Model of neural induction in the ascidian embryo.

How cell specification can be controlled in a reproducible manner is a fundamental question in developmental biology. In ascidians, a group of invertebrate chordates, geometry plays a key role in achieving this control. Here, we use mathematical modeling to demonstrate that geometry dictates the neural-epidermal cell fate choice in the 32-cell stage ascidian embryo by a two-step process involving first the modulation of ERK signaling and second, the expression of the neural marker gene, Otx. The model describes signal transduction by the ERK pathway that is stimulated by FGF and attenuated by ephrin, and ERK-mediated control of Otx gene expression, which involves both an activator and a repressor of ETS-family transcription factors. Considering the measured area of cell surface contacts with FGF- or ephrin-expressing cells as inputs, the solutions of the model reproduce the experimental observations about ERK activation and Otx expression in the different cells under normal and perturbed conditions. Sensitivity analyses and computations of Hill coefficients allow us to quantify the robustness of the specification mechanism controlled by cell surface area and to identify the respective role played by each signaling input. Simulations also predict in which conditions the dual control of gene expression by an activator and a repressor that are both under the control of ERK can induce a robust ON/OFF control of neural fate induction.

Cell geometry, signal dampening, and a bimodal transcriptional response underlie the spatial precision of an ERK-mediated embryonic induction.

Precise control of lineage segregation is critical for the development of multicellular organisms, but our quantitative understanding of how variable signaling inputs are integrated to activate lineage-specific gene programs remains limited. Here, we show how precisely two out of eight ectoderm cells adopt neural fates in response to ephrin and FGF signals during ascidian neural induction. In each ectoderm cell, FGF signals activate ERK to a level that mirrors its cell contact surface with FGF-expressing mesendoderm cells. This gradual interpretation of FGF inputs is followed by a bimodal transcriptional response of the immediate early gene, Otx, resulting in its activation specifically in the neural precursors. At low levels of ERK, Otx is repressed by an ETS family transcriptional repressor, ERF2. Ephrin signals are critical for dampening ERK activation levels across ectoderm cells so that only neural precursors exhibit above-threshold levels, evade ERF repression, and "switch on" Otx transcription.

The alternative oxidase (AOX) increases sulphide tolerance in the highly invasive marine invertebrate Ciona intestinalis.

Ecological communities and biodiversity are shaped by both abiotic and biotic factors. This is well illustrated by extreme environments and invasive species. Besides naturally occurring sulphide-rich environments, global change can lead to an increase in hydrogen sulphide episodes that threaten many multicellular organisms. With the increase in the formation, size and abundance of oxygen minimum zones and hypoxic environments, bacterial-associated sulphide production is favoured and, as such, hydrogen-sulphide-rich environments are likely to also increase in size and abundance. Many species are challenged by the inhibiting effect of sulphide on aerobic energy production via cytochrome c oxidase, ultimately causing the death of the organism. Interestingly, many protist, yeast, plant and also animal species possess a sulphide-resistant alternative oxidase (AOX). In this study, we investigated whether AOX is functionally involved in the sulphide stress response of the highly invasive marine tunicate Ciona intestinalis. At the LC50, the sulphide-induced reduction of developmental success was three times stronger in AOX knock-down embryos than in control embryos. Further, AOX mRNA levels were higher under sulphide than under control conditions, and this effect increased during embryonic development. Together, we found that AOX is indeed functionally involved in the sulphide tolerance of C. intestinalis embryos, hence, very likely contributing to its invasive potential; and that the response of AOX to sulphide seems to be controlled at the transcriptional level. We suggest that AOX-possessing species play an important role in shaping marine ecological communities, and this importance may increase under ongoing global change.

Neuromesodermal Lineage Contribution to CNS Development in Invertebrate and Vertebrate Chordates.

Ascidians are invertebrate chordates and the closest living relative to vertebrates. In ascidian embryos a large part of the central nervous system arises from cells associated with mesoderm rather than ectoderm lineages. This seems at odds with the traditional view of vertebrate nervous system development which was thought to be induced from ectoderm cells, initially with anterior character and later transformed by posteriorizing signals, to generate the entire anterior-posterior axis of the central nervous system. Recent advances in vertebrate developmental biology, however, show that much of the posterior central nervous system, or spinal cord, in fact arises from cells that share a common origin with mesoderm. This indicates a conserved role for bi-potential neuromesoderm precursors in chordate CNS formation. However, the boundary between neural tissue arising from these distinct neural lineages does not appear to be fixed, which leads to the notion that anterior-posterior patterning and neural fate formation can evolve independently.

Chromosomal Inversion Polymorphisms in Two Sympatric Ascidian Lineages.

Chromosomal rearrangements can reduce fitness of heterozygotes and can thereby prevent gene flow. Therefore, such rearrangements can play a role in local adaptation and speciation. In particular, inversions are considered to be a major potential cause for chromosomal speciation. There are two closely related, partially sympatric lineages of ascidians in the genus Ciona, which we call type-A and type-B animals in the present study. Although these invertebrate chordates are largely isolated reproductively, hybrids can be found in wild populations, suggesting incomplete prezygotic barriers. Although the genome of type-A animals has been decoded and widely used, the genome for type-B animals has not been decoded at the chromosomal level. In the present study, we sequenced the genomes of two type-B individuals from different sides of the English Channel (in the zone of sympatry with type-A individuals) and compared them at the chromosomal level with the type-A genome. Although the overall structures were well conserved between type A and type B, chromosomal alignments revealed many inversions differentiating these two types of Ciona; it is probable that the frequent inversions have contributed to separation between these two lineages. In addition, comparisons of the genomes between the two type-B individuals revealed that type B had high rates of inversion polymorphisms and nucleotide polymorphisms, and thus type B might be in the process of differentiation into multiple new types or species. Our results suggest an important role of inversions in chromosomal speciation of these broadcasting spawners.

A Nodal/Eph signalling relay drives the transition from apical constriction to apico-basal shortening in ascidian endoderm invagination.

Gastrulation is the first major morphogenetic event during animal embryogenesis. Ascidian gastrulation starts with the invagination of 10 endodermal precursor cells between the 64- and late 112-cell stages. This process occurs in the absence of endodermal cell division and in two steps, driven by myosin-dependent contractions of the acto-myosin network. First, endoderm precursors constrict their apex. Second, they shorten apico-basally, while retaining small apical surfaces, thereby causing invagination. The mechanisms that prevent endoderm cell division, trigger the transition between step 1 and step 2, and drive apico-basal shortening have remained elusive. Here, we demonstrate a conserved role for Nodal and Eph signalling during invagination in two distantly related ascidian species, Phallusia mammillata and Ciona intestinalis Specifically, we show that the transition to step 2 is triggered by Nodal relayed by Eph signalling. In addition, our results indicate that Eph signalling lengthens the endodermal cell cycle, independently of Nodal. Finally, we find that both Nodal and Eph signals are dispensable for endoderm fate specification. These results illustrate commonalities as well as differences in the action of Nodal during ascidian and vertebrate gastrulation.

Transcriptional regulation of the Ciona Gsx gene in the neural plate.

The ascidian neural plate consists of a defined number of identifiable cells organized in a grid of rows and columns, representing a useful model to investigate the molecular mechanisms controlling neural patterning in chordates. Distinct anterior brain lineages are specified via unique combinatorial inputs of signalling pathways with Nodal and Delta-Notch signals patterning along the medial-lateral axis and FGF/MEK/ERK signals patterning along the anterior-posterior axis of the neural plate. The Ciona Gsx gene is specifically expressed in the a9.33 cells in the row III/column 2 position of anterior brain lineages, characterised by a combinatorial input of Nodal-OFF, Notch-ON and FGF-ON. Here, we identify the minimal cis-regulatory element (CRE) of 376 bp, which can recapitulate the early activation of Gsx. We show that this minimal CRE responds in the same way as the endogenous Gsx gene to manipulation of FGF- and Notch-signalling pathways and to overexpression of Snail, a mediator of Nodal signals, and Six3/6, which is required to demarcate the anterior boundary of Gsx expression at the late neurula stage. We reveal that sequences proximal to the transcription start site include a temporal regulatory element required for the precise transcriptional onset of gene expression. We conclude that sufficient spatial and temporal information for Gsx expression is integrated in 376 bp of non-coding cis-regulatory sequences.

Practical Guide for Ascidian Microinjection: Phallusia mammillata.

Phallusia mammillata has recently emerged as a new ascidian model. Its unique characteristics, including the optical transparency of eggs and embryos and efficient translation of exogenously introduced mRNA in eggs, make the Phallusia system suitable for fluorescent protein (FP)-based imaging approaches. In addition, genomic and transcriptomic resources are readily available for this ascidian species, facilitating functional gene studies. Microinjection is probably the most versatile technique for introducing exogenous molecules such as plasmids, mRNAs, and proteins into ascidian eggs/embryos. However, it is not practiced widely within the community; presumably, because the system is rather laborious to set up and it requires practice. Here, we describe in as much detail as possible two microinjection methods that we use daily in the laboratory: one based on an inverted microscope and the other on a stereomicroscope. Along the stepwise description of system setup and injection procedure, we provide practical tips in the hope that this chapter might be a useful guide for introducing or improving a microinjection setup.

Patterning of brain precursors in ascidian embryos.

In terms of their embryonic origins, the anterior and posterior parts of the ascidian central nervous system (CNS) are associated with distinct germ layers. The anterior part of the sensory vesicle, or brain, originates from ectoderm lineages following a neuro-epidermal binary fate decision. In contrast, a large part of the remaining posterior CNS is generated following neuro-mesodermal binary fate decisions. Here, we address the mechanisms that pattern the anterior brain precursors along the medial-lateral axis (future ventral-dorsal) at neural plate stages. Our functional studies show that Nodal signals are required for induction of lateral genes, including Delta-like, Snail, Msxb and Trp Delta-like/Notch signalling induces intermediate (Gsx) over medial (Meis) gene expression in intermediate cells, whereas the combinatorial action of Snail and Msxb prevents the expression of Gsx in lateral cells. We conclude that despite the distinct embryonic lineage origins within the larval CNS, the mechanisms that pattern neural precursors are remarkably similar.

Antagonism between ß-catenin and Gata.a sequentially segregates the germ layers of ascidian embryos.

Many animal embryos use nuclear ß-catenin (nß-catenin) during the segregation of endomesoderm (or endoderm) from ectoderm. This mechanism is thus likely to be evolutionarily ancient. In the ascidian embryo, nß-catenin reiteratively drives binary fate decisions between ectoderm and endomesoderm at the 16-cell stage, and then between endoderm and margin (mesoderm and caudal neural) at the 32-cell stage. At the 16-cell stage, nß-catenin activates endomesoderm genes in the vegetal hemisphere. At the same time, nß-catenin suppresses the DNA-binding activity of a maternal transcription factor, Gata.a, through a physical interaction, and Gata.a thereby activates its target genes only in the ectodermal lineage. In the present study, we found that this antagonism between nß-catenin and Gata.a also operates during the binary fate switch at the 32-cell stage. Namely, in marginal cells where nß-catenin is absent, Gata.a directly activates its target, Zic-r.b (ZicL), to specify the marginal cell lineages. Thus, the antagonistic action between nß-catenin and Gata.a is involved in two consecutive stages of germ layer segregation in ascidian embryos.

Physical association between a novel plasma-membrane structure and centrosome orients cell division.

In the last mitotic division of the epidermal lineage in the ascidian embryo, the cells divide stereotypically along the anterior-posterior axis. During interphase, we found that a unique membrane structure invaginates from the posterior to the centre of the cell, in a microtubule-dependent manner. The invagination projects toward centrioles on the apical side of the nucleus and associates with one of them. Further, a cilium forms on the posterior side of the cell and its basal body remains associated with the invagination. A laser ablation experiment suggests that the invagination is under tensile force and promotes the posterior positioning of the centrosome. Finally, we showed that the orientation of the invaginations is coupled with the polarized dynamics of centrosome movements and the orientation of cell division. Based on these findings, we propose a model whereby this novel membrane structure orchestrates centrosome positioning and thus the orientation of cell division axis.

Co-expression of Foxa.a, Foxd and Fgf9/16/20 defines a transient mesendoderm regulatory state in ascidian embryos.

In many bilaterian embryos, nuclear ß-catenin (nß-catenin) promotes mesendoderm over ectoderm lineages. Although this is likely to represent an evolutionary ancient developmental process, the regulatory architecture of nß-catenin-induced mesendoderm remains elusive in the majority of animals. Here, we show that, in ascidian embryos, three nß-catenin transcriptional targets, Foxa.a, Foxd and Fgf9/16/20, are each required for the correct initiation of both the mesoderm and endoderm gene regulatory networks. Conversely, these three factors are sufficient, in combination, to produce a mesendoderm ground state that can be further programmed into mesoderm or endoderm lineages. Importantly, we show that the combinatorial activity of these three factors is sufficient to reprogramme developing ectoderm cells to mesendoderm. We conclude that in ascidian embryos, the transient mesendoderm regulatory state is defined by co-expression of Foxa.a, Foxd and Fgf9/16/20.

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.

Distinct modes of mitotic spindle orientation align cells in the dorsal midline of ascidian embryos.

The orientation of cell division can have important consequences on the choice of cell fates adopted by each daughter cell as well as on the architecture of the tissue within which the dividing cell resides. We have studied in detail the oriented cell divisions that take place in the dorsal midline of the ascidian embryo. The dorsal midline cells of the ascidian embryo emerge following an asymmetric cell division oriented along the animal-vegetal (A-V) axis. This division generates the NN (Notochord-Neural) cell at the margin and the E (Endoderm) cell more vegetally. Deviating from the default mode of cell division, these sister cells divide again along the A-V axis to generate a column of four cells. We describe these cell divisions in detail. We show that the NN cell mitotic spindle rotates 90° to align along the A-V axis while the E cell spindle forms directly along the axis following the asymmetric migration of its centrosomes. We combine live imaging, embryo manipulations and pharmacological modulation of cytoskeletal elements to address the mechanisms underlying these distinct subcellular behaviours. Our evidence suggests that, in E cells, aster asymmetry together with the E cell shape contribute to the asymmetric centrosome migration. In NN cells, an intrinsic cytoplasmic polarisation of the cell results in the accumulation of dynein to the animal pole side. Our data support a model in which a dynein-dependent directional cytoplasmic pulling force may be responsible for the NN cell spindle rotation.

Snail mediates medial-lateral patterning of the ascidian neural plate.

The ascidian neural plate exhibits a regular, grid-like arrangement of cells. Patterning of the neural plate across the medial-lateral axis is initiated by bilateral sources of Nodal signalling, such that Nodal signalling induces expression of lateral neural plate genes and represses expression of medial neural plate genes. One of the earliest lateral neural plate genes induced by Nodal signals encodes the transcription factor Snail. Here, we show that Snail is a critical downstream factor mediating this Nodal-dependent patterning. Using gain and loss of function approaches, we show that Snail is required to repress medial neural plate gene expression at neural plate stages and to maintain the lateral neural tube genetic programme at later stages. A comparison of these results to those obtained following Nodal gain and loss of function indicates that Snail mediates a subset of Nodal functions. Consistently, overexpression of Snail can partially rescue a Nodal inhibition phenotype. We conclude that Snail is an early component of the gene regulatory network, initiated by Nodal signals, that patterns the ascidian neural plate.

Ephrin-mediated restriction of ERK1/2 activity delimits the number of pigment cells in the Ciona CNS.

Recent evidence suggests that ascidian pigment cells are related to neural crest-derived melanocytes of vertebrates. Using live-imaging, we determine a revised cell lineage of the pigment cells in Ciona intestinalis embryos. The neural precursors undergo successive rounds of anterior-posterior (A-P) oriented cell divisions, starting at the blastula 64-cell stage. A previously unrecognized fourth A-P oriented cell division in the pigment cell lineage leads to the generation of the post-mitotic pigment cell precursors. We provide evidence that MEK/ERK signals are required for pigment cell specification until approximately 30min after the final cell division has taken place. Following each of the four A-P oriented cell divisions, ERK1/2 is differentially activated in the posterior sister cells, into which the pigment cell lineage segregates. Eph/ephrin signals are critical during the third A-P oriented cell division to spatially restrict ERK1/2 activation to the posterior daughter cell. Targeted inhibition of Eph/ephrin signals results in, at neurula stages, anterior expansion of both ERK1/2 activation and a pigment cell lineage marker and subsequently, at larval stages, supernumerary pigment cells. We discuss the implications of these findings with respect to the evolution of the vertebrate neural crest.

p120RasGAP mediates ephrin/Eph-dependent attenuation of FGF/ERK signals during cell fate specification in ascidian embryos.

ERK1/2 MAP kinase exhibits a highly dynamic activation pattern in developing embryos, which largely depends on fibroblast growth factor (FGF) signals. In ascidian embryos, FGF-dependent activation of ERK1/2 occurs differentially between sister cells during marginal zone and neural lineage patterning. Selective attenuation of FGF signals by localised ephrin/Eph signals accounts for this differential ERK activation, which controls the binary fate choice of each sibling cell pair. Here, we show that p120 Ras GTPase-activating protein (p120RasGAP) is a crucial mediator of these ephrin/Eph signals. First, inhibition of p120RasGAP has a similar effect to inhibition of ephrin/Eph function during marginal zone and neural patterning. Second, p120RasGAP acts epistatically to ephrin/Eph signals. Third, p120RasGAP physically associates with Eph3 in an ephrin-dependent manner. This study provides the first in vivo evidence that the functional association between Eph and RasGAP controls the spatial extent of FGF-activated ERK.

Practical tips for imaging ascidian embryos.

Decades of studies on ascidian embryogenesis have culminated in deciphering the first gene regulatory "blueprint" for the generation of all major larval tissue types in chordates. However, the current gene regulatory network (GRN) is not well integrated with the morphogenetic and cellular processes that are also taking place during embryogenesis. Describing these processes represents a major on-going challenge, aided by recent advances in imaging and fluorescent protein (FP) technologies. In this report, we describe the application of these technologies to the developmental biology of ascidians and provide a detailed practical guide on the preparation of ascidian embryos for imaging.

A dynamic history of gene duplications and losses characterizes the evolution of the SPARC family in eumetazoans.

The vertebrates share the ability to produce a skeleton made of mineralized extracellular matrix. However, our understanding of the molecular changes that accompanied their emergence remains scarce. Here, we describe the evolutionary history of the SPARC (secreted protein acidic and rich in cysteine) family, because its vertebrate orthologues are expressed in cartilage, bones and teeth where they have been proposed to bind calcium and act as extracellular collagen chaperones, and because further duplications of specific SPARC members produced the small calcium-binding phosphoproteins (SCPP) family that is crucial for skeletal mineralization to occur. Both phylogeny and synteny conservation analyses reveal that, in the eumetazoan ancestor, a unique ancestral gene duplicated to give rise to SPARC and SPARCB described here for the first time. Independent losses have eliminated one of the two paralogues in cnidarians, protostomes and tetrapods. Hence, only non-tetrapod deuterostomes have conserved both genes. Remarkably, SPARC and SPARCB paralogues are still linked in the amphioxus genome. To shed light on the evolution of the SPARC family members in chordates, we performed a comprehensive analysis of their embryonic expression patterns in amphioxus, tunicates, teleosts, amphibians and mammals. Our results show that in the chordate lineage SPARC and SPARCB family members were recurrently recruited in a variety of unrelated tissues expressing collagen genes. We propose that one of the earliest steps of skeletal evolution involved the co-expression of SPARC paralogues with collagenous proteins.

ß-Catenin-driven binary fate specification segregates germ layers in ascidian embryos.

ß-catenin is a transcriptional cofactor mediating the "canonical" Wnt signaling pathway, which activates target genes in a complex with TCF (LEF) transcription factors [1]. In many metazoans, embryos are first subdivided during early cleavage stages into nuclear ß-catenin-positive and -negative domains, with ß-catenin specifying endoderm or mesendoderm fate. This process has been demonstrated in a wide range of phyla including cnidarians, nemerteans, and invertebrate deuterostomes (echinoderms, hemichordates, and ascidians), implying that ß-catenin-dependent (mes)endoderm specification is evolutionarily ancient [2-10]. However, the mechanisms leading to the segregation of mesoderm and endoderm fates from a transient mesendodermal state are less well defined. We show that subdivision of the ascidian embryo into the three germ layers involves differential nuclear ß-catenin activity coupled with the first two animal-vegetal (A-V)-oriented cell divisions. We reveal that each of these A-V divisions operates as a binary fate choice: the first between ectoderm and mesendoderm and the second between margin (notochord and neural) and endoderm, such that a ß-catenin activation sequence of ON-to-ON specifies endoderm, OFF-to-OFF ectoderm, and ON-to-OFF margin.