Hmx gene conservation identifies the origin of vertebrate cranial ganglia.

The evolutionary origin of vertebrates included innovations in sensory processing associated with the acquisition of a predatory lifestyle1. Vertebrates perceive external stimuli through sensory systems serviced by cranial sensory ganglia, whose neurons arise predominantly from cranial placodes; however, the understanding of the evolutionary origin of placodes and cranial sensory ganglia is hampered by the anatomical differences between living lineages and the difficulty in assigning homology between cell types and structures. Here we show that the homeobox transcription factor Hmx is a constitutive component of vertebrate sensory ganglion development and that in the tunicate Ciona intestinalis, Hmx is necessary and sufficient to drive the differentiation programme of bipolar tail neurons, cells previously thought to be homologues of neural crest2,3. Using Ciona and lamprey transgenesis, we demonstrate that a unique, tandemly duplicated enhancer pair regulated Hmx expression in the stem-vertebrate lineage. We also show notably robust vertebrate Hmx enhancer function in Ciona, demonstrating that deep conservation of the upstream regulatory network spans the evolutionary origin of vertebrates. These experiments demonstrate regulatory and functional conservation between Ciona and vertebrate Hmx, and point to bipolar tail neurons as homologues of cranial sensory ganglia.

Specification of distinct cell types in a sensory-adhesive organ important for metamorphosis in tunicate larvae.

The papillae of tunicate larvae contribute sensory, adhesive, and metamorphosis-regulating functions that are crucial for the biphasic lifestyle of these marine, non-vertebrate chordates. We have identified additional molecular markers for at least 5 distinct cell types in the papillae of the model tunicate Ciona, allowing us to further study the development of these organs. Using tissue-specific CRISPR/Cas9-mediated mutagenesis and other molecular perturbations, we reveal the roles of key transcription factors and signaling pathways that are important for patterning the papilla territory into a highly organized array of different cell types and shapes. We further test the contributions of different transcription factors and cell types to the production of the adhesive glue that allows for larval attachment during settlement, and to the processes of tail retraction and body rotation during metamorphosis. With this study, we continue working towards connecting gene regulation to cellular functions that control the developmental transition between the motile larva and sessile adult of Ciona.

Optimizing CRISPR/Cas9 approaches in the polymorphic tunicate Ciona intestinalis.

CRISPR/Cas9 became a powerful tool for genetic engineering and in vivo knockout also in the invertebrate chordate Ciona intestinalis. Ciona (ascidians, tunicates) is an important model organism because it shares developmental features with the vertebrates, considered the sister group of tunicates, and offers outstanding experimental advantages: a compact genome and an invariant developmental cell lineage that, combined with electroporation mediated transgenesis allows for precise and cell type specific targeting in vivo. A high polymorphism and the mosaic expression of electroporated constructs, however, often hamper the efficient CRISPR knockout, and an optimization in Ciona is desirable. Furthermore, seasonality and artificial maintenance settings can profit from in vitro approaches that would save on animals. Here we present improvements for the CRISPR/Cas9 protocol in silico, in vitro and in vivo. Firstly, in designing sgRNAs, prior sequencing of target genomic regions from experimental animals and alignment with reference genomes of C. robusta and C. intestinalis render a correction possible of subspecies polymorphisms. Ideally, the screening for efficient and non-polymorphic sgRNAs will generate a database compatible for worldwide Ciona populations. Secondly, we challenged in vitro assays for sgRNA validation towards reduced in vivo experimentation and report their suitability but also overefficiency concerning mismatch tolerance. Thirdly, when comparing Cas9 with Cas9:Geminin, thought to synchronize editing and homology-direct repair, we could indeed increase the in vivo efficiency and notably the access to an early expressed gene. Finally, for in vivo CRISPR, genotyping by next generation sequencing (NGS) ex vivo streamlined the definition of efficient single guides. Double CRISPR then generates large deletions and reliable phenotypic excision effects. Overall, while these improvements render CRISPR more efficient in Ciona, they are useful when newly establishing the technique and very transferable to CRISPR in other organisms.

Gata is ubiquitously required for the earliest zygotic gene transcription in the ascidian embryo.

In ascidian embryos, the earliest transcription from the zygotic genome begins between the 8-cell and 16-cell stages. Gata.a, a maternally expressed Gata transcription factor, activates target genes specifically in the animal hemisphere, whereas the complex of ß-catenin and Tcf7 antagonizes the activity of Gata.a and activates target genes specifically in the vegetal hemisphere. Here, we show that genes zygotically expressed at the 16-cell stage have significantly more Gata motifs in their upstream regions. These genes included not only genes with animal hemisphere-specific expression but also genes with vegetal hemisphere-specific expression. On the basis of this finding, we performed knockdown experiments for Gata.a and reporter assays, and found that Gata.a is required for the expression of not only genes with animal hemisphere-specific expression, but also genes with vegetal hemisphere-specific expression. Our data indicated that weak Gata.a activity that cannot induce animal hemisphere-specific expression can allow ß-catenin/Tcf7 targets to be expressed in the vegetal cells. Because genes zygotically expressed at the 32-cell stage also had significantly more Gata motifs in their upstream regions, Gata.a function may not be limited to the genes expressed specifically in the animal or vegetal hemispheres at the 16-cell stage, and Gata.a may play an important role in the earliest transcription of the zygotic genome.

Papillae revisited and the nature of the adhesive secreting collocytes.

Ascidian papillae (palps) constitute a transient sensory adhesive organ that assures larval settlement and the onset of metamorphosis to the filterfeeding adult. Despite the importance of papillae for the ascidian development, their cellular composition is only roughly described. For Ciona intestinalis/robusta, a clear definition of cell numbers and discriminative molecular markers for the different cell types is missing. While some attention was given to neural cell types and their connectivity little is known about the adhesive producing collocytes. We converge serial-section electron microscopy and confocal imaging with various marker combinations to document the 3D organization of the Ciona papillae. We show the papillar development with 4 axial columnar cells (ACCs), 4 lateral primary sensory neurons (PSNs) and 12 central collocytes (CCs). We propose molecular markers for each cell type including novel ones for collocytes. The subcellular characteristics are suggestive of their role in papillar function: the ACCs featuring apical protrusions and microvilli, also contain neuroactive and endocytic vesicles indicative of a chemosensory role. They are clearly distinct from the ciliated glutamatergic PSNs. CCs encircle the ACCs and contain microvilli, small endocytic vesicles and notably a large numbers of adhesive granules that, according to element analysis and histochemistry, contain glycoproteins. Interestingly, we detect two different types of collocyte granules, one of them containing fibrous material and larger quantities of polysaccharides. Consistently, carbohydrate specific lectins label the papillar apex, the granules within CCs and the adhesive plaques upon larval attachment. We further propose CCs to derive from an evolutionary ancient neurosecretory cell type. Our findings contribute to understanding the development of the anterior ('new head') region of the Ciona larva and notably the adhesive secreting cells which has implications for developmental biology, cell differentiation and evolution, but also bioadhesion.

A Maternal System Initiating the Zygotic Developmental Program through Combinatorial Repression in the Ascidian Embryo.

Maternal factors initiate the zygotic developmental program in animal embryos. In embryos of the chordate, Ciona intestinalis, three maternal factors-Gata.a, ß-catenin, and Zic-r.a-are required to establish three domains of gene expression at the 16-cell stage; the animal hemisphere, vegetal hemisphere, and posterior vegetal domains. Here, we show how the maternal factors establish these domains. First, only ß-catenin and its effector transcription factor, Tcf7, are required to establish the vegetal hemisphere domain. Second, genes specifically expressed in the posterior vegetal domain have additional repressive cis-elements that antagonize the activity of ß-catenin/Tcf7. This antagonizing activity is suppressed by Zic-r.a, which is specifically localized in the posterior vegetal domain and binds to DNA indirectly through the interaction with Tcf7. Third, Gata.a directs specific gene expression in the animal hemisphere domain, because ß-catenin/Tcf7 weakens the Gata.a-binding activity for target sites through a physical interaction in the vegetal cells. Thus, repressive regulation through protein-protein interactions among the maternal transcription factors is essential to establish the first distinct domains of gene expression in the chordate embryo.

Correction: A Maternal System Initiating the Zygotic Developmental Program through Combinatorial Repression in the Ascidian Embryo.

[This corrects the article DOI: 10.1371/journal.pgen.1006045.].

A pipeline for the systematic identification of non-redundant full-ORF cDNAs for polymorphic and evolutionary divergent genomes: Application to the ascidian Ciona intestinalis.

Genome-wide resources, such as collections of cDNA clones encoding for complete proteins (full-ORF clones), are crucial tools for studying the evolution of gene function and genetic interactions. Non-model organisms, in particular marine organisms, provide a rich source of functional diversity. Marine organism genomes are, however, frequently highly polymorphic and encode proteins that diverge significantly from those of well-annotated model genomes. The construction of full-ORF clone collections from non-model organisms is hindered by the difficulty of predicting accurately the N-terminal ends of proteins, and distinguishing recent paralogs from highly polymorphic alleles. We report a computational strategy that overcomes these difficulties, and allows for accurate gene level clustering of transcript data followed by the automated identification of full-ORFs with correct 5'- and 3'-ends. It is robust to polymorphism, includes paralog calling and does not require evolutionary proximity to well annotated model organisms. We developed this pipeline for the ascidian Ciona intestinalis, a highly polymorphic member of the divergent sister group of the vertebrates, emerging as a powerful model organism to study chordate gene function, Gene Regulatory Networks and molecular mechanisms underlying human pathologies. Using this pipeline we have generated the first full-ORF collection for a highly polymorphic marine invertebrate. It contains 19,163 full-ORF cDNA clones covering 60% of Ciona coding genes, and full-ORF orthologs for approximately half of curated human disease-associated genes.

Stress granule-related genes during embryogenesis of an invertebrate chordate.

Controlling global protein synthesis through the assembly of stress granules represents a strategy adopted by eukaryotic cells to face various stress conditions. TIA 1-related nucleolysin (TIAR), tristetraprolin (TTP), and Ras-GTPase-activating protein SH3-domain-binding protein (G3BP) are key components of stress granules, allowing the regulation of mRNA stability, and thus controlling not only stress responses but also cell proliferation and differentiation. In this study, we aimed at investigating the roles of tiar, ttp, and g3bp during embryogenesis of the solitary ascidian Ciona robusta under both physiological and stress conditions. We carried out CRISPR/Cas9 to evaluate the effects of gene knockout on normal embryonic development, and gene reporter assay to study the time and tissue specificity of gene transcription, together with whole-mount in situ hybridization and quantitative real time PCR. To induce acute stress conditions, we used iron and cadmium as "essential" and "non-essential" metals, respectively. Our results highlight, for the first time, the importance of tiar, ttp, and g3bp in controlling the development of mesendodermal tissue derivatives during embryogenesis of an invertebrate chordate.

The ANISEED database: digital representation, formalization, and elucidation of a chordate developmental program.

Developmental biology aims to understand how the dynamics of embryonic shapes and organ functions are encoded in linear DNA molecules. Thanks to recent progress in genomics and imaging technologies, systemic approaches are now used in parallel with small-scale studies to establish links between genomic information and phenotypes, often described at the subcellular level. Current model organism databases, however, do not integrate heterogeneous data sets at different scales into a global view of the developmental program. Here, we present a novel, generic digital system, NISEED, and its implementation, ANISEED, to ascidians, which are invertebrate chordates suitable for developmental systems biology approaches. ANISEED hosts an unprecedented combination of anatomical and molecular data on ascidian development. This includes the first detailed anatomical ontologies for these embryos, and quantitative geometrical descriptions of developing cells obtained from reconstructed three-dimensional (3D) embryos up to the gastrula stages. Fully annotated gene model sets are linked to 30,000 high-resolution spatial gene expression patterns in wild-type and experimentally manipulated conditions and to 528 experimentally validated cis-regulatory regions imported from specialized databases or extracted from 160 literature articles. This highly structured data set can be explored via a Developmental Browser, a Genome Browser, and a 3D Virtual Embryo module. We show how integration of heterogeneous data in ANISEED can provide a system-level understanding of the developmental program through the automatic inference of gene regulatory interactions, the identification of inducing signals, and the discovery and explanation of novel asymmetric divisions.

Omics-based molecular analyses of adhesion by aquatic invertebrates.

Many aquatic invertebrates are associated with surfaces, using adhesives to attach to the substratum for locomotion, prey capture, reproduction, building or defence. Their intriguing and sophisticated biological glues have been the focus of study for decades. In all but a couple of specific taxa, however, the precise mechanisms by which the bioadhesives stick to surfaces underwater and (in many cases) harden have proved to be elusive. Since the bulk components are known to be based on proteins in most organisms, the opportunities provided by advancing 'omics technologies have revolutionised bioadhesion research. Time-consuming isolation and analysis of single molecules has been either replaced or augmented by the generation of massive data sets that describe the organism's translated genes and proteins. While these new approaches have provided resources and opportunities that have enabled physiological insights and taxonomic comparisons that were not previously possible, they do not provide the complete picture and continued multi-disciplinarity is essential. This review covers the various ways in which 'omics have contributed to our understanding of adhesion by aquatic invertebrates, with new data to illustrate key points. The associated challenges are highlighted and priorities are suggested for future research.

Complement system and phagocytosis in a colonial protochordate.

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

Formation of the ascidian epidermal sensory neurons: insights into the origin of the chordate peripheral nervous system.

The vertebrate peripheral nervous system (PNS) originates from neural crest and placodes. While its developmental origin is the object of intense studies, little is known concerning its evolutionary history. To address this question, we analyzed the formation of the larval tail PNS in the ascidian Ciona intestinalis. The tail PNS of Ciona is made of sensory neurons located within the epidermis midlines and extending processes in the overlying tunic median fin. We show that each midline corresponds to a single longitudinal row of epidermal cells and neurons sharing common progenitors. This simple organization is observed throughout the tail epidermis, which is made of only eight single-cell rows, each expressing a specific genetic program. We next demonstrate that the epidermal neurons are specified in two consecutive steps. During cleavage and gastrula stages, the dorsal and ventral midlines are independently induced by FGF9/16/20 and the BMP ligand ADMP, respectively. Subsequently, Delta/Notch-mediated lateral inhibition controls the number of neurons formed within these neurogenic regions. These results provide a comprehensive overview of PNS formation in ascidian and uncover surprising similarities between the fate maps and embryological mechanisms underlying formation of ascidian neurogenic epidermis midlines and the vertebrate median fin.

Identifying adhesive components in a model tunicate.

Tunicates populate a great variety of marine underwater substrates worldwide and represent a significant concern in marine shipping and aquaculture. Adhesives are secreted from the anterior papillae of their swimming larvae, which attach and metamorphose into permanently adhering, filter-feeding adults. We recently described the cellular composition of the sensory adhesive organ of the model tunicate Ciona intestinalis in great detail. Notably, the adhesive secretions of collocytes accumulate at the tip of the organ and contain glycoproteins. Here, we further explore the components of adhesive secretions and have screened for additional specificities that may influence adhesion or cohesion of the Ciona glue, including other carbohydrate moieties, catechols and substrate properties. We found a distinct set of sugar residues in the glue recognized by specific lectins with little overlap to other known marine adhesives. Surprisingly, we also detect catechol residues that likely originate from an adjacent cellular reservoir, the test cells. Furthermore, we provide information on substrate preferences where hydrophobicity outperforms charge in the attachment. Finally, we can influence the settlement process by the addition of hydrophilic heparin. The further analysis of tunicate adhesive strategies should provide a valuable knowledge source in designing physiological adhesives or green antifoulants. This article is part of the theme issue 'Transdisciplinary approaches to the study of adhesion and adhesives in biological systems'.

Embryo Microinjection and Electroporation in the Chordate Ciona intestinalis.

Simple model organisms are instrumental for in vivo studies of developmental and cellular differentiation processes. Currently, the evolutionary distance to man of conventional invertebrate model systems and the complexity of genomes in vertebrates are critical challenges to modeling human normal and pathological conditions. The chordate Ciona intestinalis is an invertebrate chordate that emerged from a common ancestor with the vertebrates and may represent features at the interface between invertebrates and vertebrates. A common body plan with much simpler cellular and genomic composition should unveil gene regulatory network (GRN) links and functional genomics readouts explaining phenomena in the vertebrate condition. The compact genome of Ciona, a fixed embryonic lineage with few divisions and large cells, combined with versatile community tools foster efficient gene functional analyses in this organism. Here, we present several crucial methods for this promising model organism, which belongs to the closest sister group to vertebrates. We present protocols for transient transgenesis by electroporation, along with microinjection-mediated gene knockdown, which together provide the means to study gene function and genomic regulatory elements. We extend our protocols to provide information on how community databases are utilized for in silico design of gene regulatory or gene functional experiments. An example study demonstrates how novel information can be gained on the interplay, and its quantification, of selected neural factors conserved between Ciona and man. Furthermore, we show examples of differential subcellular localization in embryonic cells, following DNA electroporation in Ciona zygotes. Finally, we discuss the potential of these protocols to be adapted for tissue specific gene interference with emerging gene editing methods. The in vivo approaches in Ciona overcome major shortcomings of classical model organisms in the quest of unraveling conserved mechanisms in the chordate developmental program, relevant to stem cell research, drug discovery, and subsequent clinical application.

Bioadhesion in ascidians: a developmental and functional genomics perspective.

The development of bioadhesives inspired from marine animals is a promising approach to generate new tissue-compatible medical components. A number of marine species, through their adhesive properties, also represent significant foulers that become increasingly problematic to aquaculture, shipping or local biodiversity. In order to develop more sophisticated man-made glues and/or efficient fouling resistant surfaces, it is important to understand the mechanical, structural and molecular properties of adhesive organs in selected species. Ascidians are marine invertebrates with larvae that opportunistically attach to almost any type of submerged surface to undergo metamorphosis into permanently sessile adults. Not only do they represent a globally important fouling organism, but they are becoming increasingly popular as model organisms for developmental biology. The latter is due to their phylogenetic position as the sister group to the vertebrates and their cellular and molecular accessibility for experimentation. In this paper, we review the mechanisms of larval adhesion in ascidians and draw conclusions from comparative analyses of selected species. We further discuss how knowledge from a developmental and functional genomics point of view can advance our understanding of cellular and molecular signatures and their hierarchical usage in animal adhesive organs.

Atypical Transcriptional Activation by TCF via a Zic Transcription Factor in C. elegans Neuronal Precursors.

Transcription factors of the TCF family are key mediators of the Wnt/ß-catenin pathway. TCF usually activates transcription on cis-regulatory elements containing TCF binding sites when the pathway is active and represses transcription when the pathway is inactive. However, some direct targets display an opposite regulation (activated by TCF in the absence of Wnt), but the mechanism behind this atypical regulation remains poorly characterized. Here, we use the cis-regulatory region of an opposite target gene, ttx-3, to dissect the mechanism of this atypical regulation. Using a combination of genetic, molecular, and biochemical experiments, we establish that, in the absence of Wnt pathway activation, TCF activates ttx-3 expression via a Zic binding site by forming a complex with a Zic transcription factor. This mechanism is later reinforced by specific bHLH factors. This study reveals an atypical mode of action for TCF that may apply to other binary decisions mediated by Wnt signaling.

Antagonizing retinoic acid and FGF/MAPK pathways control posterior body patterning in the invertebrate chordate Ciona intestinalis.

Vertebrate embryos exploit the mutual inhibition between the RA and FGF signalling pathways to coordinate the proliferative elongation of the main body axis with the progressive patterning and differentiation of its neuroectodermal and paraxial mesodermal structures. The evolutionary history of this patterning system is still poorly understood. Here, we investigate the role played by the RA and FGF/MAPK signals during the development of the tail structures in the tunicate Ciona intestinalis, an invertebrate chordate belonging to the sister clade of vertebrates, in which the prototypical chordate body plan is established through very derived morphogenetic processes. Ciona embryos are constituted of few cells and develop according to a fixed lineage; elongation of the tail occurs largely by rearrangement of postmitotic cells; mesoderm segmentation and somitogenesis are absent. We show that in the Ciona embryo, the antagonism of the RA and FGF/MAPK signals is required to control the anteroposterior patterning of the tail epidermis. We also demonstrate that the RA, FGF/MAPK and canonical Wnt pathways control the anteroposterior patterning of the tail peripheral nervous system, and reveal the existence of distinct subpopulations of caudal epidermal neurons with different responsiveness to the RA, FGF/MAPK and canonical Wnt signals. Our data provide the first demonstration that the use of the antagonism between the RA and FGF signals to pattern the main body axis predates the emergence of vertebrates and highlight the evolutionary plasticity of this patterning strategy, showing that in different chordates it can be used to pattern different tissues within the same homologous body region.