Gene Editing in the Ascidian Phallusia mammillata and Tail Nerve Cord Formation.

Functional approaches for studying embryonic development have greatly advanced thanks to the CRISPR-Cas9 gene editing technique. Previously practiced in just a few organisms, these knockout techniques are now widely applied. Here we describe simple techniques for applying the CRISPR-Cas9 system to study the development of the nerve cord in the ascidian Phallusia mammillata.

The ontology of the anatomy and development of the solitary ascidian Ciona: the swimming larva and its metamorphosis.

Ciona robusta (Ciona intestinalis type A), a model organism for biological studies, belongs to ascidians, the main class of tunicates, which are the closest relatives of vertebrates. In Ciona, a project on the ontology of both development and anatomy is ongoing for several years. Its goal is to standardize a resource relating each anatomical structure to developmental stages. Today, the ontology is codified until the hatching larva stage. Here, we present its extension throughout the swimming larva stages, the metamorphosis, until the juvenile stages. For standardizing the developmental ontology, we acquired different time-lapse movies, confocal microscope images and histological serial section images for each developmental event from the hatching larva stage (17.5 h post fertilization) to the juvenile stage (7 days post fertilization). Combining these data, we defined 12 new distinct developmental stages (from Stage 26 to Stage 37), in addition to the previously defined 26 stages, referred to embryonic development. The new stages were grouped into four Periods named: Adhesion, Tail Absorption, Body Axis Rotation, and Juvenile. To build the anatomical ontology, 203 anatomical entities were identified, defined according to the literature, and annotated, taking advantage from the high resolution and the complementary information obtained from confocal microscopy and histology. The ontology describes the anatomical entities in hierarchical levels, from the cell level (cell lineage) to the tissue/organ level. Comparing the number of entities during development, we found two rounds on entity increase: in addition to the one occurring after fertilization, there is a second one during the Body Axis Rotation Period, when juvenile structures appear. Vice versa, one-third of anatomical entities associated with the embryo/larval life were significantly reduced at the beginning of metamorphosis. Data was finally integrated within the web-based resource "TunicAnatO", which includes a number of anatomical images and a dictionary with synonyms. This ontology will allow the standardization of data underpinning an accurate annotation of gene expression and the comprehension of mechanisms of differentiation. It will help in understanding the emergence of elaborated structures during both embryogenesis and metamorphosis, shedding light on tissue degeneration and differentiation occurring at metamorphosis.

Structure and ultrastructure of eyes and brains of Thalia democratica (Thaliacea, Tunicata, Chordata).

Salps are marine planktonic chordates that possess an obligatory alternation of reproductive modes in subsequent generations. Within tunicates, salps represent a derived life cycle and are of interest in considerations of the evolutionary origin of complex anatomical structures and life history strategies. In the present study, the eyes and brains of both the sexual, aggregate blastozooid and the asexual, solitary oozooid stage of Thalia democratica (Forskål, ) were digitally reconstructed in detail based on serial sectioning for light and transmission electron microscopy. The blastozooid stage of T. democratica possesses three pigment cup eyes, situated in the anterior ventral part of the brain. The eyes are arranged in a way that the optical axes of each eye point toward different directions. Each eye is an inverse eye that consists of two different cell types: pigment cells (pigc) and rhabdomeric photoreceptor cells (prcs). The oozooid stage of T. democratica is equipped with a single horseshoe-shaped eye, positioned in the anterior dorsal part of the brain. The opening of the horseshoe-shaped eye points anteriorly. Similar to the eyes of the blastozooid, the eye of the oozooid consists of pigment cells and rhabdomeric photoreceptor cells. The rhabdomeric photoreceptor cells possess apical microvilli that form a densely packed presumably photosensitive receptor part adjacent to the concave side of the pigc. We suggest correspondences of the individual eyes in the blastozooid stage to respective parts of the single horseshoe-shaped eye in the oozooid stage and hypothesize that the differences in visual structures and brain anatomies evolved as a result of the aggregate life style of the blastozooid as opposed to the solitary life style of the oozooid.

Vascular budding in Symplegma brakenhielmi and the evolution of coloniality in styelid ascidians.

Individuals of colonial animals (e.g. zooids) are in continuous turnover. In ascidians colonial or solitary species have evolved by convergence multiple times. Colonial Botryllus and Botrylloides are well-studied genera that exhibit colony-wide developmental mechanisms that regulate synchronous and orchestrated cycles of budding and turnover of zooids. The origins of modular developmental mechanisms that facilitated the evolution of coloniality in this group remain unclear. To reconstruct ancestral states of coloniality we studied Symplegma brakenhielmi, a sister taxon of the botryllids. S. brakenhielmi zooids are embedded in a common tunic and present a similar vascular system as the botrylloides, however development and turnover of zooids occurs asynchronously and in a more independent manner. We generated a table of common stages of budding in Symplegma and Botryllus for comparative studies of asexual development. We tested dependent processes of budding among individuals of the colony by systemic bud or zooid removals. Although our results showed a higher degree of independence in bud development in S. brakenhielmi, we found a subtle colony-wide regulatory mechanism of modular development, i.e. new buds expedited development after the removal of all buds in the colony. Next, we characterized external morphology, ultrastructure, and abundance of circulatory blood cells in the vascular system of S. brakenhielmi. Macrophage-like cells (MLCs) are involved in zooid resorption and turnover. Proportions of MLCs in the blood of S. brakenhielmi corresponded to the peak of occurrence of this cell type during the budding cycle of B. schlosseri. We found several new blood cell types in S. brakenhielmi, including two cell types that resemble circulatory progenitor stem cells of other botryllid colonial ascidians. These cells showed features of undifferentiated cells and expressed mitotic marker Phospho-histone H3. Comparative studies of S. brakenhielmi and B. schlosseri allow us to discuss possible changes in the regulation of modular development (i.e. regulation of life and death in the colony), and a possible contribution of circulatory blood cells in budding processes. We propose that the higher degree of developmental independence in S. brakenhielmi budding is a result of its ancestral solitary mode of development.

The ascidian Styela plicata hemocytes as a potential biomarker of marine pollution: In vitro effects of seawater and organic mercury.

Toxic metals, such as mercury, contribute substantially to anthropogenic pollution in many estuarine environments. Animals living in those environments, particularly invertebrate filter feeders like tunicates, can be used as bioindicators. In an attempt to identify cellular markers for revealing pollution, this study examined in vitro the effects of different concentrations of methyl mercury on Styela plicata hemocytes. The harvested hemocytes from S. plicata that were exposed to the metal had a significant mortality, cellular count and morphometric alterations. These findings provided evidence of MeHg immunotoxic effects on S. plicata, resulting in hemocyte death and morphological changes induced by cytoskeleton alterations. Thus, a morphometric cellular parameter, such as spreading ability, was used as a complementary method for differentiation between hemocytes treated with a marine solution (as a negative control) and hemocytes incubated with methylmercury and/or Sicilian seawater samples.

Internal and external morphology of adults of the appendicularian, Oikopleura dioica: an SEM study.

The appendicularian, Oikopleura dioica, is a planktonic tunicate that retains a swimming tadpole shape throughout its life. It has relatively few cells and exhibits fast development, yet it has a basic chordate body plan. In this study, the morphology of adults was investigated using scanning electron microscopy (SEM) and fine 3D images of most organs were taken. The trunk epidermis is organized into bilateral territories secreting the house that includes the food-trapping filter. The pharynx extends ventrally and posteriorly to the gill openings and esophagus, respectively. The endostyle, with a morphologically distinct ciliated band, is embedded in the pharynx. The digestive tract showed left-right asymmetry as the connection between the pharynx and esophagus tilts leftward. The heart is located ventrally between the left stomach and the intestine and consists of a left muscular sheet and a right thin, non-muscular sheet. The brain is connected to the oral and ventral sensory organs, ciliary funnels and sensory vesicles and axons descend from it that eventually innervate the caudal ganglion. In the tail, a nerve cord with sporadically distributed neuronal somata runs along the left side of the notochord. The gonad is a single syncytium of thousands of gametes. In the ovary, an abundance of cortical membrane invaginate into the cytoplasm during oogenesis and the growing oocytes are interconnected via common cytoplasm through a ring canal. Spermatogenesis progresses synchronously within the common cytoplasm. These descriptions provide a valuable anatomical atlas for studying development and physiology using this simple organism with a chordate body plan.

Centrosomes and spindles in ascidian embryos and eggs.

During embryonic development and maternal meiotic maturation, positioning of the mitotic/meiotic spindle is subject to control mechanisms that meet the needs of the particular cell type. Here we review the methods, molecular tools, and the ascidian model we use to study three different ways in which centrosomes or spindles are positioned in three different cellular contexts. First, we review unequal cleavage in the ascidian germ lineage. In the germ cell precursors, a large macromolecular structure termed the centrosome-attracting body causes three successive rounds of unequal cleavage from the 8- to the 64-cell stage. Next, we discuss spindle positioning underlying the invariant cleavage pattern. Ascidian embryos display an invariant cleavage pattern whereby the mitotic spindle aligns in a predetermined orientation in every blastomere up to the gastrula stage (composed of 112 cells). Finally, we review methods and approaches to study meiotic spindle positioning in eggs.

3-acetylpyridine-induced degeneration in the adult ascidian neural complex: Reactive and regenerative changes in glia and blood cells.

Ascidians are interesting neurobiological models because of their evolutionary position as a sister-group of vertebrates and the high regenerative capacity of their central nervous system (CNS). We investigated the degeneration and regeneration of the cerebral ganglion complex of the ascidian Styela plicata following injection of the niacinamide antagonist 3-acetylpyridine (3AP), described as targeting the CNS of several vertebrates. For the analysis and establishment of a new model in ascidians, the ganglion complex was dissected and prepared for transmission electron microscopy (TEM), routine light microscopy (LM), immunohistochemistry and Western blotting, 1 or 10 days after injection of 3AP. The siphon stimulation test (SST) was used to quantify the functional response. One day after the injection of 3AP, CNS degeneration and recruitment of a non-neural cell type to the site of injury was observed by both TEM and LM. Furthermore, weaker immunohistochemical reactions for astrocytic glial fibrillary acidic protein (GFAP) and neuronal ßIII-tubulin were observed. In contrast, the expression of caspase-3, a protein involved in the apoptotic pathway, and the glycoprotein CD34, a marker for hematopoietic stem cells, increased. Ten days after the injection of 3AP, the expression of markers tended toward the original condition. The SST revealed attenuation and subsequent recovery of the reflexes from 1 to 10 days after 3AP. Therefore, we have developed a new method to study ascidian neural degeneration and regeneration, and identified the decreased expression of GFAP and recruitment of blood stem cells to the damaged ganglion as reasons for the success of neuroregeneration in ascidians.

Histone methylation codes involved in stemness, multipotency, and senescence in budding tunicates.

We examined the dynamics of nuclear histone H3 trimethylation related to cell differentiation and aging in a budding tunicate, Polyandrocarpa misakiensis. Throughout zooidal life, multipotent epithelial and coelomic cell nuclei showed strong trimethylation signals at H3 lysine27 (H3K27me3), consistent with the results of western blotting. Epidermal H3K27me3 repeatedly appeared in protruding buds and disappeared in senescent adult zooids. The budding-specific cytostatic factor TC14-3 allowed aging epidermal cells to restore H3K27me3 signals and mitochondrial gene activities via mitochondrial transcription factor a, all of which were made ineffective by an H3K27me3 inhibitor. Chromatin immunoprecipitation showed that TC14-3 enhances H3K27me3 of transdifferentiation-related genes and consequently downregulates the expression of these genes. In contrast, trimethylation signals at H3 lysine4 (H3K4me3) appeared transiently in transdifferentiating bud cells and stably lasted in undifferentiated adult cells without affecting H3K27me3. A transdifferentiation-related gene external signal-regulated kinase heavily underwent H3K4me3 in developing buds, which could be reproduced by retinoic acid. These results indicate that in P. misakiensis, TC14-3-driven H3K27 trimethylation is a default state of bud and zooid cells, which serves as the histone code for cell longevity. H3K27me3 and H3K4me3 double-positive signals are involved in cell stemness, and absence of signals is the indication of senescence.

Ultrastructural aspects of naturally occurring wound in the tunic of two ascidians: Ciona intestinalis and Styela plicata (Tunicata).

Efficient wound healing is essential for all animals from insects to mammals. Ciona intestinalis and Styela plicata are solitary ascidians belonging to urochordates, a subphylum that occupies a key phylogenetic position as it includes the closest relative to vertebrates. Urochordate first physical barrier against invaders is the tunic, an extracellular matrix that is constantly exposed to all kinds of insults. Thus, when damage occurs, an innate immune response is triggered to eliminate impaired tissue and potentially pathogenic microbes, and restore tissue functionality. Ultrastructural aspects of the tunic in the wound healing process of two ascidians are described. In the injured areas, we evidenced thinning of the tunic and areas of low fibre density, dense intratunic bacterial and protozoan population, and inflammatory aspects such as the increase in tunic cells, their aggregates, and phagocytosis. This is the first report on tunic physical wounding occurring in the natural habitat.

Microinjection and 4D fluorescence imaging in the eggs and embryos of the ascidian Phallusia mammillata.

Time-lapse 4D imaging of fluorescently tagged proteins to follow the dynamics of cellular structures (chromosomes, microtubules, actin, centrosomes, cortical structures like the CAB in ascidians, etc.) combined with targeted gene knockdown during embryonic development is a powerful technique to understand the mechanisms of embryonic development. The eggs and embryos of the primitive marine chordate Phallusia mammillata are an excellent model system for combining live cell imaging with gene knockdown experiments. Here we describe simple methods for microinjecting Phallusia eggs with mRNA encoding fluorescent fusion proteins combined with 4D time-lapse imaging techniques we use to follow all of embryonic development from the egg to late tailbud stage.

Evolutionary diversification of secondary mechanoreceptor cells in tunicata.

BACKGROUND: Hair cells are vertebrate secondary sensory cells located in the ear and in the lateral line organ. Until recently, these cells were considered to be mechanoreceptors exclusively found in vertebrates that evolved within this group. Evidence of secondary mechanoreceptors in some tunicates, the proposed sister group of vertebrates, has recently led to the hypothesis that vertebrate and tunicate secondary sensory cells share a common origin. Secondary sensory cells were described in detail in two tunicate groups, ascidians and thaliaceans, in which they constitute an oral sensory structure called the coronal organ. Among thaliaceans, the organ is absent in salps and it has been hypothesised that this condition is due to a different feeding system adopted by this group of animals. No information is available as to whether a comparable structure exists in the third group of tunicates, the appendicularians, although different sensory structures are known to be present in these animals. RESULTS: We studied the detailed morphology of appendicularian oral mechanoreceptors. Using light and electron microscopy we could demonstrate that the mechanosensory organ called the circumoral ring is composed of secondary sensory cells. We described the ultrastructure of the circumoral organ in two appendicularian species, Oikopleura dioica and Oikopleura albicans, and thus taxonomically completed the data collection of tunicate secondary sensory cells. To understand the evolution of secondary sensory cells in tunicates, we performed a cladistic analysis using morphological data. We constructed a matrix consisting of 19 characters derived from detailed ultrastructural studies in 16 tunicate species and used a cephalochordate and three vertebrate species as outgroups. CONCLUSIONS: Our study clearly shows that the circumoral ring is the appendicularian homologue of the coronal organ of other tunicate taxa. The cladistic analysis enabled us to reconstruct the features of the putative ancestral hair cell in tunicates, represented by a simple monociliated cell. This cell successively differentiated into the current variety of oral mechanoreceptors in the various tunicate lineages. Finally, we demonstrated that the inferred evolutionary changes coincide with major transitions in the feeding strategies in each respective lineage.

Azumiobodo hoyamushi gen. nov. et sp. nov. (Euglenozoa, Kinetoplastea, Neobodonida): a pathogenic kinetoplastid causing the soft tunic syndrome in ascidian aquaculture.

We used morphological and genetic analyses to investigate a pathogenic kinetoplastid isolated from a diseased edible ascidian Halocynthia roretzi with soft tunic syndrome. The morphological characteristics of the kinetoplastid are similar to those in the order Neobodonida in the subclass Metakinetoplastida. However, the presence of unique globular bodies distinguishes this kinetoplastid from the other polykinetoplastic genera (i.e. Cruzella, Dimastigella and Rhynchobodo) in this order. These globular bodies are cytoplasmic inclusions without an outer delimiting membrane and are composed of a homologous granular matrix containing electron-dense bands. A phylogenetic tree based on 18S rRNA gene sequences also indicated that the kinetoplastid belongs to the order Neobodonida, although it forms an independent clade in this order. From these results, we propose a new genus in the order Neobodonida, i.e. Azumiobodo gen. nov., and Azumiobodo hoyamushi as the type species for the genus.

The postbranchial digestive tract of the ascidian, Polyandrocarpa misakiensis (Tunicata: Ascidiacea). 2. Stomach.

The organization of the stomach in the compound styelid ascidian, Polyandrocarpa misakiensis, is described, and the morphology and cell types of the stomach is discussed from the phylogenetic viewpoint. The stomach is a sac-like organ whose wall is formed into longitudinal folds. The stomach consists of external and internal epithelium. The internal epithelium is simple columnar, except for the bottom of the folds. There are five cell types: absorptive cells, zymogenic cells, endocrine cells, ciliated mucous cells, and undifferentiated cells. The absorptive cells have numerous microvilli. The apical region of these cells is occupied by coated vesicles. The zymogenic cells have a conical outline and a few microvilli on their apical surfaces. There are secretory granules in the apical region of zymogenic cells. The endocrine cells have low cell height and electron-dense granules around the nucleus. Endocrine cells have one or two cilia and a few microvilli on the apical surfaces. The basolateral part of these cells often bulges into the adjoining cells. Immunoelectron microscopy revealed that some endocrine cells have serotonin-like immunoreactivity. The ciliated mucous cells are restricted to a single ventral groove. They have numerous microvilli and a few cilia on their apical surfaces. Moderately electron-dense granules are accumulated in the apical part of the ciliated mucous cells. Undifferentiated cells, filled with free ribosomes, form a pseudostratified epithelium in the base of each fold. The nucleus of undifferentiated cells has a prominent nucleolus. The pseudostratified epithelium of the pyloric caecum consists of electron-dense and electron-light cells.

Forming a tough shell via an intracellular matrix and cellular junctions in the tail epidermis of Oikopleura dioica (Chordata: Tunicata: Appendicularia).

A postanal tail is a major synapomorphy of the phylum Chordata, which is composed of three subphyla: Vertebrata, Cephalochordata, and Tunicata (Urochordata). Among tunicates, appendicularians are the only group that retains the tail in the adult, and the adult tail functions in locomotion and feeding in combination with a cellulose-based house structure. Given the phylogenetic position of tunicates, the appendicularian adult tail may possess ancestral features of the chordate tail. We assess the ultrastructural development of the tail epidermis of the appendicularian Oikopleura dioica. The epidermis of the larval tail is enclosed by the larval envelope, which is a thin sheet similar to the outer tunic layer of ascidian larvae. The epidermis of the adult tail seems to bear no tunic-like cellulosic integuments, and the tail fin is a simple folding of the epidermis. Every epidermal cell, except for the triangular cells at the edge of the tail fin, has a conspicuous matrix layer of fibrous content in the apical cytoplasm without enclosing membranes. The epidermis of the larval tail does not have a fibrous matrix layer, suggesting the production of the layer during larval development and metamorphosis. Zonulae adhaerentes firmly bind the epidermal cells of the adult tail to one another, and the dense microfilaments lining the cell borders constitute a mechanical support for the cell membranes. The intracellular matrix, cell junctions, and cytoskeletons probably make the tail epidermis a tough, flexible shell supporting the active beating of the oikopleuran adult tail.

Nephromyces, a beneficial apicomplexan symbiont in marine animals.

With malaria parasites (Plasmodium spp.), Toxoplasma, and many other species of medical and veterinary importance its iconic representatives, the protistan phylum Apicomplexa has long been defined as a group composed entirely of parasites and pathogens. We present here a report of a beneficial apicomplexan: the mutualistic marine endosymbiont Nephromyces. For more than a century, the peculiar structural and developmental features of Nephromyces, and its unusual habitat, have thwarted characterization of the phylogenetic affinities of this eukaryotic microbe. Using short-subunit ribosomal DNA (SSU rDNA) sequences as key evidence, with sequence identity confirmed by fluorescence in situ hybridization (FISH), we show that Nephromyces, originally classified as a chytrid fungus, is actually an apicomplexan. Inferences from rDNA data are further supported by the several apicomplexan-like structural features in Nephromyces, including especially the strong resemblance of Nephromyces infective stages to apicomplexan sporozoites. The striking emergence of the mutualistic Nephromyces from a quintessentially parasitic clade accentuates the promise of this organism, and the three-partner symbiosis of which it is a part, as a model for probing the factors underlying the evolution of mutualism, pathogenicity, and infectious disease.

Filtration of submicrometer particles by pelagic tunicates.

Salps are common in oceanic waters and have higher per-individual filtration rates than any other zooplankton filter feeder. Although salps are centimeters in length, feeding via particle capture occurs on a fine, mucous mesh (fiber diameter d approximately 0.1 microm) at low velocity (U = 1.6 +/- 0.6 cmxs(-1), mean +/- SD) and is thus a low Reynolds-number (Re approximately 10(-3)) process. In contrast to the current view that particle encounter is dictated by simple sieving of particles larger than the mesh spacing, a low-Re mathematical model of encounter rates by the salp feeding apparatus for realistic oceanic particle-size distributions shows that submicron particles, due to their higher abundances, are encountered at higher rates (particles per time) than larger particles. Data from feeding experiments with 0.5-, 1-, and 3-microm diameter polystyrene spheres corroborate these findings. Although particles larger than 1 microm (e.g., flagellates, small diatoms) represent a larger carbon pool, smaller particles in the 0.1- to 1-microm range (e.g., bacteria, Prochlorococcus) may be more quickly digestible because they present more surface area, and we find that particles smaller than the mesh size (1.4 microm) can fully satisfy salp energetic needs. Furthermore, by packaging submicrometer particles into rapidly sinking fecal pellets, pelagic tunicates can substantially change particle-size spectra and increase downward fluxes in the ocean.

Transfer of prokaryotic algal symbionts from a tropical ascidian (Lissoclinum punctatum) colony to its larvae.

Lissoclinum punctatum is a colonial ascidian that harbors the symbiotic prokaryotic alga Prochloron in its tunic and in the peribranchial and common cloacal cavities. Most symbiotic cells in the tunic are intracellular (tunic phycocytes), while those in the cavities are extracellular. We found that neither gametes nor embryos brooded in the tunic were associated with photosymbionts. We determined that algal cells attach to posterior parts of the trunk of hatching larvae swimming in the common cloacal cavity. No symbiont cells were found intracellularly in larval tissues. Thus, extracellular Prochloron cells in the cloacal cavities were transferred to the larvae, but intracellular photosymbionts in the tunic were not. The intracellular symbiosis must be reestablished in each generation after larval settlement.

Natural apoptosis during the blastogenetic cycle of the colonial ascidian Botryllus schlosseri: a morphological analysis.

Colonies of the compound ascidian Botryllus schiosseri undergo regular generation changes, during which adult zooids are progressively resorbed and replaced by growing buds. The generation change, or take-over, is characterized by massive cell death by apoptosis, as indicated by nuclear condensation, activation of caspases, overexpression of molecules recognized by antibodies against mammalian Bax, Fas, and FasL, changes in the expression of surface molecules by senescent cells of zooid tissues, and recruitment of circulating phagocytes in zooid tissues which ensure the complete clearing of dying cells. The entire process lasts 24-36 h at 20 degrees C and has been subdivided, on the basis of the degree of contraction of old zooids, into four substages. In the present work, we carried out a detailed morphological analysis of the events occurring in zooid tissues during the take-over substages. Results Indicate that traces of apoptosis can be found in the epidermis, peribranchial epithelium, and heart in the late substage but are easily found in the branchial basket 2-4 h after the beginning of the generation change, thus confirming the antero-posterior progression of cell death, at least in the alimentary system.

Oikopleura dioica alcohol dehydrogenase class 3 provides new insights into the evolution of retinoic acid synthesis in chordates.

Enzymes that synthesize retinoic acid (RA) constitute the first level of regulation of RA action. In vertebrates, enzymes of the medium-chain alcohol dehydrogenase (MDR-Adh) family catalyze the first step of the RA synthetic pathway by oxidizing retinol. Among MDR-Adh enzymes, Adh3 is the only member present in non-vertebrates, and whether Adh3 is actually involved in RA biosynthesis remains uncertain. Here, we investigate the MDR-Adh family in Oikopleura dioica, a urochordate representing the sister group to vertebrates. Oikopleura is of special interest because it has lost the classical RA role in development, which relaxed evolutionary constraints to preserve the RA-genetic machinery, leading to the loss of RA-system components. The hypothesis that Adh3 plays a role in RA synthesis predicts that the relaxation of selection in Oikopleura should have led to the loss of Adh3, or changes in residues related to retinol oxidation. The hypothesis also predicts changes in the expression pattern of Oikopleura Adh3 compared to other chordates that preserved RA-signaling. Our results, however, revealed the presence of a highly conserved Adh3 gene in Oikopleura, with no significant changes in functional residues. Our results also revealed that the Oikopleura Adh3 expression remains unchanged in comparison to other non-vertebrate chordates, restricted to specific compartments of the digestive system. Because Adh3 has been highly conserved in an animal that has dismantled the RA system, we conclude that Adh3 preservation is not due to a conserved role in RA synthesis. Thereby, if Adh3 plays a role in RA synthesis in vertebrates, it might be a lineage-specific neofunctionalization.