Molecular control of cellulosic fin morphogenesis in ascidians.

BACKGROUND: The tunicates form a group of filter-feeding marine animals closely related to vertebrates. They share with them a number of features such as a notochord and a dorsal neural tube in the tadpole larvae of ascidians, one of the three groups that make tunicates. However, a number of typical chordate characters have been lost in different branches of tunicates, a diverse and fast-evolving phylum. Consequently, the tunic, a sort of exoskeleton made of extracellular material including cellulose secreted by the epidermis, is the unifying character defining the tunicate phylum. In the larva of ascidians, the tunic differentiates in the tail into a median fin (with dorsal and ventral extended blades) and a caudal fin. RESULTS: Here we have performed experiments in the ascidian Phallusia mammillata to address the molecular control of tunic 3D morphogenesis. We have demonstrated that the tail epidermis medio-lateral patterning essential for peripheral nervous system specification also controls tunic elongation into fins. More specifically, when tail epidermis midline identity was abolished by BMP signaling inhibition, or CRISPR/Cas9 inactivation of the transcription factor coding genes Msx or Klf1/2/4/17, median fin did not form. We postulated that this genetic program should regulate effectors of tunic secretion. We thus analyzed the expression and regulation in different ascidian species of two genes acquired by horizontal gene transfer (HGT) from bacteria, CesA coding for a cellulose synthase and Gh6 coding for a cellulase. We have uncovered an unexpected dynamic history of these genes in tunicates and high levels of variability in gene expression and regulation among ascidians. Although, in Phallusia, Gh6 has a regionalized expression in the epidermis compatible with an involvement in fin elongation, our functional studies indicate a minor function during caudal fin formation only. CONCLUSIONS: Our study constitutes an important step in the study of the integration of HGT-acquired genes into developmental networks and a cellulose-based morphogenesis of extracellular material in animals.

Genomic Resources and Annotations for a Colonial Ascidian, the Light-Bulb Sea Squirt Clavelina lepadiformis.

Ascidian embryos have been studied since the birth of experimental embryology at the end of the 19th century. They represent textbook examples of mosaic development characterized by a fast development with very few cells and invariant cleavage patterns and lineages. Ascidians belong to tunicates, the vertebrate sister group, and their study is essential to shed light on the emergence of vertebrates. Importantly, deciphering developmental gene regulatory networks has been carried out mostly in two of the three ascidian orders, Phlebobranchia and Stolidobranchia. To infer ancestral developmental programs in ascidians, it is thus essential to carry out molecular embryology in the third ascidian order, the Aplousobranchia. Here, we present genomic resources for the colonial aplousobranch Clavelina lepadiformis: a transcriptome produced from various embryonic stages, and an annotated genome. The assembly consists of 184 contigs making a total of 233.6 Mb with a N50 of 8.5 Mb and a L50 of 11. The 32,318 predicted genes capture 96.3% of BUSCO orthologs. We further show that these resources are suitable to study developmental gene expression and regulation in a comparative framework within ascidians. Additionally, they will prove valuable for evolutionary and ecological studies.

Regulation of anterior neurectoderm specification and differentiation by BMP signaling in ascidians.

The most anterior structure of the ascidian larva is made of three palps with sensory and adhesive functions essential for metamorphosis. They derive from the anterior neural border and their formation is regulated by FGF and Wnt. Given that they also share gene expression profiles with vertebrate anterior neural tissue and cranial placodes, their study should shed light on the emergence of the unique vertebrate telencephalon. We show that BMP signaling regulates two phases of palp formation in Ciona intestinalis. During gastrulation, the anterior neural border is specified in a domain of inactive BMP signaling, and activating BMP prevented its formation. During neurulation, BMP defines ventral palp identity and indirectly specifies the inter-papilla territory separating the ventral and dorsal palps. Finally, we show that BMP has similar functions in the ascidian Phallusia mammillata, for which we identified novel palp markers. Collectively, we provide a better molecular description of palp formation in ascidians that will be instrumental for comparative studies.

Highly distinct genetic programs for peripheral nervous system formation in chordates.

BACKGROUND: Vertebrates develop their peripheral nervous system (PNS) from transient unique embryonic structures, the neural crest, and the ectodermal placodes that are located at the border of the forming central nervous system. By contrast, in the invertebrate chordates, amphioxus and ascidians, a large part of the PNS originates at the opposite of the embryo, in the ventral ectoderm. In both groups, a biphasic mechanism regulates ventral PNS formation: high BMP levels specify a neurogenic territory within which glutamatergic epidermal sensory neuron formation is controlled by the Notch pathway. Given these similarities and the phylogenetic relationships within chordates, it is likely that ventral PNS is an ancestral feature in chordates and that it has been lost in vertebrates. RESULTS: In order to get insights into the molecular control of ventral PNS formation and to test the hypothesis of their homology and potential contribution to the emergence of vertebrate PNS, we undertook a close comparison of ventral PNS formation in the ascidian Phallusia mammillata and the amphioxus Branchiostoma lanceolatum. Using timed RNA-seq series, we identified novel markers of the ventral PNS during different phases of its development in both species. By extensively determining the expression of paralogous and orthologous genes, we observed that only a minority of genes have a shared expression in the ventral PNS. However, a large fraction of ventral PNS orthologous genes are expressed in the dorsally forming PNS of vertebrates. CONCLUSIONS: Our work has significantly increased the molecular characterization of ventral PNS formation in invertebrate chordates. The low observed conservation of gene expression in the ventral PNS suggests that the amphioxus and ascidian ventral PNS are either not homologous, or alternatively extensive drift has occurred in their regulatory mechanisms following a long period (600 My) of separate evolution and accelerated evolution in the ascidian lineage. The homology to genes expressed in the dorsally forming PNS of vertebrates suggests that ancestral sensory neurons gene networks have been redeployed in vertebrates.

En masse DNA Electroporation for in vivo Transcriptional Assay in Ascidian Embryos.

Ascidian embryos are powerful models for functional genomics, in particular, due to the ease of generating a large number of transgenic embryos by electroporation. In addition, the small size of their genome makes them an attractive model for studying cis-regulatory elements that control gene expression during embryonic development. Here, I describe the adaptation of the seminal method developed 25 years ago in Ciona robusta for en masse DNA electroporation for in vivo transcription to additional species belonging to three genera. It is likely that similar optimizations would make electroporation successful in other ascidian species, where in vitro fertilization can be performed on a large number of eggs.

Conservation of peripheral nervous system formation mechanisms in divergent ascidian embryos.

Ascidians with very similar embryos but highly divergent genomes are thought to have undergone extensive developmental system drift. We compared, in four species (Ciona and Phallusia for Phlebobranchia, Molgula and Halocynthia for Stolidobranchia), gene expression and gene regulation for a network of six transcription factors regulating peripheral nervous system (PNS) formation in Ciona. All genes, but one in Molgula, were expressed in the PNS with some differences correlating with phylogenetic distance. Cross-species transgenesis indicated strong levels of conservation, except in Molgula, in gene regulation despite lack of sequence conservation of the enhancers. Developmental system drift in ascidians is thus higher for gene regulation than for gene expression and is impacted not only by phylogenetic distance, but also in a clade-specific manner and unevenly within a network. Finally, considering that Molgula is divergent in our analyses, this suggests deep conservation of developmental mechanisms in ascidians after 390 My of separate evolution.

Antero-posterior ectoderm patterning by canonical Wnt signaling during ascidian development.

Wnt/ß-catenin signaling is an ancient pathway in metazoans and controls various developmental processes, in particular the establishment and patterning of the embryonic primary axis. In vertebrates, a graded Wnt activity from posterior to anterior endows cells with positional information in the central nervous system. Recent studies in hemichordates support a conserved role for Wnt/ß-catenin in ectoderm antero-posterior patterning at the base of the deuterostomes. Ascidians are marine invertebrates and the closest relatives of vertebrates. By combining gain- and loss-of-function approaches, we have determined the role of Wnt/ß-catenin in patterning the three ectoderm derivatives of the ascidian Ciona intestinalis, central nervous system, peripheral nervous system and epidermis. Activating Wnt/ß-catenin signaling from gastrulation led to a dramatic transformation of the ectoderm with a loss of anterior identities and a reciprocal anterior extension of posterior identities, consistent with studies in other metazoans. Surprisingly, inhibiting Wnt signaling did not produce a reciprocal anteriorization of the embryo with a loss of more posterior identities like in vertebrates and hemichordate. Epidermis patterning was overall unchanged. Only the identity of two discrete regions of the central nervous system, the anteriormost and the posteriormost regions, were under the control of Wnt. Finally, the caudal peripheral nervous system, while being initially Wnt dependent, formed normally. Our results show that the Ciona embryonic ectoderm responds to Wnt activation in a manner that is compatible with the proposed function for this pathway at the base of the deuterostomes. However, possibly because of its fast and divergent mode of development that includes extensive use of maternal determinants, the overall antero-posterior patterning of the Ciona ectoderm is Wnt independent, and Wnt/ß-catenin signaling controls the formation of some sub-domains. Our results thus indicate that there has likely been a drift in the developmental systems controlling ectoderm patterning in the lineage leading to ascidians.

Convergent Acquisition of Nonembryonic Development in Styelid Ascidians.

Asexual propagation and whole body regeneration are forms of nonembryonic development (NED) widespread across animal phyla and central in life history and evolutionary diversification of metazoans. Whereas it is challenging to reconstruct the gains or losses of NED at large phylogenetic scale, comparative studies could benefit from being conducted at more restricted taxonomic scale, in groups for which phylogenetic relationships are well established. The ascidian family of Styelidae encompasses strictly sexually reproducing solitary forms as well as colonial species that combine sexual reproduction with different forms of NED. To date, the phylogenetic relationships between colonial and solitary styelids remain controversial and so is the pattern of NED evolution. In this study, we built an original pipeline to combine eight genomes with 18 de novo assembled transcriptomes and constructed data sets of unambiguously orthologous genes. Using a phylogenomic super-matrix of 4,908 genes from these 26 tunicates we provided a robust phylogeny of this family of chordates, which supports two convergent acquisitions of NED. This result prompted us to further describe the budding process in the species Polyandrocarpa zorritensis, leading to the discovery of a novel mechanism of asexual development. Whereas the pipeline and the data sets produced can be used for further phylogenetic reconstructions in tunicates, the phylogeny provided here sets an evolutionary framework for future experimental studies on the emergence and disappearance of complex characters such as asexual propagation and whole body regeneration.

Anteroposterior axis patterning by early canonical Wnt signaling during hemichordate development.

The Wnt family of secreted proteins has been proposed to play a conserved role in early specification of the bilaterian anteroposterior (A/P) axis. This hypothesis is based predominantly on data from vertebrate embryogenesis as well as planarian regeneration and homeostasis, indicating that canonical Wnt (cWnt) signaling endows cells with positional information along the A/P axis. Outside of these phyla, there is strong support for a conserved role of cWnt signaling in the repression of anterior fates, but little comparative support for a conserved role in promotion of posterior fates. We further test the hypothesis by investigating the role of cWnt signaling during early patterning along the A/P axis of the hemichordate Saccoglossus kowalevskii. We have cloned and investigated the expression of the complete Wnt ligand and Frizzled receptor complement of S. kowalevskii during early development along with many secreted Wnt modifiers. Eleven of the 13 Wnt ligands are ectodermally expressed in overlapping domains, predominantly in the posterior, and Wnt antagonists are localized predominantly to the anterior ectoderm in a pattern reminiscent of their distribution in vertebrate embryos. Overexpression and knockdown experiments, in combination with embryological manipulations, establish the importance of cWnt signaling for repression of anterior fates and activation of mid-axial ectodermal fates during the early development of S. kowalevskii. However, surprisingly, terminal posterior fates, defined by posterior Hox genes, are unresponsive to manipulation of cWnt levels during the early establishment of the A/P axis at late blastula and early gastrula. We establish experimental support for a conserved role of Wnt signaling in the early specification of the A/P axis during deuterostome body plan diversification, and further build support for an ancestral role of this pathway in early evolution of the bilaterian A/P axis. We find strong support for a role of cWnt in suppression of anterior fates and promotion of mid-axial fates, but we find no evidence that cWnt signaling plays a role in the early specification of the most posterior axial fates in S. kowalevskii. This posterior autonomy may be a conserved feature of early deuterostome axis specification.

Coupling molecular data and experimental crosses sheds light about species delineation: a case study with the genus Ciona.

Molecular studies sometimes reveal evolutionary divergence within accepted species. Such findings can initiate taxonomic revision, as exemplified in the formerly recognized species Ciona intestinalis. While an increasing number of studies have examined the ecology, reproductive barriers and genetics of C. intestinalis and C. robusta, there are still much uncertainties regarding other species of this genus. Using experimental crosses and mitochondrial data, we investigated the evolutionary relationships among four native and introduced Ciona spp., found in sympatry in the Mediterranean Sea or English Channel. Outcome of 62 bi-parental reciprocal crosses between C. intestinalis, C. robusta, C. roulei and C. edwardsi showed that C. edwardsi is reproductively isolated from the other taxa, which is in agreement with its distinct location in the phylogenetic tree. Conversely, hybrids are easily obtained in both direction when crossing C. intestinalis and C. roulei, reinforcing the hypothesis of two genetically differentiated lineages but likely being from a same species. Altogether, this study sheds light on the evolutionary relationship in this complex genus. It also calls for further investigation notably based on genome-wide investigation to better describe the evolutionary history within the genus Ciona, a challenging task in a changing world where biological introductions are shuffling species distribution.

Msxb is a core component of the genetic circuitry specifying the dorsal and ventral neurogenic midlines in the ascidian embryo.

The tail ascidian larval peripheral nervous system is made up of epidermal sensory neurons distributed more or less regularly in ventral and dorsal midlines. Their formation occurs in two-steps: the ventral and dorsal midlines are induced as neurogenic territories by Fgf9/16/20 and Admp respectively. The Delta2/Notch interaction then controls the number of neurons that form. The genetic machinery acting between the inductive processes taking place before gastrulation and neuron specification at tailbud stages are largely unknown. The analysis of seven transcription factors expressed in the forming midlines revealed an unexpected complexity and dynamic of gene expression. Their systematic overexpression confirmed that these genes do not interact following a linear cascade of activation. However, the integration of our data revealed the distinct key roles of the two upstream factors Msxb and Nkx-C that are the earliest expressed genes and the only ones able to induce neurogenic midline and ESN formation. Our data suggest that Msxb would be the primary midline gene integrating inputs from the ventral and dorsal inducers and launching a pan-midline transcriptional program. Nkx-C would be involved in tail tip specification, in maintenance of the pan-midline network and in a posterior to anterior wave controlling differentiation.

An otx/nodal regulatory signature for posterior neural development in ascidians.

In chordates, neural induction is the first step of a complex developmental process through which ectodermal cells acquire a neural identity. In ascidians, FGF-mediated neural induction occurs at the 32-cell stage in two blastomere pairs, precursors respectively of anterior and posterior neural tissue. We combined molecular embryology and cis-regulatory analysis to unveil in the ascidian Ciona intestinalis the remarkably simple proximal genetic network that controls posterior neural fate acquisition downstream of FGF. We report that the combined action of two direct FGF targets, the TGFß factor Nodal, acting via Smad- and Fox-binding sites, and the transcription factor Otx suffices to trigger ascidian posterior neural tissue formation. Moreover, we found that this strategy is conserved in the distantly related ascidian Phallusia mammillata, in spite of extreme sequence divergence in the cis-regulatory sequences involved. Our results thus highlight that the modes of gene regulatory network evolution differ with the evolutionary scale considered. Within ascidians, developmental regulatory networks are remarkably robust to genome sequence divergence. Between ascidians and vertebrates, major fate determinants, such as Otx and Nodal, can be co-opted into different networks. Comparative developmental studies in ascidians with divergent genomes will thus uncover shared ascidian strategies, and contribute to a better understanding of the diversity of developmental strategies within chordates.

ß-catenin specifies the endomesoderm and defines the posterior organizer of the hemichordate Saccoglossus kowalevskii.

The canonical Wnt/ß-catenin pathway is a key regulator of body plan organization and axis formation in metazoans, being involved in germ layer specification, posterior growth and patterning of the anteroposterior axis. Results from animals spanning a wide phylogenetic range suggest that a unifying function of ß-catenin in metazoans is to define the posterior/vegetal part of the embryo. Although the specification of vegetal territories (endoderm) by ß-catenin has been demonstrated in distantly related animals (cnidarians, a protostome, echinoderms and ascidians), the definition of the posterior part of the embryo is well supported only for vertebrates and planarians. To gain insights into ß-catenin functions during deuterostome evolution, we have studied the early development of the direct developing hemichordate Saccoglossus kowalevskii. We show that the zygote is polarized after fertilization along the animal-vegetal axis by cytoplasmic rearrangements resembling the ascidian vegetal contraction. This early asymmetry is translated into nuclear accumulation of ß-catenin at the vegetal pole, which is necessary and sufficient to specify endomesoderm. We show that endomesoderm specification is crucial for anteroposterior axis establishment in the ectoderm. The endomesoderm secretes as yet unidentified signals that posteriorize the ectoderm, which would otherwise adopt an anterior fate. Our results point to a conserved function at the base of deuterostomes for ß-catenin in germ layer specification and to a causal link in the definition of the posterior part of the embryonic ectoderm by way of activating posteriorizing endomesodermal factors. Consequently, the definition of the vegetal and the posterior regions of the embryo by ß-catenin should be distinguished and carefully re-examined.

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.

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.

From experimental imaging techniques to virtual embryology.

Modern embryology increasingly relies on descriptive and functional three dimensional (3D) and four dimensional (4D) analysis of physically, optically, or virtually sectioned specimens. To cope with the technical requirements, new methods for high detailed in vivo imaging, as well as the generation of high resolution digital volume data sets for the accurate visualisation of transgene activity and gene product presence, in the context of embryo morphology, were recently developed and are under construction. These methods profoundly change the scientific applicability, appearance and style of modern embryo representations. In this paper, we present an overview of the emerging techniques to create, visualise and administrate embryo representations (databases, digital data sets, 3-4D embryo reconstructions, models, etc.), and discuss the implications of these new methods on the work of modern embryologists, including, research, teaching, the selection of specific model organisms, and potential collaborators.

A conserved role for the MEK signalling pathway in neural tissue specification and posteriorisation in the invertebrate chordate, the ascidian Ciona intestinalis.

Ascidians are invertebrate chordates with a larval body plan similar to that of vertebrates. The ascidian larval CNS is divided along the anteroposterior axis into sensory vesicle, neck, visceral ganglion and tail nerve cord. The anterior part of the sensory vesicle comes from the a-line animal blastomeres, whereas the remaining CNS is largely derived from the A-line vegetal blastomeres. We have analysed the role of the Ras/MEK/ERK signalling pathway in the formation of the larval CNS in the ascidian, Ciona intestinalis. We show evidence that this pathway is required, during the cleavage stages, for the acquisition of: (1) neural fates in otherwise epidermal cells (in a-line cells); and (2) the posterior identity of tail nerve cord precursors that otherwise adopt a more anterior neural character (in A-line cells). Altogether, the MEK signalling pathway appears to play evolutionary conserved roles in these processes in ascidians and vertebrates, suggesting that this may represent an ancestral chordate strategy.

Notch signaling is involved in nervous system formation in ascidian embryos.

Notch signaling plays crucial roles during embryogenesis in various metazoans. HrNotch, a Notch homologue in the ascidian Halocynthia roretzi, has been previously cloned, and its expression pattern suggests that HrNotch signaling is involved in nervous system formation. To determine the function of HrNotch signaling, in the present study we examined the effects of the constitutively activated forms of HrNotch. Overexpression resulted in larvae with defects in neural tube closure and brain vesicle formation. In embryos expressing the activated HrNotch, the expression of a neural marker gene, HrETR-1, was enhanced and expanded in the central nervous system, although ectopic expression decreased during the tailbud stage. The activated HrNotch also suppressed the formation of the adhesive organ (palps) and the peripheral nervous system, which consists of ciliary mechanosensory neurons, whereas it promoted epidermal differentiation. The suppression and promotion of the formation of these respective cell types were confirmed by examination of the expression of relevant tissue-specific markers. We also cloned Hrdelta, an ascidian homologue of DSL family genes, which encode ligands for which Notch acts as a receptor. The expression of Hrdelta was observed in the precursors of palps and peripheral neurons in addition to the CNS. These results suggest that Notch signaling is important for ascidian nervous system formation and that it affects the fate choice between palps and epidermis and between peripheral neurons and epidermis within the neurogenic regions of the surface ectoderm by suppressing the formations of palps and peripheral neurons and promoting epidermal differentiation.