Identification of LINE retrotransposons and long non-coding RNAs expressed in the octopus brain.

BACKGROUND: Transposable elements (TEs) widely contribute to the evolution of genomes allowing genomic innovations, generating germinal and somatic heterogeneity, and giving birth to long non-coding RNAs (lncRNAs). These features have been associated to the evolution, functioning, and complexity of the nervous system at such a level that somatic retrotransposition of long interspersed element (LINE) L1 has been proposed to be associated to human cognition. Among invertebrates, octopuses are fascinating animals whose nervous system reaches a high level of complexity achieving sophisticated cognitive abilities. The sequencing of the genome of the Octopus bimaculoides revealed a striking expansion of TEs which were proposed to have contributed to the evolution of its complex nervous system. We recently found a similar expansion also in the genome of Octopus vulgaris. However, a specific search for the existence and the transcription of full-length transpositionally competent TEs has not been performed in this genus. RESULTS: Here, we report the identification of LINE elements competent for retrotransposition in Octopus vulgaris and Octopus bimaculoides and show evidence suggesting that they might be transcribed and determine germline and somatic polymorphisms especially in the brain. Transcription and translation measured for one of these elements resulted in specific signals in neurons belonging to areas associated with behavioral plasticity. We also report the transcription of thousands of lncRNAs and the pervasive inclusion of TE fragments in the transcriptomes of both Octopus species, further testifying the crucial activity of TEs in the evolution of the octopus genomes. CONCLUSIONS: The neural transcriptome of the octopus shows the transcription of thousands of putative lncRNAs and of a full-length LINE element belonging to the RTE class. We speculate that a convergent evolutionary process involving retrotransposons activity in the brain has been important for the evolution of sophisticated cognitive abilities in this genus.

A cis-regulatory change underlying the motor neuron-specific loss of Ebf expression in immotile tunicate larvae.

Many species in the tunicate family Molgulidae have independently lost their swimming larval form and instead develop as tailless, immotile larvae. These larvae do not develop structures that are essential for swimming such as the notochord, otolith, and tail muscles. However, little is known about neural development in these nonswimming larvae. Here, we studied the patterning of the Motor Ganglion (MG) of Molgula occulta, a nonswimming species. We found that spatial patterns of MG neuron regulators in this species are conserved, compared with species with swimming larvae, suggesting that the gene networks regulating their expression are intact despite the loss of swimming. However, expression of the key motor neuron regulatory gene Ebf (Collier/Olf/EBF) was reduced in the developing MG of M. occulta when compared with molgulid species with swimming larvae. This was corroborated by measuring allele-specific expression of Ebf in hybrid embryos from crosses of M. occulta with the swimming species M. oculata. Heterologous reporter construct assays in the model tunicate species Ciona robusta revealed a specific cis-regulatory sequence change that reduces expression of Ebf in the MG, but not in other cells. Taken together, these data suggest that MG neurons are still specified in M. occulta larvae, but their differentiation might be impaired due to reduction of Ebf expression levels.

Transcriptional regulation of Rab32/38, a specific marker of pigment cell formation in Ciona robusta.

Through a myriad of pigments stored in different cells, animal pigmentation represents a crucial process to face disparate environmental and ecological challenges. In vertebrates, the small GTPase Rab32 and Rab38 have a conserved role in the transport of key melanogenic enzymes, as tyrosinase (tyr) and tyrosinase-related protein (tyrp), to the melanosomes in formation. We provide a survey on Rab32/38 evolution and its regulatory logics during pigment cell formation in Ciona robusta. Our phylogeny supports the existence of a single Rab32/38 gene in tunicates, which is probably the unique transporter for tyrosinase family members in this clade. Different deletions allow us to identify the minimal cis-regulatory element able to recapitulate the endogenous gene expression during pigment cell development in C. robusta. In this conserved region, we identified two putative binding sites for the transcription factor Mitf, which is known for its role as regulator of pigmentation in vertebrates. Mutational analysis revealed that both Mitf binding sites are essential for the activity of this regulatory region and we demonstrated that Mitf misexpression is able to induce ectopic activation of the Rab32/38 regulatory region in vivo. Our results strongly indicate that Mitf is involved in the regulation of Rab32/38 activity during Ciona pigment cell development.

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.

The evolutionary landscape of the Rab family in chordates.

Intracellular traffic amongst organelles represents a key feature for eukaryotes and is orchestrated principally by members of Rab family, the largest within Ras superfamily. Given that variations in Rab repertoire have been fundamental in animal diversification, we provided the most exhaustive survey regarding the Rab toolkit of chordates. Our findings reveal the existence of 42 metazoan conserved subfamilies exhibiting a univocal intron/exon structure preserved from cnidarians to vertebrates. Since the current view does not capture the Rab complexity, we propose a new Rab family classification in three distinct monophyletic clades. The Rab complement of chordates shows a dramatic diversification due to genome duplications and independent gene duplications and losses with sharp differences amongst cephalochordates, tunicates and gnathostome vertebrates. Strikingly, the analysis of the domain architecture of this family highlighted the existence of chimeric calcium-binding Rabs, which are animal novelties characterized by a complex evolutionary history in gnathostomes and whose role in cellular metabolism is obscure. This work provides novel insights in the knowledge of Rab family: our hypothesis is that chordates represent a hotspot of Rab variability, with many events of gene gains and losses impacting intracellular traffic capabilities. Our results help to elucidate the role of Rab members in the transport amongst endomembranes and shed light on intracellular traffic routes in vertebrates. Then, since the predominant role of Rabs in the molecular communication between different cellular districts, this study paves to way to comprehend inherited or acquired human disorders provoked by dysfunctions in Rab genes.

ANISEED 2015: a digital framework for the comparative developmental biology of ascidians.

Ascidians belong to the tunicates, the sister group of vertebrates and are recognized model organisms in the field of embryonic development, regeneration and stem cells. ANISEED is the main information system in the field of ascidian developmental biology. This article reports the development of the system since its initial publication in 2010. Over the past five years, we refactored the system from an initial custom schema to an extended version of the Chado schema and redesigned all user and back end interfaces. This new architecture was used to improve and enrich the description of Ciona intestinalis embryonic development, based on an improved genome assembly and gene model set, refined functional gene annotation, and anatomical ontologies, and a new collection of full ORF cDNAs. The genomes of nine ascidian species have been sequenced since the release of the C. intestinalis genome. In ANISEED 2015, all nine new ascidian species can be explored via dedicated genome browsers, and searched by Blast. In addition, ANISEED provides full functional gene annotation, anatomical ontologies and some gene expression data for the six species with highest quality genomes. ANISEED is publicly available at: http://www.aniseed.cnrs.fr.

Expression of caspase 3 in ovarian follicle cells of the lizard Podarcis sicula.

In this study, our aim was to determine whether caspase 3 plays a role, during previtellogenesis, in the ovarian follicular epithelium of the lizard Podarcis sicula. We investigated the presence and localization of proform and active caspase 3 by enzyme assay, Western blotting and immunocytochemistry. In parallel, a fragment of caspase 3 was cloned for the first time in this species, sequenced and used for in situ hybridization to localize messengers and analysed by a phylogenetic survey to shed light on its homology with reptilian caspases. Results demonstrated that: (1) the follicle cells expressed a caspase of the 3/7 group and the mRNA for caspase 3 was transcribed in the stem phase and was completely translated during cell differentiation; (2) the proform protein was stored during the differentiated (nurse) stage and activated at the end of previtellogenesis provoking the degeneration of cells; (3) the predicted protein sequence, although partial, had a strong similarity with the known reptilian caspases 3. The epithelial cells of the ovarian follicle, therefore, do not employ caspase 3 during the nurse stage but, instead, prepare for apoptosis long before the process actually begins. The relevance of this strategy is discussed.

Rab32 and Rab38 genes in chordate pigmentation: an evolutionary perspective.

BACKGROUND: The regulation of cellular membrane trafficking in all eukaryotes is a very complex mechanism, mostly regulated by the Rab family proteins. Among all membrane-enclosed organelles, melanosomes are the cellular site for synthesis, storage and transport of melanin granules, making them an excellent model for studies on organelle biogenesis and motility. Specific Rab proteins, as Rab32 and Rab38, have been shown to play a key role in melanosome biogenesis. We analysed the Rab32 and Rab38 genes in the teleost zebrafish and in the cephalochordate amphioxus, gaining insight on their evolutionary history following gene and genome duplications. RESULTS: We studied the molecular evolution of Rab supergroup III in deuterostomes by phylogenetic reconstruction, intron and synteny conservation. We discovered a novel amino acid stretch, named FALK, shared by three related classes belonging to Rab supergroup III: Rab7L1, Rab32LO and Rab32/Rab38. Among these, we demonstrated that the Rab32LO class, already present in the last common eukaryotic ancestor, was lost in urochordates and vertebrates. Synteny shows that one zebrafish gene, Rab38a, which is expressed in pigmented cells, retained the linkage with tyrosinase, a protein essential for pigmentation. Moreover, the chromosomal linkage of Rab32 or Rab38 with a member of the glutamate receptor metabotropic (Grm) family has been retained in all analysed gnathostomes, suggesting a conserved microsynteny in the vertebrate ancestor. Expression patterns of Rab32 and Rab38 genes in zebrafish, and Rab32/38 in amphioxus, indicate their involvement in development of pigmented cells and notochord. CONCLUSIONS: Phylogenetic, intron conservation and synteny analyses point towards an evolutionary scenario based on a duplication of a single invertebrate Rab32/38 gene giving rise to vertebrate Rab32 and Rab38. The expression patterns of Rab38 paralogues highlight sub-functionalization event. Finally, the discovery of a chromosomal linkage between the Rab32 or Rab38 gene with a Grm opens new perspectives on possible conserved bystander gene regulation across the vertebrate evolution.

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.

FGF/MAPK/Ets signaling renders pigment cell precursors competent to respond to Wnt signal by directly controlling Ci-Tcf transcription.

FGF and Wnt pathways constitute two fundamental signaling cascades, which appear to crosstalk in cooperative or antagonistic fashions in several developmental processes. In vertebrates, both cascades are involved in pigment cell development, but the possible interplay between FGF and Wnt remains to be elucidated. In this study, we have investigated the role of FGF and Wnt signaling in development of the pigment cells in the sensory organs of C. intestinalis. This species possesses the basic features of an ancestral chordate, thus sharing conserved molecular developmental mechanisms with vertebrates. Chemical and targeted perturbation approaches revealed that a FGF signal, spreading in time from early gastrulation to neural tube closure, is responsible for pigment cell precursor induction. This signal is transmitted via the MAPK pathway, which activates the Ci-Ets1/2 transcription factor. Targeted perturbation of Ci-TCF, a downstream factor of the canonical Wnt pathway, indicated its contribution to pigment cell differentiation Furthermore, analyses of the Ci-Tcf regulatory region revealed the involvement of the FGF effector, Ci-Ets1/2, in Ci-Tcf transcriptional regulation in pigment cell precursors. Our results indicate that both FGF and the canonical Wnt pathways are involved in C. intestinalis pigment cell induction and differentiation. Moreover, we present a case of direct transcriptional regulation exerted by the FGF signaling cascade, via the MAPK-ERK-Ets1/2, on the Wnt downstream gene Ci-Tcf. Several examples of FGF/Wnt signaling crosstalk have been described in different developmental processes; however, to our knowledge, FGF-Wnt cross-interaction at the transcriptional level has never been previously reported. These findings further contribute to clarifying the multitude of FGF-Wnt pathway interactions.

Investigating sperm cryopreservation in a model tunicate, Ciona intestinalis sp. A.

In cryopreservation procedures, the capacity to protect the cells from freezing and thawing processes is sensitive to the choice of the cryoprotective agent (CPA) and to its optimal concentration. The advancement of research on Tunicate model species has raised interest in liquid nitrogen cryopreservation for the storage and distribution of genetic resources. Ciona intestinalis (Linnè, 1767) consists of a complex of cryptic taxa that are central to several areas of investigation, from comparative genomics to invasive biology. Here we investigated how five CPAs, three chilling rates and two freezing rates influence semen cryopreservation in C. intestinalis sp. A. By using larval morphology and motility as endpoints, we estimated that long term semen storage requires 10% dimethyl sulfoxide as a protective agent, -1°C/min chilling rate (18°C to 5°C) and -13°C/min freezing rate (5°C to -80°C), followed by immersion in liquid nitrogen.

Gene expression in notochord and nuclei pulposi: a study of gene families across the chordate phylum.

The transition from notochord to vertebral column is a crucial milestone in chordate evolution and in prenatal development of all vertebrates. As ossification of the vertebral bodies proceeds, involutions of residual notochord cells into the intervertebral discs form the nuclei pulposi, shock-absorbing structures that confer flexibility to the spine. Numerous studies have outlined the developmental and evolutionary relationship between notochord and nuclei pulposi. However, the knowledge of the similarities and differences in the genetic repertoires of these two structures remains limited, also because comparative studies of notochord and nuclei pulposi across chordates are complicated by the gene/genome duplication events that led to extant vertebrates. Here we show the results of a pilot study aimed at bridging the information on these two structures. We have followed in different vertebrates the evolutionary trajectory of notochord genes identified in the invertebrate chordate Ciona, and we have evaluated the extent of conservation of their expression in notochord cells. Our results have uncovered evolutionarily conserved markers of both notochord development and aging/degeneration of the nuclei pulposi.

Brain Sensory Organs of the Ascidian Ciona robusta: Structure, Function and Developmental Mechanisms.

During evolution, new characters are designed by modifying pre-existing structures already present in ancient organisms. In this perspective, the Central Nervous System (CNS) of ascidian larva offers a good opportunity to analyze a complex phenomenon with a simplified approach. As sister group of vertebrates, ascidian tadpole larva exhibits a dorsal CNS, made up of only about 330 cells distributed into the anterior sensory brain vesicle (BV), connected to the motor ganglion (MG) and a caudal nerve cord (CNC) in the tail. Low number of cells does not mean, however, low complexity. The larval brain contains 177 neurons, for which a documented synaptic connectome is now available, and two pigmented organs, the otolith and the ocellus, controlling larval swimming behavior. The otolith is involved in gravity perception and the ocellus in light perception. Here, we specifically review the studies focused on the development of the building blocks of ascidians pigmented sensory organs, namely pigment cells and photoreceptor cells. We focus on what it is known, up to now, on the molecular bases of specification and differentiation of both lineages, on the function of these organs after larval hatching during pre-settlement period, and on the most cutting-edge technologies, like single cell RNAseq and genome editing CRISPR/CAS9, that, adapted and applied to Ciona embryos, are increasingly enhancing the tractability of Ciona for developmental studies, including pigmented organs formation.

The Cis-Regulatory Code for Kelch-like 21/30 Specific Expression in Ciona robusta Sensory Organs.

The tunicate Ciona robusta is an emerging model system to study the evolution of the nervous system. Due to their small embryos and compact genomes, tunicates, like Ciona robusta, have great potential to comprehend genetic circuitry underlying cell specific gene repertoire, among different neuronal cells. Their simple larvae possess a sensory vesicle comprising two pigmented sensory organs, the ocellus and the otolith. We focused here on Klhl21/30, a gene belonging to Kelch family, that, in Ciona robusta, starts to be expressed in pigmented cell precursors, becoming specifically maintained in the otolith precursor during embryogenesis. Evolutionary analyses demonstrated the conservation of Klhl21/30 in all the chordates. Cis-regulatory analyses and CRISPR/Cas9 mutagenesis of potential upstream factors, revealed that Klhl21/30 expression is controlled by the combined action of three transcription factors, Mitf, Dmrt, and Msx, which are downstream of FGF signaling. The central role of Mitf is consistent with its function as a fundamental regulator of vertebrate pigment cell development. Moreover, our results unraveled a new function for Dmrt and Msx as transcriptional co-activators in the context of the Ciona otolith.

Rimbp, a New Marker for the Nervous System of the Tunicate Ciona robusta.

Establishment of presynaptic mechanisms by proteins that regulate neurotransmitter release in the presynaptic active zone is considered a fundamental step in animal evolution. Rab3 interacting molecule-binding proteins (Rimbps) are crucial components of the presynaptic active zone and key players in calcium homeostasis. Although Rimbp involvement in these dynamics has been described in distantly related models such as fly and human, the role of this family in most invertebrates remains obscure. To fill this gap, we defined the evolutionary history of Rimbp family in animals, from sponges to mammals. We report, for the first time, the expression of the two isoforms of the unique Rimbp family member in Ciona robusta in distinct domains of the larval nervous system. We identify intronic enhancers that are able to drive expression in different nervous system territories partially corresponding to Rimbp endogenous expression. The analysis of gene expression patterns and the identification of regulatory elements of Rimbp will positively impact our understanding of this family of genes in the context of Ciona embryogenesis.

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.