Zic-r.b controls cell numbers in Ciona embryos by activating CDKN1B.

The control of cell numbers and the establishment of cell types are two processes that are essential in early embryonic development. We have a reasonable understanding of how these processes occur individually, but we have considerably less sophisticated understanding of how these processes are linked. Tunicates have fixed cell lineages with predictable cell cycles, making them well suited to investigate these processes. In the ascidian Ciona, we show that the transcription factor Zic-r.b, known to be involved in establishing several cell types in early development also activates the expression of the cell cycle inhibitor CDKN1B. Zic-r.b is a major missing component of the cell division clock establishing specific cell numbers. We also show that a larvacean homolog of Zic-r.b is expressed one cell cycle earlier than its Ciona counterpart. The early expression in larvaceans may explain why they have half as many notochord cells as ascidians and may illustrate a general mechanism to evolve changes in morphology.

Germline development during embryogenesis of the larvacean, Oikopleura dioica.

Germ cells develop into eggs and sperms and represent a lineage that survives through multiple generations. Germ cell specification during embryogenesis proceeds through one of two basic modes: either the cell-autonomous mode or the inductive mode. In the cell-autonomous mode, specification of germ cell fate involves asymmetric partitioning of the specialized maternal cytoplasm, known as the germplasm. Oikopleura dioica is a larvacean (class Appendicularia) and a chordate. It is regarded as a promising animal model for studying chordate development because of its short life cycle (5 days) and small genome size (∼60 â€‹Mb). We show that their embryos possess germplasm, as observed in ascidians (class Ascidiacea). The vegetal cytoplasm shifted towards the future posterior pole before the first cleavage occurred. A bilateral pair of primordial germ cells (PGC, B11 â€‹cells) was formed at the posterior pole at the 32-cell stage through two rounds of unequal cleavage. These B11 â€‹cells did not undergo further division before hatching of the tadpole-shaped larvae. The centrosome-attracting body (CAB) is a subcellular structure that contains the germplasm and plays crucial roles in germ cell development in ascidians. The presence of CAB with germplasm was observed in the germline lineage cells of larvaceans via electron microscopy and using extracted embryos. The CAB appeared at the 8-cell stage and persisted until the middle stage of embryogenesis. The antigen for the phosphorylated histone 3 antibody was localized to the CAB and persisted in the PGC until hatching after the CAB disappeared. Maternal snail mRNA, which encodes a transcription factor, was co-localized with the antigen for the H3S28p antibody. Furthermore, we found a novel PGC-specific subcellular structure that we call the germ body (GB). This study thus highlights the conserved and non-conserved features of germline development between ascidians and larvaceans. The rapid development and short life cycle (five days) of O. dioica would open the way to genetically analyze germ cell development in the future.

Formation of the brain by stem cell divisions of large neuroblasts in Oikopleura dioica, a simple chordate.

Stem cell division contributes to the generation of various cell types during animal development, especially a diverse pool of neural cells in the nervous system. One example is reiterated unequal stem cell divisions, in which a large stem cell undergoes a series of oriented unequal divisions to produce a chain of small daughter cells that differentiate. We show that reiterated unequal stem cell divisions are involved in the formation of the brain in simple chordate appendicularians (larvaceans). Two large neuroblasts in the anterior and middle of the brain-forming region of hatched larvae were observed. They produced at least 30 neural cells out of 96 total brain cells before completion of brain formation at 10 hours after fertilization by reiterated unequal stem cell divisions. The daughter cells of the anterior neuroblast were postmitotic, and the number was at least 19. The neuroblast produced small daughter neural cells posteriorly every 20 min. The neural cells first moved toward the dorsal side, turned in the anterior direction, aligned in a single line according to their birth order, and showed collective movement to accumulate in the anterior part of the brain. The anterior neuroblast originated from the right-anterior blastomeres of the eight-cell embryos and the right a222 blastomere of the 64-cell embryo. The posterior neuroblast also showed reiterated unequal stem cell divisions, and generated at least 11 neural cells. Sequential unequal stem cell divisions without stem cell growth have been observed in protostomes, such as insects and annelids. The results provide the first examples of this kind of stem cell division during brain formation in non-vertebrate deuterostomes.

A chordate species lacking Nodal utilizes calcium oscillation and Bmp for left-right patterning.

Larvaceans are chordates with a tadpole-like morphology. In contrast to most chordates of which early embryonic morphology is bilaterally symmetric and the left-right (L-R) axis is specified by the Nodal pathway later on, invariant L-R asymmetry emerges in four-cell embryos of larvaceans. The asymmetric cell arrangements exist through development of the tailbud. The tail thus twists 90° in a counterclockwise direction relative to the trunk, and the tail nerve cord localizes on the left side. Here, we demonstrate that larvacean embryos have nonconventional L-R asymmetries: 1) L- and R-cells of the two-cell embryo had remarkably asymmetric cell fates; 2) Ca2+ oscillation occurred through embryogenesis; 3) Nodal, an evolutionarily conserved left-determining gene, was absent in the genome; and 4) bone morphogenetic protein gene (Bmp) homolog Bmp.a showed right-sided expression in the tailbud and larvae. We also showed that Ca2+ oscillation is required for Bmp.a expression, and that BMP signaling suppresses ectopic expression of neural genes. These results indicate that there is a chordate species lacking Nodal that utilizes Ca2+ oscillation and Bmp.a for embryonic L-R patterning. The right-side Bmp.a expression may have arisen via cooption of conventional BMP signaling in order to restrict neural gene expression on the left side.

Nkx2-1 and FoxE regionalize glandular (mucus-producing) and thyroid-equivalent traits in the endostyle of the chordate Oikopleura dioica.

The endostyle is a ventral pharyngeal organ used for internal filter feeding of basal chordates and is considered homologous to the follicular thyroid of vertebrates. It contains mucus-producing (glandular) and thyroid-equivalent regions organized along the dorsoventral (DV) axis. Although thyroid-related genes (Nkx2-1, FoxE, and thyroid peroxidase (TPO)) are known to be expressed in the endostyle, their roles in establishing regionalization within the organ have not been demonstrated. We report that Nkx2-1 and FoxE are essential for establishing DV axial identity in the endostyle of Oikopleura dioica. Genome and expression analyses showed von Willebrand factor-like (vWFL) and TPO/dual oxidase (Duox)/Nkx2-1/FoxE as orthologs of glandular and thyroid-related genes, respectively. Knockdown experiments showed that Nkx2-1 is necessary for the expression of glandular and thyroid-related genes, whereas FoxE is necessary only for thyroid-related genes. Moreover, Nkx2-1 expression is necessary for FoxE expression in larvae during organogenesis. The results demonstrate the essential roles of Nkx2-1 and FoxE in establishing regionalization in the endostyle, including (1) the Nkx2-1-dependent glandular region, and (2) the Nkx2-1/FoxE-dependent thyroid-equivalent region. DV axial regionalization may be responsible for organizing glandular and thyroid-equivalent traits of the pharynx along the DV axis.

Differentiation of endostyle cells by Nkx2-1 and FoxE in the ascidian Ciona intestinalis type A: insights into shared gene regulation in glandular- and thyroid-equivalent elements of the chordate endostyle.

Due to similarities in iodine concentrations and peroxidase activities, the thyroid in vertebrates is considered to originate from the endostyle of invertebrate chordates even though it is a glandular (mucus-producing) organ for aquatic suspension feeding. Among chordates with an endostyle, urochordates are useful evolutionary research models for the study of vertebrate traits. The ascidian Ciona intestinalis forms an endostyle with specific components of glandular- and thyroid-related elements, and molecular markers have been identified for these components. Since we previously examined a simple endostyle in the larvacean Oikopleura dioica, the expression of the thyroid-related transcription factor genes, Ciona Nkx2-1 and FoxE, was perturbed by TALEN-mediated gene knockout in the present study to elucidate the shared and/or divergent features of a complex ascidian endostyle. The knockout of Ciona Nkx2-1 and FoxE exerted different effects on the morphology of the developing endostyle. The knockout of Nkx2-1 eliminated the expression of both glandular and thyroidal differentiation marker genes, e.g., vWFL1, vWFL2, CiEnds1, TPO, and Duox, while that of FoxE eliminated the expression of the differentiation marker genes, TPO and CiEnds1. The supporting element-related expression of Pax2/5/8a, Pax2/5/8b, FoxQ1, and ß-tubulin persisted in the hypoplastic endostyles of Nkx2-1- and FoxE-knockout juveniles. Although the gene regulation of ascidian-specific CiEnds1 remains unclear, these results provide insights into the evolution of the vertebrate thyroid as well as the urochordate endostyle.

Developmental biology of the larvacean Oikopleura dioica: Genome resources, functional screening, and imaging.

The larvacean Oikopleura dioica is a cosmopolitan planktonic chordate and is closely related to vertebrates. It is characterized by a tadpole-shaped morphology with notochord flanked by muscle in the tail and brain on the dorsal side, a short life cycle of five days, a compact genome of approximately 56 Mb, a simple and transparent body with a small number of cells (~4000 in functional juveniles), invariant embryonic cell lineages, and fast development that ensures complete morphogenesis and organ formation 10 h after fertilization. With these features, this marine chordate is a promising and advantageous animal model in which genetic manipulation is feasible. In this review, we introduce relevant resources and modern techniques that have been developed: (1) Genome and transcriptomes. Oikopleura dioica has the smallest genome among non-parasitic metazoans. Its genome databases have been generated using three geographically distant O. dioica populations, and several intra-species sequence differences are becoming evident; (2) Functional genetic knockdown techniques. Comprehensive screening of genes is feasible using ovarian microinjection and double-strand DNA-induced gene knockdown; and (3) Live imaging of embryos and larvae. Application of these techniques has uncovered novel aspects of development, including meiotic cell arrest, left-right patterning, epidermal cell patterning, and mouth formation involving the connection of ectoderm and endoderm sheets. Oikopleura dioca has become very useful for developmental and evolutionary studies in chordates.

ANISEED 2019: 4D exploration of genetic data for an extended range of tunicates.

ANISEED (https://www.aniseed.cnrs.fr) is the main model organism database for the worldwide community of scientists working on tunicates, the vertebrate sister-group. Information provided for each species includes functionally-annotated gene and transcript models with orthology relationships within tunicates, and with echinoderms, cephalochordates and vertebrates. Beyond genes the system describes other genetic elements, including repeated elements and cis-regulatory modules. Gene expression profiles for several thousand genes are formalized in both wild-type and experimentally-manipulated conditions, using formal anatomical ontologies. These data can be explored through three complementary types of browsers, each offering a different view-point. A developmental browser summarizes the information in a gene- or territory-centric manner. Advanced genomic browsers integrate the genetic features surrounding genes or gene sets within a species. A Genomicus synteny browser explores the conservation of local gene order across deuterostome. This new release covers an extended taxonomic range of 14 species, including for the first time a non-ascidian species, the appendicularian Oikopleura dioica. Functional annotations, provided for each species, were enhanced through a combination of manual curation of gene models and the development of an improved orthology detection pipeline. Finally, gene expression profiles and anatomical territories can be explored in 4D online through the newly developed Morphonet morphogenetic browser.

Mother-to-embryo vitellogenin transport in a viviparous teleost Xenotoca eiseni.

Vitellogenin (Vtg), a yolk nutrient protein that is synthesized in the livers of female animals, and subsequently carried into the ovary, contributes to vitellogenesis in oviparous animals. Thus, Vtg levels are elevated during oogenesis. In contrast, Vtg proteins have been genetically lost in viviparous mammals, thus the yolk protein is not involved in their oogenesis and embryonic development. In this study, we identified Vtg protein in the livers of females during the gestation of the viviparous teleost, Xenotoca eiseni Although vitellogenesis is arrested during gestation, biochemical assays revealed that Vtg protein was present in ovarian tissues and lumen fluid. The Vtg protein was also detected in the trophotaeniae of the intraovarian embryo. Immunoelectron microscopy revealed that Vtg protein is absorbed into intracellular vesicles in the epithelial cells of the trophotaeniae. Furthermore, extraneous Vtg protein injected into the abdominal cavity of a pregnant female was subsequently detected in the trophotaeniae of the intraovarian embryo. Our data suggest that the yolk protein is one of the matrotrophic factors supplied from the mother to the intraovarian embryo during gestation in X. eiseni.

Protein phosphatase 2A is essential to maintain meiotic arrest, and to prevent Ca2+ burst at spawning and eventual parthenogenesis in the larvacean Oikopleura dioica.

Unfertilized eggs of most animals are arrested at a certain point in the meiotic cell cycles. Reinitiation of meiosis and the start of embryogenesis are triggered by fertilization. This arrest is essential for preventing parthenogenetic activation and for promoting proper initiation of development by fertilization. In the larvacean Oikopleura dioica, which is a simple model organism for studies of chordate development, the unfertilized egg is arrested at metaphase of meiosis I. We show here that protein phosphatase 2A (PP2A) is essential for maintenance of meiotic arrest after spawning of oocytes. Knockdown (KD) of the maternal PP2A catalytic subunit, which was found in functional screening of maternal factors, caused unfertilized eggs to spontaneously release polar bodies after spawning, and then start pseudo-cleavages without fertilization, namely, parthenogenesis. Parthenogenetic embryos failed to undergo proper mitosis and cytokinesis because of lack of a centrosome, which is to be brought into the egg by a sperm. Activation of the KD oocytes was triggered by possible rise of ambient and intracellular pH upon their release from the gonad into seawater at spawning. Live recording of intracellular calcium level of the KD oocytes indicated that the pH rise caused an aberrant Ca2+ burst, which mimicked the Ca2+ burst that occurs at fertilization. Then, the aberrant Ca2+ burst triggered meiosis resumption through Calcium/calmodulin-dependent protein kinase (CaMK II). Therefore, PP2A is essential for maintenance of meiotic arrest and prevention of parthenogenesis by suppressing the aberrant Ca2+ burst at spawning.

Wavy movements of epidermis monocilia drive the neurula rotation that determines left-right asymmetry in ascidian embryos.

Tadpole larvae of the ascidian, Halocynthia roretzi, show morphological left-right asymmetry in the brain structures and the orientation of tail bending within the vitelline membrane. Neurula embryos rotate along the anterior-posterior axis in a counterclockwise direction, and then this rotation stops when the left side of the embryo is oriented downwards. Contact of the left-side epidermis with the vitelline membrane promotes nodal gene expression in the left-side epidermis. This is a novel mechanism in which rotation of whole embryos provides the initial cue for breaking left-right symmetry. Here we show that epidermal monocilia, which appear at the neurula rotation stage, generate the driving force for rotation. A ciliary protein, Arl13b, fused with Venus YFP was used for live imaging of ciliary movements. Although overexpression of wild-type Arl13b fusion protein resulted in aberrant movements of the cilia and abrogation of neurula rotation, mutant Arl13b fusion protein, in which the GTPase and coiled-coil domains were removed, did not affect the normal ciliary movements and neurula rotation. Epidermis cilia moved in a wavy and serpentine way like sperm flagella but not in a rotational way or beating way with effective stroke and recovery stroke. They moved very slowly, at 1/7 Hz, consistent with the low angular velocity of neurula rotation (ca. 43°/min). The tips of most cilia pointed in the opposite direction of embryonic rotation. Similar motility was also observed in Ciona robusta embryos. When embryos were treated with a dynein inhibitor, Ciliobrevin D, both ciliary movements and neurula rotation were abrogated, showing that ciliary movements drive neurula rotation in Halocynthia. The drug also inhibited Ciona neurula rotation. Our observations suggest that the driving force of rotation is generated using the vitelline membrane as a substrate but not by making a water current around the embryo. It is of evolutionary interest that ascidians use ciliary movements to break embryonic left-right symmetry, like in many vertebrates. Meanwhile, ascidian embryos rotate as a whole, similar to embryos of non-vertebrate deuterostomes, such as echinoderm, hemichordate, and amphioxus, while swimming.

Mouth opening is mediated by separation of dorsal and ventral daughter cells of the lip precursor cells in the larvacean, Oikopleura dioica.

Mouth formation involves the processes of mouth opening, formation of the oral cavity, and the development of associated sensory organs. In deuterostomes, the surface ectoderm and the anterior part of the archenteron are reconfigured and reconnected to make a mouth opening. This study of the larval development of the larvacean, Oikopleura dioica, investigates the cellular organization of the oral region, the developmental processes of the mouth, and the formation of associated sensory cells. O. dioica is a simple chordate whose larvae are transparent and have a small number of constituent cells. It completes organ morphogenesis in 7 h, between hatching 3 h after fertilization and the juvenile stage at 10 h, when it attains adult form and starts to feed. It has two types of mechanosensory cell embedded in the oral epithelium, which is a single layer of cells. There are twenty coronal sensory cells in the circumoral nerve ring and two dorsal sensory organ cells. Two bilateral lip precursor cells (LPCs), facing the anterior surface, divide dorsoventrally and make a wedge-shaped cleft between the two daughter cells named the dorsal lip cell (DLC) and the ventral lip cell (VLC). Eventually, the DLC and VLC become detached and separated into dorsal and ventral lips, triggering mouth opening. This is an intriguing example of cell division itself contributing to morphogenesis. The boundary between the ectoderm and endoderm is present between the lip cells and coronal sensory cells. All oral sensory cells, including dorsal sensory organ cells, were of endodermal origin and were not derived from the ectodermal placode. These observations on mouth formation provide a cellular basis for further studies at a molecular level, in this simple chordate.

A genome database for a Japanese population of the larvacean Oikopleura dioica.

The larvacean Oikopleura dioica is a planktonic chordate and is a tunicate that belongs to the closest relatives to vertebrates. Its simple and transparent body, invariant embryonic cell lineages, and short life cycle of 5 days make it a promising model organism for the study of developmental biology. The genome browser OikoBase was established in 2013 using Norwegian O. dioica. However, genome information for other populations is not available, even though many researchers have studied local populations. In the present study, we sequenced using Illumina and PacBio RSII technologies the genome of O. dioica from a southwestern Japanese population that was cultured in our laboratory for 3 years. The genome of Japanese O. dioica was assembled into 576 scaffold sequences with a total length and N50 length of 56.6 and 1.5 Mb, respectively. A total of 18,743 gene models (transcript models) were predicted in the genome assembly, named OSKA2016. In addition, 19,277 non-redundant transcripts were assembled using RNA-seq data. The OSKA2016 has global sequence similarity of only 86.5% when compared with the OikoBase, highlighting the sequence difference between the two far distant O. dioica populations on the globe. The genome assembly, transcript assembly, and transcript models were incorporated into ANISEED (https://www.aniseed.cnrs.fr/) for genome browsing and BLAST searches. Mapping of reads obtained from male- or female-specific genome libraries yielded male-specific scaffolds in the OSKA2016 and revealed that over 2.6 Mb of sequence were included in the male-specific Y-region. The genome and transcriptome resources from two distinct populations will be useful datasets for developmental biology, evolutionary biology, and molecular ecology using this model organism.

Patterning and morphogenesis of the intricate but stereotyped oikoplastic epidermis of the appendicularian, Oikopleura dioica.

Mechanisms for morphogenetic processes that generate complex patterns in a reproducible manner remain elusive. Live imaging provides a powerful tool to record cell behaviors. The appendicularian, Oikopleura dioica, is a planktonic tunicate that has a rapid developmental speed, small number of cells (less than 3500 cells in a juvenile), and a transparent body. The trunk epidermis, called the oikoplastic epithelium (OE), has elaborate cellular arrangements showing a complex pattern to secrete so-called "house" made of extracellular components. The OE is characterized by invariant number, size, and shape of the monolayer epithelial cells. Pattern formation is achieved during 5h of larval development without growth of the body, making this a suitable system for live imaging of a two-dimensional (2D) sheet. First, we subdivided the OE and defined several domains by cellular resolution, and systematically gave names to the constituent cells, since there is no variation among individuals. Time-lapse imaging of the epidermal cells revealed region-specific pattern formation processes. Each identified domain served as a compartment into which distribution of descendant cells of founder cells is restricted. Regulation of orientation, timing, and the number of rounds of cell divisions, but not cell death and migration, was a critical mechanism for determination of final cell arrangement and size. In addition, displacement of epithelial sheet plates was observed in the Eisen domain. Stem-cell-like cell divisions, whereby large mother stem cells generate a chain of small daughter cells, were involved in formation of the Nasse region and ventral sensory organ. These are the first examples of this kind of stem-cell-like cell division in deuterostomes. Furthermore, labeling of the left or right blastomere of the two-cell-stage embryo, which roughly gives rise to the left or right side of the body, respectively, revealed that the boundary of the descendant cells does not match with the midline of the trunk epidermis. Left and right descendants largely invade into the opposite side in an invariant way, suggesting the possibility that specification of the OE cell identities may occur later in development, most probably around hatching, and depending on cell position in the OE epithelial sheet. These detailed descriptions of OE patterning processes provide basic and essential information to analyze further cell behaviors in the generation of elaborate and intricate but stereotyped 2D cellular patterns in this advantageous model system for developmental and cell biological studies in chordates.

Redundant mechanisms are involved in suppression of default cell fates during embryonic mesenchyme and notochord induction in ascidians.

During embryonic induction, the responding cells invoke an induced developmental program, whereas in the absence of an inducing signal, they assume a default uninduced cell fate. Suppression of the default fate during the inductive event is crucial for choice of the binary cell fate. In contrast to the mechanisms that promote an induced cell fate, those that suppress the default fate have been overlooked. Upon induction, intracellular signal transduction results in activation of genes encoding key transcription factors for induced tissue differentiation. It is elusive whether an induced key transcription factor has dual functions involving suppression of the default fates and promotion of the induced fate, or whether suppression of the default fate is independently regulated by other factors that are also downstream of the signaling cascade. We show that during ascidian embryonic induction, default fates were suppressed by multifold redundant mechanisms. The key transcription factor, Twist-related.a, which is required for mesenchyme differentiation, and another independent transcription factor, Lhx3, which is dispensable for mesenchyme differentiation, sequentially and redundantly suppress the default muscle fate in induced mesenchyme cells. Similarly in notochord induction, Brachyury, which is required for notochord differentiation, and other factors, Lhx3 and Mnx, are likely to suppress the default nerve cord fate redundantly. Lhx3 commonly suppresses the default fates in two kinds of induction. Mis-activation of the autonomously executed default program in induced cells is detrimental to choice of the binary cell fate. Multifold redundant mechanisms would be required for suppression of the default fate to be secure.

Genome-wide survey of miRNAs and their evolutionary history in the ascidian, Halocynthia roretzi.

BACKGROUND: miRNAs play essential roles in the modulation of cellular functions via degradation and/or translation attenuation of target mRNAs. They have been surveyed in a single ascidian genus, Ciona. Recently, an annotated draft genome sequence for a distantly related ascidian, Halocynthia roretzi, has become available, but miRNAs in H. roretzi have not been previously studied. RESULTS: We report the prediction of 319 candidate H. roretzi miRNAs, obtained through three complementary methods. Experimental validation suggests that more than half of these candidate miRNAs are expressed during embryogenesis. The majority of predicted H. roretzi miRNAs appear specific to ascidians or tunicates, and only 32 candidates, belonging to 25 families, are widely conserved across metazoans. CONCLUSION: Our study presents a comprehensive identification of candidate H. roretzi miRNAs. This resource will facilitate the study of the mechanisms for miRNA-controlled gene regulatory networks during ascidian development. Further, our analysis suggests that the majority of Halocynthia miRNAs are specific to ascidian or tunicates, with only a small number of widely conserved miRNAs. This result is consistent with the general notion that animal miRNAs are less conserved between taxa than plant ones.

Modified whole-mount in situ hybridisation and immunohistochemistry protocols without removal of the vitelline membrane in the appendicularian Oikopleura dioica.

The appendicularian Oikopleura dioica is a planktonic chordate that retains a tadpole shape throughout its life. Its simple and transparent body, invariant cell lineages, fast development and available genome and transcriptome resources make it a promising model organism for research in developmental biology. However, large-scale analysis of gene expression in O. dioica is limited owing to the laborious and time-consuming process of manual removal of the vitelline membrane, because devitellinisation of pre-hatching embryos causes failure of normal development. Therefore, in this study, modified procedures were developed for whole-mount in situ hybridisation (WISH) and immunohistochemistry (WIHC). This protocol enables rapid mRNA or protein detection without a manual devitellination step for each specimen. The critical procedure is brief treatment of the vitelline membrane of living embryos with 0.05% actinase E before fixation. Two minutes of treatment was optimal for the penetration of antisense RNA probes and antibodies through the vitelline membrane. This WISH protocol was applicable for chromogenic and fluorescent tyramide signal amplification reactions. Using the new protocol, we found eight genes with tissue-specific expression in the tail muscle, trunk epidermis, heart, pharynx, oesophagus, stomach or gill openings of developing larvae. This procedure also allowed for the detection of exogenous FLAG-tagged histone-enhanced green fluorescent protein by WIHC using anti-FLAG antibody. This study provides a useful and convenient tool for studying spatial and temporal gene expression patterns in this simple chordate model and should facilitate handling large amounts of genetic data from transcriptome-based approaches and other techniques such as treatments with chemical inhibitors.

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.

Long-distance cell migration during larval development in the appendicularian, Oikopleura dioica.

The appendicularian, Oikopleura dioica, is a planktonic chordate. Its simple and transparent body, invariant cell lineages and short life cycle of 5 days make it a promising model organism for studies of chordate development. Here we describe the cell migration that occurs during development of the O. dioica larva. Using time-lapse imaging facilitated by florescent labeling of cells, three cell populations exhibiting long-distance migration were identified and characterized. These included (i) a multinucleated oral gland precursor that migrates anteriorly within the trunk region and eventually separates into the left and right sides, (ii) endodermal strand cells that are collectively retracted from the tail into the trunk in a tractor movement, and (iii) two subchordal cell precursors that individually migrate out from the trunk to the tip of the tail. The migration of subchordal cell precursors starts when all of the endodermal strand cells enter the trunk, and follows the same path but in a direction opposite to that of the latter. Labeling of these cells with a photoconvertible fluorescent protein, Kaede, demonstrated that the endodermal strand cells and subchordal cell precursors have distinct origins and eventual fates. Surgical removal of the trunk from the tail demonstrated that the endodermal strand cells do not require the trunk for migration, and that the subchordal cell precursors would be attracted by the distal part of the tail. This well-defined, invariant and traceable long-distance cell migration provides a unique experimental system for exploring the mechanisms of versatile cell migration in this simple organism with a chordate body plan.

Maternal and zygotic transcriptomes in the appendicularian, Oikopleura dioica: novel protein-encoding genes, intra-species sequence variations, and trans-spliced RNA leader.

RNA sequencing analysis was carried out to characterize egg and larval transcriptomes in the appendicularian, Oikopleura dioica, a planktonic chordate, which is characterized by rapid development and short life cycle of 5 days, using a Japanese population of the organism. De novo transcriptome assembly matched with 16,423 proteins corresponding to 95.4% of the protein-encoding genes deposited in the OikoBase, the genome database of the Norwegian population. Nucleotide and amino acid sequence identities between the Japanese and Norwegian O. dioica were estimated to be around 91.0 and 94.8%, respectively. We discovered 175 novel protein-encoding genes: 144 unigenes were common to both the Japanese and Norwegian populations, whereas 31 unigenes were not found in the OikoBase genome reference. Among the total 12,311 unigenes, approximately 63% were detected in egg-stage RNAs, whereas 99% were detected in larval stage RNAs; 3772 genes were up-regulated, and 1336 genes were down-regulated more than four-fold in the larvae. Gene ontology analyses characterized gene activities in these two developmental stages. We found a messenger RNA (mRNA) 5' trans-spliced leader, which was observed in 40.8% of the total unique transcripts. It showed preferential linkage to adenine at the 5' ends of the downstream exons. Trans-splicing was observed more frequently in egg mRNAs compared with larva-specific mRNAs.