathway. Adaptation of already existing proteins in order to perform new roles is a common feature observed during the course of evolution. Through sequence similarity approaches, we investigated the presence of bona fide thyroid peroxidase (TPO), iodothyronine deiodinase (DIO), and thyroid hormone receptors (THRs) in non-vertebrate and vertebrate chordates. Additionally, we determined both the conservation and divergence degrees of functional domains at the protein level. This study supports the hypothesis that non-vertebrate chordates have a functional thyroid hormone signaling system and provides additional information about its possible evolutionary adaptation.

The Ascidia Ciona robusta Provides Novel Insights on the Evolution of the AP-1 Transcriptional Complex.

The Activator Protein-1 transcription factor family (AP-1) transcriptional complex is historically defined as an early response group of transcription factors formed by dimeric complexes of the Jun, Fos, Atf, and Maf bZIP proteins that control cell proliferation and differentiation by regulating gene expression. It has been greatly investigated in many model organisms across metazoan evolution. Nevertheless, its complexity and variability of action made its multiple functions difficult to be defined. Here, we place the foundations for understanding the complexity of AP-1 transcriptional members in tunicates. We investigated the gene members of this family in the ascidian Ciona robusta and identified single copies of Jun, Fos, Atf3, Atf2/7, and Maf bZIP-related factors that could have a role in the formation of the AP-1 complex. We highlight that mesenchyme is a common cellular population where all these factors are expressed during embryonic development, and that, moreover, Fos shows a wider pattern of expression including also notochord and neural cells. By ectopic expression in transgenic embryos of Jun and Fos genes alone or in combination, we investigated the phenotypic alterations induced by these factors and highlighted a degree of functional conservation of the AP-1 complex between Ciona and vertebrates. The lack of gene redundancy and the first pieces of evidence of conserved functions in the control of cell movements and structural organization exerted by these factors open the way for using Ciona as a helpful model system to uncover the multiple potentialities of this highly complex family of bZIP transcription factors.

Onecut Regulates Core Components of the Molecular Machinery for Neurotransmission in Photoreceptor Differentiation.

Photoreceptor cells (PRC) are neurons highly specialized for sensing light stimuli and have considerably diversified during evolution. The genetic mechanisms that underlie photoreceptor differentiation and accompanied the progressive increase in complexity and diversification of this sensory cell type are a matter of great interest in the field. A role of the homeodomain transcription factor Onecut (Oc) in photoreceptor cell formation is proposed throughout multicellular organisms. However, knowledge of the identity of the Oc downstream-acting factors that mediate specific tasks in the differentiation of the PRC remains limited. Here, we used transgenic perturbation of the Ciona robusta Oc protein to show its requirement for ciliary PRC differentiation. Then, transcriptome profiling between the trans-activation and trans-repression Oc phenotypes identified differentially expressed genes that are enriched in exocytosis, calcium homeostasis, and neurotransmission. Finally, comparison of RNA-Seq datasets in Ciona and mouse identifies a set of Oc downstream genes conserved between tunicates and vertebrates. The transcription factor Oc emerges as a key regulator of neurotransmission in retinal cell types.

Transphyletic conservation of nitric oxide synthase regulation in cephalochordates and tunicates.

Nitric oxide synthase is ubiquitously present in metazoans and is involved in a wide range of biological processes. Three distinct Nos genes have been so far identified in vertebrates exhibiting a complex expression pattern and transcriptional regulation. Nevertheless, although independent events of Nos duplication have been observed in several taxa, only few studies described the regulatory mechanisms responsible for their activation in non-vertebrate animals. To shed light on the mechanisms underlying neuronal-type Nos expression, we focused on two non-vertebrate chordates: the cephalochordate Branchiostoma lanceolatum and the tunicate Ciona robusta. Here, throughout transphyletic and transgenic approaches, we identified genomic regions in both species acting as Nos functional enhancers during development. In vivo analyses of Nos genomic fragments revealed their ability to recapitulate the endogenous expression territories. Therefore, our results suggest the existence of evolutionary conserved mechanisms responsible for neuronal-type Nos regulation in non-vertebrate chordates. In conclusion, this study paves the way for future characterization of conserved transcriptional logic underlying the expression of neuronal-type Nos genes in chordates.

Sea as a color palette: the ecology and evolution of fluorescence.

Fluorescence and luminescence are widespread optical phenomena exhibited by organisms living in terrestrial and aquatic environments. While many underlying mechanistic features have been identified and characterized at the molecular and cellular levels, much less is known about the ecology and evolution of these forms of bioluminescence. In this review, we summarize recent findings in the evolutionary history and ecological functions of fluorescent proteins (FP) and pigments. Evidence for green fluorescent protein (GFP) orthologs in cephalochordates and non-GFP fluorescent proteins in vertebrates suggests unexplored evolutionary scenarios that favor multiple independent origins of fluorescence across metazoan lineages. Several context-dependent behavioral and physiological roles have been attributed to fluorescent proteins, ranging from communication and predation to UV protection. However, rigorous functional and mechanistic studies are needed to shed light on the ecological functions and control mechanisms of fluorescence.

An indoor study of the combined effect of industrial pollution and turbulence events on the gut environment in a marine invertebrate.

Natural storms are able to determine reworking of seabed up to considerable depths and favour suspension of sediment-associated chemicals. Yet, a direct link between exposure to resuspended contaminants and the biological effects on marine organisms have to be fully established. We exposed adults of a suspension feeder, the ascidian Ciona robusta, to polluted sediment (e.g., containing mixtures of polycyclic aromatic hydrocarbons and heavy metals) from the industrial area of Bagnoli-Coroglio under two temporal patterns ('aggregated' vs. 'spaced') of turbulence events. Then, we assessed the impact of resuspended pollutants on the ascidian gut environment via four broad categories: oxidative stress, innate immunity, host-microbiota interactions, and epithelium. An early oxidative stress response was seen after a week of exposure to static sediment. Instead, water turbulence had no effect on the antioxidant defence. The first episode of turbulent suspension induced a minimal pro-inflammatory response in the 'spaced' pattern. Mucus overproduction and a complete occlusion of the crypt lumen were found following sediment reworking. This study suggests a protective response of the gut environment in marine invertebrates exposed to environmental extremes, leading to increased susceptibility to disease and to concerns on the combined effects of chronic environmental contamination and acute disturbance events possibly associated with climate change.

Functional conserved non-coding elements among tunicates and chordates.

Non-coding regions with dozens to several hundred base pairs of extreme conservation have been found in all metazoan genomes. The distribution of these conserved non-coding elements (CNE) within and across genomes has suggested that many of them may have roles as transcriptional regulatory elements. A combination of bioinformatics and experimental approaches can be used to identify CNEs with regulatory activity in phylogenetically distant species. Nevertheless, the high divergent rate of genomic sequences of several organisms, such as tunicates, complicates the characterization of these conserved elements and very few examples really may prove their functional activity. We used a comparative approach to facilitate the identification of CNEs among distantly related or highly divergent species and experimentally demonstrated the functional significance of these novel CNEs. We first experimentally tested, in C. robusta and D. rerio transgenic embryos, the regulatory activity of conserved elements associated to genes involved in developmental control among different chordates (Homo sapiens and Danio rerio for vertebrates, Ciona robusta and Ciona savignyi for tunicates and Branchiostoma floridae for cephalochordates). Once demonstrated the cross-species functional conservation of these CNEs, the same gene loci were used as references to locate homologous regions and possible CNEs in available tunicate genomes. Comparison of tunicate-specific and chordate-specific CNEs revealed absence of conservation of the regulatory elements in spite of conservation of regulatory patterns, likely due to evolutionary specification of the respective developmental networks. This result highlights the importance of an integrative in-silico/in-vivo approach to CNEs investigation, encompassing both bioinformatics, essential for putative CNEs identification, and laboratory experiments, pivotal for the understanding of CNEs functionality.

A comprehensive analysis of neurotrophins and neurotrophin tyrosine kinase receptors expression during development of zebrafish.

Neurotrophins (NTF) are a family of secreted nerve growth factors with affinity for tyrosine kinase (Ntrk) and p75 receptors. To fully understand the variety of developmental roles played by NTFs, it is critical to know when and where genes encoding individual ligands and receptors are transcribed. Identification of ntf and ntrk transcripts in zebrafish development remains to be fully characterized for further uncovering the potential function(s) of the NTF signal transduction pathway. Here, we conducted a systematic analysis of the expression profiles of four ntf and five ntrk genes during zebrafish development using whole-mount in situ hybridization. Our study unveils new expression domains in the developing embryo, confirms those previously known, and shows that ntf and ntrk genes have different degrees of cell- and tissue-type specificity. The unique and overlapping expression patterns here depicted indicate the coordination of the redundant and divergent functions of NTFs and represent valuable tools for deciphering the molecular pathways involved in the specification and function of embryonic cell types.

A Rapid and Cheap Methodology for CRISPR/Cas9 Zebrafish Mutant Screening.

The introduction of new genome editing tools such as ZFNs, TALENs and, more recently, the CRISPR/Cas9 system, has greatly expanded the ability to knock-out genes in different animal models, including zebrafish. However, time and costs required for the screening of a huge number of animals, aimed to identify first founder fishes (F0), and then carriers (F1) are still a bottleneck. Currently, high-resolution melting (HRM) analysis is the most efficient technology for large-scale InDels detection, but the very expensive equipment demanded for its application may represent a limitation for research laboratories. Here, we propose a rapid and cheap method for high-throughput genotyping that displays efficiency rate similar to the HRM. In fact, using a common ViiA™7 real-time PCR system and optimizing the parameters of the melting analysis, we demonstrated that it is possible to discriminate between the mutant and the wild type melting curves. Due to its simplicity, rapidity and cheapness, our method can be used as a preliminary one-step approach for massive screening, in order to restrict the scope at a limited number of embryos and to focus merely on them for the next sequencing step, necessary for the exact sequence identification of the induced mutation. Moreover, thanks to its versatility, this simple approach can be readily adapted to the detection of any kind of genome editing approach directed to genes or regulatory regions and can be applied to many other animal models.

Regulatory elements retained during chordate evolution: coming across tunicates.

Understanding the role of conserved noncoding elements (CNEs) throughout the genome is taking advantage of the improved efficiency of genome-sequencing techniques and bioinformatics tools. Tunicates diverged before the vertebrate whole genome duplications and, therefore, represent an optimal model system to study the evolution of complex regulatory networks. Here, we review the current knowledge on the characterization of CNEs during embryonic development, focusing on the evolutionary similarity and divergence between tunicates and other chordates. Many vertebrate specific CNEs that regulate developmental processes were identified based on high level of sequence conservation, but only few of them have been recognized in tunicates or other invertebrates because of genomic sequences divergence. We discuss recent studies demonstrating that a combination of different methodologies, based not only on high sequence identity, can collectively be used to identify CNEs with regulatory activity in phylogenetically distant species. Here, a low sequence constraints approach was successfully used to search orthologous chordate gene regions for cross-species conserved regulatory elements that control developmental genes.

Mutation studies in ascidians: a review.

Historically, mutations have had a significant impact on the study of developmental processes and phenotypic evolution. Lesions in DNA are created by artificial methods or detected by natural genetic variation. Random mutations are then ascribed to genetic change by direct sequencing or positional cloning. Tunicate species of the ascidian genus Ciona represent nearly fully realized model systems in which gene function can be investigated in depth. Additionally, tunicates are valuable organisms for the study of naturally occurring mutations due to the capability to exploit genetic variation down to the molecular level. Here, we summarize the available information about how mutations are studied in ascidians with examples of insights that have resulted from these applications. We also describe notions and methodologies that might be useful for the implementation of easy and tight procedures for mutations studies in Ciona.

Auto and cross regulatory elements control Onecut expression in the ascidian nervous system.

The expression pattern of Onecut genes in the central and peripheral nervous systems is highly conserved in invertebrates and vertebrates but the regulatory networks in which they are involved are still largely unknown. The presence of three gene copies in vertebrates has revealed the functional roles of the Onecut genes in liver, pancreas and some populations of motor neurons. Urochordates have only one Onecut gene and are the closest living relatives of vertebrates and thus represent a good model system to understand its regulatory network and involvement in nervous system formation. In order to define the Onecut genetic cascade, we extensively characterized the Onecut upstream cis-regulatory DNA in the ascidian Ciona intestinalis. Electroporation experiments using a 2.5kb genomic fragment and of a series of deletion constructs identified a small region of 262bp able to reproduce most of the Onecut expression profile during embryonic development. Further analyses, both bioinformatic and in vivo using transient transgenes, permitted the identification of transcription factors responsible for Onecut endogenous expression. We provide evidence that Neurogenin is a direct activator of Onecut and that an autoregulatory loop is responsible for the maintenance of its expression. Furthermore, for the first time we propose the existence of a direct connection among Neurogenin, Onecut and Rx transcription factors in photoreceptor cell formation.

Evolution of anterior Hox regulatory elements among chordates.

BACKGROUND: The Hox family of transcription factors has a fundamental role in segmentation pathways and axial patterning of embryonic development and their clustered organization is linked with the regulatory mechanisms governing their coordinated expression along embryonic axes. Among chordates, of particular interest are the Hox paralogous genes in groups 1-4 since their expression is coupled to the control of regional identity in the anterior nervous system, where the highest structural diversity is observed. RESULTS: To investigate the degree of conservation in cis-regulatory components that form the basis of Hox expression in the anterior nervous system, we have used assays for transcriptional activity in ascidians and vertebrates to compare and contrast regulatory potential. We identified four regulatory sequences located near the CiHox1, CiHox2 and CiHox4 genes of the ascidian Ciona intestinalis which direct neural specific domains of expression. Using functional assays in Ciona and vertebrate embryos in combination with sequence analyses of enhancer fragments located in similar positions adjacent to Hox paralogy group genes, we compared the activity of these four Ciona cis-elements with a series of neural specific enhancers from the amphioxus Hox1-3 genes and from mouse Hox paralogous groups 1-4. CONCLUSIONS: This analysis revealed that Kreisler and Krox20 dependent enhancers critical in segmental regulation of the hindbrain appear to be specific for the vertebrate lineage. In contrast, neural enhancers that function as Hox response elements through the action of Hox/Pbx binding motifs have been conserved during chordate evolution. The functional assays reveal that these Hox response cis-elements are recognized by the regulatory components of different and extant species. Together, our results indicate that during chordate evolution, cis-elements dependent upon Hox/Pbx regulatory complexes, are responsible for key aspects of segmental Hox expression in neural tissue and appeared with urochordates after cephalochordate divergence.

Onecut is a direct neural-specific transcriptional activator of Rx in Ciona intestinalis.

Retinal homeobox (Rx) genes play a crucial and conserved role in the development of the anterior neural plate of metazoans. During chordate evolution, they have also acquired a novel function in the control of eye formation and neurogenesis. To characterize the Rx genetic cascade and shed light on the mechanisms that led to the acquisition of this new role in eye development, we studied Rx transcriptional regulation using the ascidian, Ciona intestinalis. Through deletion analysis of the Ci-Rx promoter, we have identified two distinct enhancer elements able to induce Ci-Rx specific expression in the anterior part of the CNS and in the photosensory organ at tailbud and larva stages. Bioinformatic analysis highlighted the presence of two Onecut binding sites contained in these enhancers, so we explored the role of this transcription factor in the regulation of Ci-Rx. By in situ hybridization, we first confirmed that these genes are co-expressed in the same cells. Through a series of in vivo and in vitro experiments, we then demonstrated that the two Onecut sites are responsible for enhancer activation in Ci-Rx endogenous territories. We also demonstrated in vivo that Onecut misexpression is able to induce ectopic activation of the Rx promoter. Finally, we demonstrated that Ci-Onecut is able to promote Ci-Rx expression in the sensory vesicle. Together, these results support the conclusion that in Ciona embryogenesis, Ci-Rx expression is under the control of the Onecut transcription factor and that this factor is necessary and sufficient to specifically activate Ci-Rx through two enhancer elements.

Expression of Prothymosin alpha during the spermatogenesis of the spotted ray Torpedo marmorata.

In this study, we show that Prothymosin alpha (Ptma), a small, unfolded, negatively charged protein, is present in the cartilaginous fish Torpedo marmorata. The ptma gene is functional and peculiarly controlled during the male spermatogenesis of T. marmorata, as revealed by in situ hybridization and by immunocytochemistry studies. The data show that the ptma transcript is present in stage-specific germ cells, i.e. spermatocytes II and round spermatids. The Ptma protein is detectable in spermatocytes II, in round and elongated spermatids as well as in spermatozoa before their release from cysts, while it is not evident in spermatozoa located in male genital tracts. The ptma transcript and protein are also evident in some Leydig cells, located among maturing cysts containing meiotic and differentiating male cells. No expression for ptma is observed within Sertoli cells. Furthermore, immunolocalization procedures demonstrate that the protein is preferentially localized in the cytoplasm, whereas a nuclear localization is observed in round and elongated spermatids. The possibility that Ptma is involved in testis activity is discussed.