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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 five distinct cell types in the papillae of the model tunicateCiona,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 ofCiona.

Vertebrate myoblast fusion allows for multinucleated muscle fibers to compound the size and strength of mononucleated cells, but the evolution of this important process is unknown. We investigated the evolutionary origins and function of membrane-coalescing agents Myomaker and Myomixer in various groups of chordates. Here, we report that Myomaker likely arose through gene duplication in the last common ancestor of tunicates and vertebrates, while Myomixer appears to have evolved de novo in early vertebrates. Functional tests revealed a complex evolutionary history of myoblast fusion. A prevertebrate phase of muscle multinucleation driven by Myomaker was followed by the later emergence of Myomixer that enables the highly efficient fusion system of vertebrates. Evolutionary comparisons between vertebrate and nonvertebrate Myomaker revealed key structural and mechanistic insights into myoblast fusion. Thus, our findings suggest an evolutionary model of chordate fusogens and illustrate how new genes shape the emergence of novel morphogenetic traits and mechanisms.

Background The evolution of vertebrate smooth muscles is obscured by lack of identifiable smooth muscle-like cells in tunicates, the invertebrates most closely related to vertebrates. A recent evolutionary model was proposed in which smooth muscles arose before the last bilaterian common ancestor, and were later diversified, secondarily lost or modified in the branches leading to extant animal taxa. However, there is currently no data from tunicates to support this scenario. Methods and results Here, we show that the axial columnar cells, a unique cell type in the adhesive larval papillae of the tunicate Ciona, are enriched for orthologs of vertebrate smooth/non-muscle-specific effectors of contractility, in addition to developing from progenitors that express conserved cardiomyocyte regulatory factors. We show that these cells contract during the retraction of the Ciona papillae during larval settlement and metamorphosis. Conclusions We propose that the axial columnar cells of Ciona are a myoepithelial cell type required for transducing external stimuli into mechanical forces that aid in the attachment of the motile larva to its final substrate. Furthermore, they share developmental and functional features with vertebrate myoepithelial cells, vascular smooth muscle cells, and cardiomyocytes. We discuss these findings in the context of the proposed models of vertebrate smooth muscle and cardiomyocyte evolution.

The central nervous system of the Ciona larva contains only 177 neurons. The precise regulation of neuron subtype-specific morphogenesis and differentiation observed during the formation of this minimal connectome offers a unique opportunity to dissect gene regulatory networks underlying chordate neurodevelopment. Here we compare the transcriptomes of two very distinct neuron types in the hindbrain/spinal cord homolog of Ciona, the Motor Ganglion (MG): the Descending decussating neuron (ddN, proposed homolog of Mauthner Cells in vertebrates) and the MG Interneuron 2 (MGIN2). Both types are invariantly represented by a single bilaterally symmetric left/right pair of cells in every larva. Supernumerary ddNs and MGIN2s were generated in synchronized embryos and isolated by fluorescence-activated cell sorting for transcriptome profiling. Differential gene expression analysis revealed ddN- and MGIN2-specific enrichment of a wide range of genes, including many encoding potential “effectors” of subtype-specific morphological and functional traits. More specifically, we identified the upregulation of centrosome-associated, microtubule-stabilizing/bundling proteins and extracellular guidance cues part of a single intrinsic regulatory program that might underlie the unique polarization of the ddNs, the only descending MG neurons that cross the midline. Consistent with our predictions, CRISPR/Cas9-mediated, tissue-specific elimination of two such candidate effectors, Efcab6-related and Netrin1, impaired ddN polarized axon outgrowth across the midline.

The size of an animal is determined by the size of its musculoskeletal system. Myoblast fusion is an innovative mechanism that allows for multinucleated muscle fibers to compound the size and strength of individual mononucleated cells. However, the evolutionary history of the control mechanism underlying this important process is currently unknown. The phylum Chordata hosts closely related groups that span distinct myoblast fusion states: no fusion in cephalochordates, restricted fusion and multinucleation in tunicates, and extensive, obligatory fusion in vertebrates. To elucidate how these differences may have evolved, we studied the evolutionary origins and function of membrane-coalescing agents Myomaker and Myomixer in various groups of chordates. Here we report that Myomaker likely arose through gene duplication in the last common ancestor of tunicates and vertebrates, while Myomixer appears to have evolved de novo in early vertebrates. Functional tests revealed an unexpectedly complex evolutionary history of myoblast fusion in chordates. A pre-vertebrate phase of muscle multinucleation driven by Myomaker was followed by the later emergence of Myomixer that enables the highly efficient fusion system of vertebrates. Thus, our findings reveal the evolutionary origins of chordate-specific fusogens and illustrate how new genes can shape the emergence of novel morphogenetic traits and mechanisms.

Ascidians are invertebrate chordates, with swimming chordate tadpole larvae that have distinct heads and tails. The head contains the small brain, sensory organs, including the ocellus (light) and otolith (gravity) and the presumptive endoderm, while the tail has a notochord surrounded by muscle cells and a dorsal nerve cord. One of the chordate features is a post-anal tail. Ascidian tadpoles are nonfeeding, and their tails are critical for larval locomotion. After hatching the larvae swim up toward light and are carried by the tide and ocean currents. When competent to settle, ascidian tadpole larvae swim down, away from light, to settle and metamorphose into a sessile adult. Tunicates are classified as chordates because of their chordate tadpole larvae; in contrast, the sessile adult has a U-shaped gut and very derived body plan, looking nothing like a chordate. There is one group of ascidians, the Molgulidae, where many species are known to have tailless larvae. The Swalla Lab has been studying the evolution of tailless ascidian larvae in this clade for over 30 years and has shown that tailless larvae have evolved independently several times in this clade. Comparison of the genomes of two closely related species, the tailed Molgula oculata and tailless Molgula occulta reveals much synteny, but there have been multiple insertions and deletions that have disrupted larval genes in the tailless species. Genomics and transcriptomics have previously shown that there are pseudogenes expressed in the tailless embryos, suggesting that the partial rescue of tailed features in their hybrid larvae is due to the expression of intact genes from the tailed parent. Yet surprisingly, we find that the notochord gene regulatory network is mostly intact in the tailless M. occulta, although the notochord does not converge and extend and remains as an aggregate of cells we call the “notoball.” We expect that eventually many of the larval gene networks will become evolutionarily lost in tailless ascidians and the larval body plan abandoned, with eggs developing directly into an adult. Here we review the current evolutionary and developmental evidence on how the molgulids lost their tails.

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.

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.

Neurogenesis is a complex sequence of cellular processes and behaviors driven by the coordinated expression of conserved effectors. The bipolar tail neurons (BTNs) of Ciona develop according to a highly dynamic, yet highly stereotyped developmental program and thus could serve as an accessible model system for neurogenesis, including underlying cell behaviors like neuronal delamination, migration, and polarized axon outgrowth. Here we investigate both the upstream events that shape BTN neurogenesis through spatiotemporal regulation of the conserved proneural factor Neurog, spatiotemporal, and the gene expression profile of differentiating BTNs downstream of Neurog activity. We show that, although early FGF signaling is required for Neurog expression and BTN specification, Fgf8/17/18 is expressed in tail tip cells at later stages and suppresses sustained Neurog expression in the anterior BTN (aBTN) lineage, such that only one cell (the one furthest from the source of Fgf8/17/18) maintains Neurog expression and becomes a neuron. Curiously, Fgf8/17/18 might not affect neurogenesis of the posterior BTNs (pBTNs), which are in direct contact with the Fgf8/17/18-expressing cells. Finally, to profile gene expression associated with BTN neurogenesis we performed RNAseq of isolated BTN lineage cells in which BTN neurogenesis was enhanced or suppressed by perturbing Neurog function. This allowed us to identify several candidate genes that might play conserved roles in neurogenesis and neuronal migration in other animals, including mammals.

Larvae of the tunicate Ciona intestinalis possess a central nervous system of 177 neurons. This simplicity has facilitated the generation of a complete synaptic connectome. As chordates and the closest relatives of vertebrates, tunicates promise insight into the organization and evolution of vertebrate nervous systems. Ciona larvae have several sensory systems, including the ocellus and otolith, which are sensitive to light and gravity, respectively. Here we describe circuitry by which these two are integrated into a complex behavior: the rapid reorientation of the body followed by upward swimming in response to dimming. Significantly, the gravity response causes an orienting behavior consisting of curved swims in downward-facing larvae, but only when triggered by dimming. In contrast, the majority of larvae facing upward do not respond to dimming with orientation swims – but instead swim directly upward. Under constant light conditions, the gravity circuit appears to be inoperable, and both upward and downward swims were observed. Using connectomic and neurotransmitter data we propose a circuit model that can account for these behaviors. The otolith consists of a statocyst cell and projecting excitatory sensory neurons (antenna cells). Postsynaptic to the antenna cells are a group of inhibitory primary interneurons, the antenna relay neurons (antRNs), which then project asymmetrically to the right and left motor units, thereby mediating curved orientation swims. Also projecting to the antRNs are inhibitory photoreceptor relay interneurons. These interneurons appear to antagonize the otolith circuit until they themselves are inhibited by photoreceptors in response to dimming, thus providing a triggering circuit.

The larval nervous system of the solitary tunicateCionais a simple model for the study of chordate neurodevelopment. The development and connectivity of theCionaMotor Ganglion (MG) has been studied in fine detail, but how this important structure develops in other tunicates is not well known. By comparing gene expression patterns in the developing MG of the distantly related tunicateMolgula occidentalis, we found that its patterning is highly conserved compared to theCionaMG. MG neuronal subtypes inMolgulawere specified in the exact same positions as inCiona, though the timing of subtype-specific gene expression onset was slightly shifted to begin earlier, relative to mitotic exit and differentiation. In transgenicMolgulaembryos electroporated withDmbxreporter plasmids, we were also able to characterize the morphology of the lone pair of descending decussating neurons (ddNs) inMolgula, revealing the same unique contralateral projection seen inCionaddNs and their putative vertebrate homologs the Mauthner cells. AlthoughDmbxexpression labels the ddNs in both species, cross-species transgenic assays revealed significant changes to thecis-regulatory logic underlyingDmbxtranscription. We found thatDmbx cis-regulatory DNAs fromCionacan drive highly specific reporter gene expression inMolguladdNs, butMolgulasequences are not active inCionaddNs. This acute divergence in the molecular mechanisms that underlie otherwise functionally conservedcis-regulatory DNAs supports the recently proposed idea that the extreme genetic plasticity observed in tunicates may be attributed to the extremerigidityof the spatial organization of their embryonic cell lineages.

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.

For more than a century, studies on tunicate muscle formation have revealed many principles of cell fate specification, gene regulation, morphogenesis, and evolution. Here, we review the key studies that have probed the development of all the various muscle cell types in a wide variety of tunicate species. We seize this occasion to explore the implications and questions raised by these findings in the broader context of muscle evolution in chordates.

The bipolar tail neurons (BTNs) of Ciona develop according to a highly dynamic, yet highly stereotyped developmental program and thus could serve as an accessible model system for neuronal delamination, migration, and polarized axon outgrowth. Here we used FACS/RNAseq to profile the transcriptional output of Neurogenin in the BTNs, searching for candidate effectors of BTN cell behaviors. We identified several candidate genes that might play conserved roles in similar cell behaviors in other animals, including mammals. Among the more interesting candidates were several microtubule-binding proteins and TGFβ pathway antagonists. A small Gαi subunit was also found to be upregulated in migrating BTNs, and interfering with its function through expression of a dominant negative inhibited delamination and a complete epithelial-to-mesenchymal transition. We propose models for the regulation of BTN behaviors by the identified candidate effectors, establishing a foundation for testing effector gene functions that might be conserved in chordate neurodevelopment.

The central nervous system of theCionalarva contains only 177 neurons. The precise regulation of neuron subtype-specific morphogenesis and differentiation observed in during the formation of this minimal connectome offers a unique opportunity to dissect gene regulatory networks underlying chordate neurodevelopment. Here we compare the transcriptomes of two very distinct neuron types in the hindbrain/spinal cord homolog ofCiona, the Motor Ganglion (MG): the Descending decussating neuron (ddN, proposed homolog of Mauthner Cells in vertebrates) and the MG Interneuron 2 (MGIN2). Both types are invariantly represented by a single bilaterally symmetric left/right pair of cells in every larva. Supernumerary ddNs and MGIN2s were generated in synchronized embryos and isolated by fluorescence-activated cell sorting for transcriptome profiling. Differential gene expression analysis revealed ddN- and MGIN2-specific enrichment of a wide range of genes, including many encoding potential “effectors” of subtype-specific morphological and functional traits. More specifically, we identified the upregulation of centrosome-associated, microtubule-stabilizing/bundling proteins and extracellular matrix proteins and axon guidance cues as part of a single intrinsic regulatory program that might underlie the unique polarization of the ddNs, the only descending MG neurons that cross the midline.

The evolutionary history of animal body plans cannot be fully reconstructed without considering the roles of both novelties and losses. Some of the more remarkable examples of massively parallel evolutionary losses in animals comes from many species in the tunicate genusMolgulathat have independently lost the swimming larva and instead develop as tail-less, immotile larvae that bypass the period of swimming and dispersal observed in other tunicates, marine invertebrate chordates that alternate between motile larval and sessile adult life cycle stages. The larvae ofMolgula occultaand other tail-less species do not fully develop structures that are essential for swimming behavior, including notochord, tail muscles, and otolith, and loss-of-function mutations have been identified in various genes required for the differentiation of these tissues. However, little is known about the extent of development of the larval nervous system inM. occulta. While differentiated neurons might in principle be entirely dispensable to the non-swimming larva, the adult has a fully functional nervous system like any other tunicate. To further investigate this conundrum, we studied the specification and patterning of theM. occultaMotor Ganglion, which is the key central nervous system compartment that drives the motor movements of swimming tunicate larvae. We found that the expression patterns of important regulators of MG neuron subtype specification are highly conserved during the development of the non-swimming larvae ofM. occulta, suggesting that the gene networks regulating their expression are largely intact in this species, despite the loss of swimming ability. However, we identified aM. occulta-specific reduction in expression of the important motor neuron terminal selector geneEbf (Collier/Olf/EBF or COE)in the Motor Ganglion. AlthoughM. occulta Ebfis predicted to encode a fully functional protein, its expression was reduced in developing motor neurons when compared to species with swimming larvae, which was corroborated by measuring allele-specific expression ofEbfin interspecific hybrid embryos produced by crossingM. occultawith the closely related swimming speciesM. oculata. Comparative reporter construct experiments also revealed a specificcis-regulatory sequence change that underlies the reduced expression ofM. occulta Ebfin motor neurons, but not in other tissues and cell types. This points to a potential mechanism for arresting larval motor neuron differentiation in the non-swimming larvae of this species.

The tadpole-type larva ofCionahas emerged as an intriguing model system for the study of neurodevelopment. TheCiona intestinalisconnectome has been recently mapped, revealing the smallest central nervous system (CNS) known in any chordate, with only 177 neurons. This minimal CNS is highly reminiscent of larger CNS of vertebrates, sharing many conserved developmental processes, anatomical compartments, neuron subtypes, and even specific neural circuits. Thus, theCionatadpole offers a unique opportunity to understand the development and wiring of a chordate CNS at single-cell resolution. Here we report the use of single-cell RNAseq to profile the transcriptomes of single cells isolated by fluorescence-activated cell sorting (FACS) from the whole brain ofCiona robusta(formerlyintestinalis Type A)larvae. We have also compared these profiles to bulk RNAseq data from specific subsets of brain cells isolated by FACS using cell type-specific reporter plasmid expression. Taken together, these datasets have begun to reveal the compartment- and cell-specific gene expression patterns that define the organization of theCionalarval brain.

Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 has emerged as a revolutionary tool for fast and efficient targeted gene knockouts and genome editing in almost any organism. The laboratory model tunicate Ciona is no exception. Here, we describe our latest protocol for the design, implementation, and evaluation of successful CRISPR/Cas9-mediated gene knockouts in somatic cells of electroporated Ciona embryos. Using commercially available reagents, publicly accessible plasmids, and free web-based software applications, any Ciona researcher can easily knock out any gene of interest in their favorite embryonic cell lineage.

Neuronal precursor cells in the tunicate Ciona intestinalis are shown to delaminate and undergo directed cell migration along either side of the neural tube before differentiating into bipolar neurons, suggesting that vertebrate neural-crest-derived sensory neurons have much deeper evolutionary roots. Many of the features that make vertebrates distinctive derive from an embryonic tissue called the neural crest, which starts life on the borders of the developing neural plate and migrates through the body. How did such a distinctive embryonic tissue evolve? Until recently no trace of it was known in the invertebrates closest to vertebrates such as tunicates and amphioxus. However, signs of neural plate border cells expressing cognates of vertebrate-specific neural-crest-related genes have been found. Here Lionel Christiaen and colleagues take the research a step further by showing how neuronal cells in the 'tadpole' larva of the tunicate Ciona intestinalis (the sea squirt) share features with neural-crest-derived spinal ganglia neurons in vertebrates. The precursors of these cells derive from caudal neural plate border cells and delaminate and undergo directed cell migration along either side of the neural tube before differentiating into axons, suggesting that migration — previously thought to be unique to vertebrate neural crests — has much deeper evolutionary roots. The neural crest is an evolutionary novelty that fostered the emergence of vertebrate anatomical innovations such as the cranium and jaws1. During embryonic development, multipotent neural crest cells are specified at the lateral borders of the neural plate before delaminating, migrating and differentiating into various cell types. In invertebrate chordates (cephalochordates and tunicates), neural plate border cells express conserved factors such as Msx, Snail and Pax3/7 and generate melanin-containing pigment cells2,3,4, a derivative of the neural crest in vertebrates. However, invertebrate neural plate border cells have not been shown to generate homologues of other neural crest derivatives. Thus, proposed models of neural crest evolution postulate vertebrate-specific elaborations on an ancestral neural plate border program, through acquisition of migratory capabilities and the potential to generate several cell types5,6,7. Here we show that a particular neuronal cell type in the tadpole larva of the tunicate Ciona intestinalis, the bipolar tail neuron, shares a set of features with neural-crest-derived spinal ganglia neurons in vertebrates. Bipolar tail neuron precursors derive from caudal neural plate border cells, delaminate and migrate along the paraxial mesoderm on either side of the neural tube, eventually differentiating into afferent neurons that form synaptic contacts with both epidermal sensory cells and motor neurons. We propose that the neural plate borders of the chordate ancestor already produced migratory peripheral neurons and pigment cells, and that the neural crest evolved through the acquisition of a multipotent progenitor regulatory state upstream of multiple, pre-existing neural plate border cell differentiation programs.

Background Analyzing close species with diverse developmental modes is instrumental for investigating the evolutionary significance of physiological, anatomical and behavioral features at a molecular level. Many examples of trait loss are known in metazoan populations living in dark environments. Tunicates are the closest living relatives of vertebrates and typically present a lifecycle with distinct motile larval and sessile adult stages. The nervous system of the motile larva contains melanized cells associated with geotactic and light-sensing organs. It has been suggested that these are homologous to vertebrate neural crest-derived melanocytes. Probably due to ecological adaptation to distinct habitats, several species of tunicates in the Molgulidae family have tailless (anural) larvae that fail to develop sensory organ-associated melanocytes. Here we studied the evolution of Tyrosinase family genes, indispensible for melanogenesis, in the anural, unpigmented Molgula occulta and in the tailed, pigmented Molgula oculata by using phylogenetic, developmental and molecular approaches. Results We performed an evolutionary reconstruction of the tunicate Tyrosinase gene family: in particular, we found that M. oculata possesses genes predicted to encode one Tyrosinase (Tyr) and three Tyrosinase-related proteins (Tyrps) while M. occulta has only Tyr and Tyrp.a pseudogenes that are not likely to encode functional proteins. Analysis of Tyr sequences from various M. occulta individuals indicates that different alleles independently acquired frameshifting short indels and/or larger mobile genetic element insertions, resulting in pseudogenization of the Tyr locus. In M. oculata, Tyr is expressed in presumptive pigment cell precursors as in the model tunicate Ciona robusta. Furthermore, a M. oculata Tyr reporter gene construct was active in the pigment cell precursors of C. robusta embryos, hinting at conservation of the regulatory network underlying Tyr expression in tunicates. In contrast, we did not observe any expression of the Tyr pseudogene in M. occulta embryos. Similarly, M. occulta Tyr allele expression was not rescued in pigmented interspecific M. occulta × M. oculata hybrid embryos, suggesting deleterious mutations also to its cis-regulatory sequences. However, in situ hybridization for transcripts from the M. occulta Tyrp.a pseudogene revealed its expression in vestigial pigment cell precursors in this species. Conclusions We reveal a complex evolutionary history of the melanogenesis pathway in tunicates, characterized by distinct gene duplication and loss events. Our expression and molecular data support a tight correlation between pseudogenization of Tyrosinase family members and the absence of pigmentation in the immotile larvae of M. occulta. These results suggest that relaxation of purifying selection has resulted in the loss of sensory organ-associated melanocytes and core genes in the melanogenesis biosynthetic pathway in M. occulta.

Tunicates are invertebrate members of the chordate phylum, and are considered to be the sister group of vertebrates. Tunicates are composed of ascidians, thaliaceans, and appendicularians. With the advent of inexpensive high-throughput sequencing, the number of sequenced tunicate genomes is expected to rise sharply within the coming years. To facilitate comparative genomics within the tunicates, and between tunicates and vertebrates, standardized rules for the nomenclature of tunicate genetic elements need to be established. Here we propose a set of nomenclature rules, consensual within the community, for predicted genes, pseudogenes, transcripts, operons, transcriptional cis-regulatory regions, transposable elements, and transgenic constructs. In addition, the document proposes guidelines for naming transgenic and mutant lines.

SummaryIn vertebrates, pluripotent pharyngeal mesoderm progenitors produce the cardiac precursors of the second heart field as well as the branchiomeric head muscles and associated stem cells. However, the mechanisms underlying the transition from multipotent progenitors to distinct muscle precursors remain obscured by the complexity of vertebrate embryos. Using Ciona intestinalis as a simple chordate model, we show that bipotent cardiopharyngeal progenitors are primed to activate both heart and pharyngeal muscle transcriptional programs, which progressively become restricted to corresponding precursors. The transcription factor COE (Collier/OLF/EBF) orchestrates the transition to pharyngeal muscle fate both by promoting an MRF-associated myogenic program in myoblasts and by maintaining an undifferentiated state in their sister cells through Notch-mediated lateral inhibition. The latter are stem cell-like muscle precursors that form most of the juvenile pharyngeal muscles. We discuss the implications of our findings for the development and evolution of the chordate cardiopharyngeal mesoderm.

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.

The CRISPR/Cas9 system has emerged as an important tool for a wide variety of genome engineering applications, including reverse genetic screens. Previously, we described the implementation of the CRISPR/Cas9 system to induce tissue-specific mutations at targeted locations in the genome of the sea squirt Ciona (STOLFI et al. 2014). In the present study, we designed 83 single guide RNA (sgRNA) vectors targeting 23 genes expressed in the cardiopharyngeal progenitors and surrounding tissues in the Ciona embryo and measured their mutagenesis efficacy rates by massively parallel indel detection at the targeted loci using highthroughput sequencing. We show that the combined activity of two highly active sgRNAs allows us to generate large (>3 kbp) deletions of intervening genomic DNA in somatic cells of electroporated embryos, permitting tissue-specific gene knockouts. Additionally, we employed L1-regularized regression modeling to develop an optimal sgRNA design algorithm (TuniCUT), based on correlations between target sequence features and mutagenesis rates. Using this algorithm, we have predicted mutagenesis rates for sgRNAs targeting all 4,853,589 sites in the Ciona genome, which we have compiled into a "CRISPR/Cas9-induced Ciona Knock-Out" (Ci2KO) sgRNA sequence library. Finally, we describe a new method for the assembly of sgRNA expression cassettes using a simple one-step overlap PCR (OSO-PCR) protocol. These cassettes can be electroporated directly into Ciona embryos as unpurified PCR products to drive sgRNA expression, bypassing the need for time-consuming cloning and plasmid DNA preparations. We anticipate that this method will be used in combination with genome-wide sgRNA predictions to systematically investigate tissue-specific gene functions in Ciona.

The experimental malleability and unique phylogenetic position of the sea squirt Ciona intestinalis as part of the sister group to the vertebrates have helped establish these marine chordates as model organisms for the study of developmental genetics and evolution. Here we summarize the tools, techniques, and resources available to the Ciona geneticist, citing examples of studies that employed such strategies in the elucidation of gene function in Ciona. Genetic screens, germline transgenesis, electroporation of plasmid DNA, and microinjection of morpholinos are all routinely employed, and in the near future we expect these to be complemented by targeted mutagenesis, homologous recombination, and RNAi. The genomic resources available will continue to support the design and interpretation of genetic experiments and allow for increasingly sophisticated approaches on a high-throughput, whole-genome scale.

The motor ganglion (MG) controls the rhythmic swimming behavior of the Ciona intestinalis tadpole. Despite its cellular simplicity (five pairs of neurons), the MG exhibits conservation of transcription factor expression with the spinal cord of vertebrates. Evidence is presented that the developing MG is patterned by sequential Ephrin/FGF/MAPK and Delta/Notch signaling events. FGF/MAPK attenuation by a localized EphrinAb signal specifies posterior neuronal subtypes, which in turn relay a Delta2/Notch signal that specifies anterior fates. This short-range relay is distinct from the patterning of the vertebrate spinal cord, which is a result of opposing BMP and Shh morphogen gradients. Nonetheless, both mechanisms lead to localized expression of related homeodomain codes for the specification of distinct neuronal subtypes. This MG regulatory network provides a foundation for elucidating the genetic and cellular basis of a model chordate central pattern generator.

Cholinergic motor neurons are defined by the coexpression of a battery of genes encoding proteins that act sequentially to synthesize, package and degrade acetylcholine and reuptake its breakdown product, choline. How expression of these critical motor neuron identity determinants is controlled and coordinated is not understood. We show here that, in the nematode Caenorhabditis elegans, all members of the cholinergic gene battery, as well as many other markers of terminal motor neuron fate, are co-regulated by a shared cis-regulatory signature and a common trans-acting factor, the phylogenetically conserved COE (Collier, Olf, EBF)-type transcription factor UNC-3. UNC-3 initiated and maintained expression of cholinergic fate markers and was sufficient to induce cholinergic fate in other neuron types. UNC-3 furthermore operated in negative feedforward loops to induce the expression of transcription factors that repress individual UNC-3-induced terminal fate markers, resulting in diversification of motor neuron differentiation programs in specific motor neuron subtypes. A chordate ortholog of UNC-3, Ciona intestinalis COE, was also both required and sufficient for inducing a cholinergic fate. Thus, UNC-3 is a terminal selector for cholinergic motor neuron differentiation whose function is conserved across phylogeny.

The CRISPR/Cas9 system has ushered in a new era of targeted genetic manipulations. Here, we report the use of CRISPR/Cas9 to induce double-stranded breaks in the genome of the sea squirt Ciona intestinalis. We use electroporation to deliver CRISPR/Cas9 components for tissue-specific disruption of the Ebf (Collier/Olf/EBF) gene in hundreds of synchronized Ciona embryos. Phenotyping of transfected embryos in the ‘F0’ generation revealed that endogenous Ebf function is required for specification of Islet-expressing motor ganglion neurons and atrial siphon muscles. We demonstrate that CRISPR/Cas9 is sufficiently effective and specific to generate large numbers of embryos carrying mutations in a targeted gene of interest, which should allow for rapid screening of gene function in Ciona.

Ascidians present a striking dichotomy between conserved phenotypes and divergent genomes: embryonic cell lineages and gene expression patterns are conserved between distantly related species. Much research has focused on Ciona or Halocynthia spp. but development in other ascidians remains poorly characterized. In this study, we surveyed the multipotent myogenic B7.5 lineage in Molgula spp. Comparisons to the homologous lineage in Ciona revealed identical cell division and fate specification events that result in segregation of larval, cardiac, and pharyngeal muscle progenitors. Moreover, the expression patterns of key regulators are conserved, but cross-species transgenic assays uncovered incompatibility, or ‘unintelligibility’, of orthologous cis-regulatory sequences between Molgula and Ciona. These sequences drive identical expression patterns that are not recapitulated in cross-species assays. We show that this unintelligibility is likely due to changes in both cis- and trans-acting elements, hinting at widespread and frequent turnover of regulatory mechanisms underlying otherwise conserved aspects of ascidian embryogenesis. DOI: http://dx.doi.org/10.7554/eLife.03728.001, eLife digest When two species have features that look similar, this may be because the features arise by the same processes during development. Other features may look similar yet develop by different mechanisms. ‘Developmental system drift’ refers to the process where a physical feature remains unaltered during evolution, but the underlying pathway that controls its development is changed. However, to date, there have been only a few experimental studies that support this idea. Ascidians—also commonly known as sea squirts—are vase-like marine creatures, which start off as tadpole-like larvae that swim around until they find a place to settle down and attach themselves. Once attached, the sea squirts lose the ability to swim and start feeding, typically by filtering material out of the seawater. Sea squirts and their close relatives are the invertebrates (animals without backbones) that are most closely related to all vertebrates (animals with backbones), including humans. Furthermore, although different species of sea squirt have almost identical embryos, their genomes are very different. Stolfi et al. have now studied whether developmental system drift may have occurred during the evolution of ascidians, by analyzing different species of sea squirt named Molgula and Ciona. Stolfi et al. compared the genomes of Molgula and Ciona and studied the expression of genes in the cells that give rise to the heart and the muscles of the head. As an embryo develops, specific genes are switched on or off, and these patterns of gene activation were broadly identical in the two species of sea squirt examined. Enhancers are sequences of DNA that control when and how a gene is switched on. Given the similarities between the development of heart and head muscle cells in the different sea squirts, Stolfi et al. looked to see if the mechanisms of gene expression, and therefore the enhancers, were also conserved. Unexpectedly, this was not the case. When enhancers from Molgula were introduced into Ciona (and vice versa), these sequences were unable to switch on gene expression—thus enhancers from one sea squirt species could not function in the other. Stolfi et al. conclude that the developmental systems may have drifted considerably during evolution of the sea squirts, in spite of their nearly identical embryos. This reinforces the view that different paths can lead to the formation of similar physical features. DOI: http://dx.doi.org/10.7554/eLife.03728.002

The anterior-most ectoderm of ascidian larvae contains the adhesive papillae, or palps, which play an important role in triggering the metamorphosis of swimming tadpoles. In Ciona intestinalis, the palps consist of three conical protrusions within a field of thickened epithelium that form late in embryogenesis, as tailbuds mature into larvae. The palp protrusions express the LIM-homeodomain transcription factor Islet. Protrusion occurs through differential cell elongation, probably mediated by Islet, as we find that ectopic expression of Islet is sufficient to promote cell lengthening. FGF signaling is required for both Islet expression and palp morphogenesis. Importantly, we show that Islet expression can rescue the palp-deficient phenotype that results from inhibition of FGF signaling. We conclude that Islet is a key regulatory factor governing morphogenesis of the palps. It is conceivable that Islet is also essential for the cellular morphogenesis of placode-derived sensory neurons in vertebrates.

Astrocytes, one of the most common cell types in the brain, are essential for processes ranging from neural development through potassium homeostasis to synaptic plasticity. Surprisingly, the developmental origins of astrocytes in the neocortex are still controversial. To investigate the patterns of astrocyte development in the neocortex we examined cortical development in a transgenic mouse in which a random, sparse subset of neural progenitors undergoes CRE/lox recombination, permanently labeling their progeny. We demonstrate that neural progenitors in neocortex generate discrete columnar structures that contain both projection neurons and protoplasmic astrocytes. Ninety-five percent of developmental cortical columns labeled in our system contained both astrocytes and neurons. The astrocyte to neuron ratio of labeled cells in a developmental column was 1:7.4, similar to the overall ratio of 1:8.4 across the entire gray matter of the neocortex, indicating that column-associated astrocytes account for the majority of protoplasmic astrocytes in neocortex. Most of the labeled columns contained multiple clusters of several astrocytes. Dividing cells were found at the base of neuronal columns at the beginning of gliogenesis, and later within the cortical layers, suggesting a mechanism by which astrocytes could be distributed within a column. These data indicate that radial glia are the source of both neurons and astrocytes in the neocortex, and that these two cell types are generated in a spatially restricted manner during cortical development.

The identification of subpharyngeal cardiac precursors has had a strong influence on the way we think about early cardiac development. From this discovery was born the concept of multiple heart fields. Early support for the concept came from gene expression, genetic retrospective fate mapping, and gene targeting studies, which collectively suggested the existence of a second heart field (SHF) on the basis of specific Islet-1 (Isl-1) expression, presence of two cardiac ancestral lineages, and compatible cardiac knockout phenotypes, respectively. A decade after the original studies, support for the SHF concept is dwindling. This is because in all bilaterian models studied, Isl expression in heart progenitors is not SHF-specific, because lineage data are best explained by alternative models including an older, truly ancestral, lineage of cardiac pioneers with unrestricted contribution to all cardiac segments and, finally, because the inflow-to-outflow segmental nature of the early vertebrate peristaltic heart has been reaffirmed with novel, less invasive, methodologies. Altogether, the paradigms derived from the discovery of subpharyngeal cardiac progenitors helped us shift from relatively simple models, which rely predominantly either on patterning, gene expression patterns or lineages, to a much more sophisticated body of knowledge in which all these parameters must be accounted. Thus, it is well possible that due consideration of the key elements contained in the inflow/outflow, pioneer/scaffold, ballooning, and SHF hypotheses may provide us with a unified framework of the early stages of cardiac development. Here, we advance into this direction by suggesting an intuitive model of early heart development based on the concept of an inflow/outflow scaffold erected by cardiac pioneers, one that is required to assemble all the subsequent cell contribution that emigrates from cardiac progenitor areas.

The identification of subpharyngeal cardiac precursors has had a strong influence on the way we think about early cardiac development. From this discovery was born the concept of multiple heart fields. Early support for the concept came from gene expression, genetic retrospective fate mapping, and gene targeting studies, which collectively suggested the existence of a second heart field (SHF) on the basis of specific Islet-1 (Isl-1) expression, presence of two cardiac ancestral lineages, and compatible cardiac knockout phenotypes, respectively. A decade after the original studies, support for the SHF concept is dwindling. This is because in all bilaterian models studied, Isl expression in heart progenitors is not SHF-specific, because lineage data are best explained by alternative models including an older, truly ancestral, lineage of cardiac pioneers with unrestricted contribution to all cardiac segments and, finally, because the inflow-to-outflow segmental nature of the early vertebrate peristaltic heart has been reaffirmed with novel, less invasive, methodologies. Altogether, the paradigms derived from the discovery of subpharyngeal cardiac progenitors helped us shift from relatively simple models, which rely predominantly either on patterning, gene expression patterns or lineages, to a much more sophisticated body of knowledge in which all these parameters must be accounted. Thus, it is well possible that due consideration of the key elements contained in the inflow/outflow, pioneer/scaffold, ballooning, and SHF hypotheses may provide us with a unified framework of the early stages of cardiac development. Here, we advance into this direction by suggesting an intuitive model of early heart development based on the concept of an inflow/outflow scaffold erected by cardiac pioneers, one that is required to assemble all the subsequent cell contribution that emigrates from cardiac progenitor areas.