Ascidian Tadpole Larvae Research Articles

Molecular control of cellulosic fin morphogenesis in ascidians

BackgroundThe 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.ResultsHere 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.ConclusionsOur 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.

Bilaterally Asymmetric Helical Myofibrils in Ascidian Tadpole Larvae.

The locomotor system is highly bilateral at the macroscopic level. Homochirality of biological molecules is fully compatible with the bilateral body. However, whether and how single-handed cells contribute to the bilateral locomotor system is obscure. Here, exploiting the small number of cells in the swimming tadpole larva of the ascidian Ciona, we analyzed morphology of the tail at cellular and subcellular scales. Quantitative phase-contrast X-ray tomographic microscopy revealed a high-density midline structure ventral to the notochord in the tail. Muscle cell nuclei on each side of the notochord were roughly bilaterally aligned. However, fluorescence microscopy detected left-right asymmetry of myofibril inclination relative to the longitudinal axis of the tail. Zernike phase-contrast X-ray tomographic microscopy revealed the presence of left-handed helices of myofibrils in muscle cells on both sides. Therefore, the locomotor system of ascidian larvae harbors symmetry-breaking left-handed helical cells, while maintaining bilaterally symmetrical cell alignment. These results suggest that bilateral animals can override cellular homochirality to generate the bilateral locomotor systems at the supracellular scale.

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

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

The Degenerate Tale of Ascidian Tails.

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.

Neurobiology: Swimming at the Intersection of Light and Gravity.

Many animals use gravity as a spatial reference to help navigate their surroundings, but how they do so is not well understood. A new study reveals that a representative of our closest invertebrate relatives, the tunicate Ciona, processes light and gravity cues through a simple neural circuit to decide when and how to swim.

The regulatory pathway from genes directly activated by maternal factors to muscle structural genes in ascidian embryos

Striated muscle cells in the tail of ascidian tadpole larvae differentiate cell-autonomously. Although several key regulatory factors have been identified, the genetic regulatory pathway is not fully understood; comprehensive understanding of the regulatory pathway is essential for accurate modeling in order to deduce principles for gene regulatory network dynamics, and for comparative analysis on how ascidians have evolved the cell-autonomous gene regulatory mechanism. Here, we reveal regulatory interactions among three key regulatory factors, Zic-r.b, Tbx6-r.b and Mrf, and elucidate the mechanism by which these factors activate muscle structural genes. We reveal a cross-regulatory circuit among these regulatory factors, which maintains the expression of Tbx6-r.b and Mrf during gastrulation. Although these two factors combinatorially activate muscle structural genes in late-stage embryos, muscle structural genes are activated mainly by Tbx6-r.b before gastrulation. Time points when expression of muscle structural genes become first detectable are strongly correlated with the degree of Tbx6-r.b occupancy. Thus, the genetic pathway, starting with Tbx6-r.b and Zic-r.b, which are activated by maternal factors, and ending with expression of muscle structural genes, has been revealed.

Distinct regulation of Snail in two muscle lineages of the ascidian embryo achieves temporal coordination of muscle development.

The transcriptional repressor Snail is required for proper differentiation of the tail muscle of ascidian tadpole larvae. Two muscle lineages (B5.1 and B6.4) contribute to the anterior tail muscle cells, and are consecutively separated from a transcriptionally quiescent germ cell lineage at the 16- and 32-cell stages. Concomitantly, cells of these lineages begin to express Tbx6.b (Tbx6-r.b) at the 16- and 32-cell stages, respectively. Meanwhile, Snail expression begins in these two lineages simultaneously at the 32-cell stage. Here, we show that Snail expression is regulated differently between these two lineages. In the B5.1 lineage, Snail was activated through Tbx6.b, which is activated by maternal factors, including Zic-r.a. In the B6.4 lineage, the MAPK pathway was cell-autonomously activated by a constitutively active form of Raf, enabling Zic-r.a to activate Snail independently of Tbx6.b As a result, Snail begins to be expressed at the 32-cell stage simultaneously in these two lineages. Such shortcuts might be required for coordinating developmental programs in embryos in which cells become separated progressively from stem cells, including germline cells.

The peripheral nervous system of the ascidian tadpole larva: Types of neurons and their synaptic networks.

Physical and chemical cues from the environment are used to direct animal behavior through a complex network of connections originating in exteroceptors. In chordates, mechanosensory and chemosensory neurons of the peripheral nervous system (PNS) must signal to the motor circuits of the central nervous system (CNS) through a series of pathways that integrate and regulate the output to motor neurons (MN); ultimately these drive contraction of the tail and limb muscles. We used serial-section electron microscopy to reconstruct PNS neurons and their hitherto unknown synaptic networks in the tadpole larva of a sibling chordate, the ascidian, Ciona intestinalis. The larva has groups of neurons in its apical papillae, epidermal neurons in the rostral and apical trunk, caudal neurons in the dorsal and ventral epidermis, and a single tail tip neuron. The connectome reveals that the PNS input arises from scattered groups of these epidermal neurons, 54 in total, and has three main centers of integration in the CNS: in the anterior brain vesicle (which additionally receives input from photoreceptors of the ocellus), the motor ganglion (which contains five pairs of MN), and the tail, all of which in turn are themselves interconnected through important functional relay neurons. Some neurons have long collaterals that form autapses. Our study reveals interconnections with other sensory systems, and the exact inputs to the motor system required to regulate contractions in the tail that underlie larval swimming, or to the CNS to regulate substrate preference prior to the induction of larval settlement and metamorphosis.

The CNS connectome of a tadpole larva of Ciona intestinalis (L.) highlights sidedness in the brain of a chordate sibling.

Left-right asymmetries in brains are usually minor or cryptic. We report brain asymmetries in the tiny, dorsal tubular nervous system of the ascidian tadpole larva, Ciona intestinalis. Chordate in body plan and development, the larva provides an outstanding example of brain asymmetry. Although early neural development is well studied, detailed cellular organization of the swimming larva's CNS remains unreported. Using serial-section EM we document the synaptic connectome of the larva's 177 CNS neurons. These formed 6618 synapses including 1772 neuromuscular junctions, augmented by 1206 gap junctions. Neurons are unipolar with at most a single dendrite, and few synapses. Some synapses are unpolarised, others form reciprocal or serial motifs; 922 were polyadic. Axo-axonal synapses predominate. Most neurons have ciliary organelles, and many features lack structural specialization. Despite equal cell numbers on both sides, neuron identities and pathways differ left/right. Brain vesicle asymmetries include a right ocellus and left coronet cells.

Neuronal map reveals the highly regionalized pattern of the juvenile central nervous system of the ascidian Ciona intestinalis.

The dorsally located central nervous system (CNS) is an important hallmark of chordates. Among chordates, tunicate ascidians change their CNS remarkably by means of a metamorphosis from a highly regionalized larval CNS to an oval-shaped juvenile CNS without prominent morphological features. The neuronal organization of the CNS of ascidian tadpole larvae has been well described, but that in the CNS of postmetamorphosis juveniles has not been characterized well. We investigated the number of neural cells, the number and position of differentiated neurons, and their axonal trajectories in the juvenile CNS of the ascidian Ciona intestinalis. The cell bodies of cholinergic, glutamatergic, and GABAergic/glycinergic neurons exhibited different localization patterns along the anterior-posterior axis in the juvenile CNS. Cholinergic neurons extended their axons toward the oral, atrial and body wall muscles and pharyngeal gill to regulate muscle contraction and ciliary movement. Unlike its featureless shape, the juvenile CNS is highly patterned along the anterior-posterior axis. This patterning may be necessary for exerting multiple roles in the regulation of adult tissues distributed throughout the body. This basic information of the juvenile CNS of Ciona will allow in-depth studies of molecular mechanisms underlying the reconstruction of the CNS during ascidian metamorphosis.

Neurula rotation determines left-right asymmetry in ascidian tadpole larvae

Tadpole larvae of the ascidian Halocynthia roretzi show morphological left-right asymmetry. The tail invariably bends towards the left side within the vitelline membrane. The structure of the larval brain is remarkably asymmetric. nodal, a conserved gene that shows left-sided expression, is also expressed on the left side in H. roretzi but in the epidermis unlike in vertebrates. We show that nodal signaling at the late neurula stage is required for stereotypic morphological left-right asymmetry at later stages. We uncover a novel mechanism to break embryonic symmetry, in which rotation of whole embryos provides the initial cue for left-sided expression of nodal. Two hours prior to the onset of nodal expression, the neurula embryo rotates along the anterior-posterior axis in a counterclockwise direction when seen in posterior view, and then this rotation stops when the left side of the embryo is oriented downwards. It is likely that epidermis monocilia, which appear at the neurula rotation stage, generate the driving force for the rotation. When the embryo lies on the left side, protrusion of the neural fold physically prevents it from rotating further. Experiments in which neurula rotation is perturbed by various means, including centrifugation and sandwiching between glass, indicate that contact of the left epidermis with the vitelline membrane as a consequence of neurula rotation promotes nodal expression in the left epidermis. We suggest that chemical, and not mechanical, signals from the vitelline membrane promote nodal expression. Neurula rotation is also conserved in other ascidian species.

On some historical and theoretical foundations of the concept of chordates

The concept of chordates arose from the alliance between embryology and evolution in the second half of the nineteenth century, as a result of a theoretical elaboration on Kowalevsky's discoveries about some fundamental similarities between the ontogeny of the lancelet, a putative primitive fish, and that of ascidians, then classified as molluscs. Carrying out his embryological studies in the light of Darwin's theory and von Baer's account of the germ layers, Kowalevsky was influenced by the German tradition of idealistic morphology that was concerned with transformations driven by laws of form, rather than with a gradual evolution occurring by means of variation, selection and adaptation. In agreement with this tradition, Kowalevsky interpreted the vertebrate-like structures of the ascidian larva according to von Kölliker's model of heterogeneous generation. Then, he asserted the homology of the germ layers and their derivatives in different types of animals and suggested a common descent of annelids and vertebrates, in agreement with Saint-Hilaire's hypothesis of the unity of composition of body plans, but in contrast with Haeckel's idea of the Chordonia (chordates). In The Descent of Man Darwin quoted Kowalevsky's discoveries, but accepted Haeckel's interpretation of the ascidian embryology within the frame of a monophyletic tree of life that was produced by the fundamental biogenetic law. Joining embryology to evolution in the light of idealistic morphology, the biogenetic law turned out to be instrumental in bringing forth different evolutionary hypotheses: it was used by Haeckel and Darwin to link vertebrates to invertebrates by means of the concept of chordates, and by Kowalevsky to corroborate the annelid theory of the origin of vertebrates. Yet, there was still another interpretation of Kowalevsky's discoveries. As an adherent to empiricism and to Cuvier's theory of types, von Baer asserted that these discoveries did not prove convincingly a dorsal position of the nervous system in the ascidian tadpole larva; hence, they could not support a homology between different animal types suggesting a kinship between ascidians and vertebrates.

Texture preferences of ascidian tadpole larvae during settlement

Ascidian tadpole larvae settle on hard surfaces and undergo metamorphosis into sessile adults. To test whether tadpoles evaluate the texture of surfaces they settle upon, we presented tadpoles with surfaces that were divided into halves; each half had one of four different textures: smooth, fine sandpaper, coarse sandpaper, and sandblasted. In all cases, twice as many individuals settled on one side over the other, but this was not consistently the smooth side or the rough side. More tadpoles settled on a smooth surface than one scoured by sandpaper, but more tadpoles settled on a sandblasted surface than smooth one. This indicates tadpoles are capable of finer tactile discrimination than merely detecting a hard surface, and supports the hypothesis that ascidian tadpoles have mechanoreceptive sensory neurons.

FGF9/16/20 and Wnt-5α signals are involved in specification of secondary muscle fate in embryos of the ascidian, Halocynthia roretzi

The tail muscle cells of the ascidian tadpole larva originate from two different lineages, the B- (primary) and A- and b- (secondary) line blastomeres of the eight-cell stage embryo. The primary muscle cells assume muscle fate cell-autonomously with the involvement of a localized muscle determinant, macho-1. On the other hand, fate determination of secondary muscle cells is a non-cell-autonomous process that depends on cellular interactions. In this paper, we investigated the mechanisms underlying fate specification of secondary muscle cells in Halocynthia roretzi. We found that FGF and Wnt5 signals were required. In contrast, the Nodal signal, which is required for specification of A-line muscle cells in another ascidian, Ciona intestinalis, was not necessary for the formation of any secondary muscle cells in Halocynthia embryo. Therefore, Halocynthia and Ciona show distinctly different mechanisms for generation of the secondary lineages, despite the fact that embryogenesis appears very similar between these species. We also found that the mechanisms involved in specification of A- and b-line muscle cells were distinct in that the required timing of the FGF signal for the A-line muscle cells preceded that for the b-line. Moreover, the inducer blastomeres for specification of these two lineages were different.

Ascidian embryonic development: An emerging model system for the study of cell fate specification in chordates

The ascidian tadpole larva represents the basic body plan of all chordates in a relatively small number of cells and tissue types. Although it had been considered that ascidians develop largely in a determinative way, whereas vertebrates develop in an inductive way, recent studies at the molecular and cellular levels have uncovered several similarities in the way developmental fates are specified. In this review, we describe ascidian embryogenesis and its cell lineages, introduce several characteristics of ascidian embryos, describe recent advances in understanding of the mechanisms of cell fate specification, and discuss them in the context of what is known in vertebrates and other organisms.

Neurons of the ascidian larval nervous system in Ciona intestinalis: II. Peripheral nervous system

The peripheral nervous system of the ascidian tadpole larva comprises a distributed population of isolated receptor neurons, most of unproved function, organized along the trunk or tail epithelium. Previous reports using immunocytochemical methods failed to resolve the detailed morphology of the neurons and their axon pathways. Precleavage embryos of Ciona intestinalis transfected with the promoter of the neuron-specific synaptotagmin gene fused to a green fluorescent protein (GFP) gene yielded clearly labelled GFP profiles. These we examined in confocal image stacks of 31 larvae. Anchor cells, at least eight in each adhesive apical papilla, contribute axons to the papillar nerves that terminate in the sensory vesicle of the central nervous system. Two nerve bundles projected from each papilla, suggesting that at least two subpopulations of papillar neurons exist. Each bundle fasciculated with axons of the rostral trunk epidermal neurons (RTEN) in a stereotyped pattern. The RTEN had a hitherto unreported elaborate arbor of sensory dendrites within the tunic, suggesting that each has an extended sensorial field. Two subpopulations of apical trunk epidermal neurons (ATEN), anterior and posterior, were distinguished. As with the RTEN, these neurons extended dendritic arbors into the tunic. Two additional types of tail neuron, the caudal epidermal neurons (dorsal and ventral) as well as a novel bipolar interneuron, were identified. These identified neuron types are the substrate for the ascidian larva's entire peripheral sensory input, important during larval swimming and settlement.

우렁쉥이(Halocynthia roretzi) 유생의 뇌포 구조와 수압수용쳬의 형성

The tadpole larvae of most ascidians have two sensory pigment cells in their brain vesicle. The anterior otolith pigment cell is sensitive to gravity, whereas the posterior ocellus pigment cell responds to light. Besides these two sensory cells, the larvae also possess another type of sensory receptor cell: hydrostatic pressure receptor (Hpr) cells. The Hpr cells have been presumed to sense hydrostatic water pressure, although no functional analysis has been performed. In larvae of the ascidian Halocynthia reretzi, the development of the Hpr cells and their structure in the brain vesicle are poorly understood. To investigate the morphology and formation of the Hpr cells, we established a monoclonal antibody, Hpr-1, that specifically recognizes Hpr cells. The Hpr-1 antigens became detectable in the brain vesicle at the late tailbud stage. Each Hpr cell projected a small globular body, connected by a short stalk, into the lumen of the brain vesicle. The brain vesicle showed remarkable left-right asymmetry. Pigment cells were located on the right side in the lumen of the brain vesicle, whereas Hpr cells were present in the left side. After metamorphosis, the Hpr cells were observed near the rudimental siphons of the juvenile.

Effect of emetine, a protein synthesis inhibitor, on larval tail resorption in the ascidian Halocynthia roretzi

Ascidian tadpole larvae settle and metamorphose after a free-swimming period, and one of the typical morphological changes during metamorphosis is tail resorption. Halocynthia roretzi larvae cannot metamorphose for 6hrs after hatching even in the presence of metamorphosis inducers, which suggests that some internal changes occur in the larvae during this period. Larval protein synthesis was examined as one possibility by using a protein synthesis inhibitor, emetine. When tadpole larvae just after hatching were incubated with 5-10μM emetine, tail resorption did not occur. In contrast, larvae that were treated after a free-swimming period of 8 or 24hrs underwent tail resorption, although some were abnormal. These results suggest that protein synthesis during the swimming period is necessary for tail resorption.

Structure of ocellus photoreceptors in the ascidian Ciona intestinalis larva as revealed by an anti‐arrestin antibody

Although there have been several studies on the structure of the ocellus photoreceptors in ascidian tadpole larvae using electron microscopy, the overall structure of these photoreceptor cells, especially the projection sites of the axons, has not been revealed completely. The number of photoreceptor cells is also controversial. Here, the whole structure of the ocellus photoreceptors in the larvae of the ascidian Ciona intestinalis was revealed by using an anti-arrestin (anti-Ci-Arr) antibody. The cell bodies of 30 photoreceptor cells covered the right side of the ocellus pigment cell and their outer segments extended through the pigment cell into the pigment cup. The axons of the photoreceptor cells were bundled together ventro-posteriorly in a single tract extending towards the midline. The nerve terminals diverged antero-posteriorly at the midline of the posterior sensory vesicle (SV). The Ci-arr gene was expressed throughout the SV at the embryonic mid-tailbud stage and it became restricted to the neighborhood of the ocellus pigment when ocellus pigmentation occurred. At the same time, the Ci-Arr protein was first detected, suggesting that the photoreceptor cells began to differentiate. The development of photoreceptor cells after hatching was also investigated using the anti-Ci-Arr antibody. Three hours after hatching, the photoreceptor terminals began to ramify and then expanded. Previous behavioral analysis showed that the larvae did not respond to the step-down of light until 2 h after hatching and then the photoresponse became robust. Accordingly, our results suggest that growth of the photoreceptor terminal is critical for the larvae to become photoresponsive.

The neurobiology of the ascidian tadpole larva: recent developments in an ancient chordate.

With little more than 330 cells, two thirds within the sensory vesicle, the CNS of the tadpole larva of the ascidian Ciona intestinalis provides us with a chordate nervous system in miniature. Neurulation, neurogenesis and its genetic bases, as well as the gene expression territories of this tiny constituency of cells all follow a chordate plan, giving rise in some cases to frank structural homologies with the vertebrate brain. Recent advances are fueled by the release of the genome and EST expression databases and by the development of methods to transfect embryos by electroporation. Immediate prospects to test the function of neural genes are based on the isolation of mutants by classical genetics and insertional mutagenesis, as well as by the disruption of gene function by morpholino antisense oligo-nucleotides. Coupled with high-speed video analysis of larval swimming, optophysiological methods offer the prospect to analyze at single-cell level the function of a CNS built on a vertebrate plan.