Takeshi A. Onuma

RNA interference in the appendicularian Oikopleura dioica reveals the function of the Brachyury gene

The appendicularian Oikopleura dioica is a chordate that has a remarkably simple adult body with small cell number. Its transparency, stereotyped cell lineages, short life cycle, and small genome make it a promising new experimental model of chordate developmental biology. However, the functions of its various genes are still poorly understood due to lack of a tool for suppression of gene expression. Here, we applied a double-stranded RNA (dsRNA)-based-RNA interference (RNAi) method in O. dioica. For introducing dsRNA into eggs and embryos, we injected dsRNAs into the ovary. dsRNA, which is specific to EGFP or mCherry mRNA, decreased the exogenous mRNA-derived fluorescence in both eggs and embryos. dsRNA specific to the Brachyury gene of O. dioica, which is a homologous gene of a key notochord transcriptional factor in ascidians, triggered degradation of endogenous Brachyury mRNA and induced malformation or loss of the notochord in the tail. This effect was Brachyury sequence specific, as three dsRNAs covering different sequences produced the same phenotype. The result is in accordance with its expression site and also with the key regulatory function of Brachyury in notochord formation in other chordates. RNAi in O. dioica would be a useful tool for gaining insight into the oogenesis and early developmental processes in chordates.

DNA interference: DNA-induced gene silencing in the appendicularian Oikopleura dioica.

RNA interference is widely employed as a gene-silencing system in eukaryotes for host defence against invading nucleic acids. In response to invading double-stranded RNA (dsRNA), mRNA is degraded in sequence-specific manner. So far, however, DNA interference (DNAi) has been reported only in plants, ciliates and archaea, and has not been explored in Metazoa. Here, we demonstrate that linear double-stranded DNA promotes both sequence-specific transcription blocking and mRNA degradation in developing embryos of the appendicularian Oikopleura dioica. Introduced polymerase chain reaction (PCR) products or linearized plasmids encoding Brachyury induced tail malformation and mRNA degradation. This malformation was also promoted by DNA fragments of the putative 5′-flanking region and intron without the coding region. PCR products encoding Zic-like1 and acetylcholine esterase also induced loss of sensory organ and muscle acetylcholinesterase activity, respectively. Co-injection of mRNA encoding EGFP and mCherry, and PCR products encoding these fluorescent proteins, induced sequence-specific decrease in the green or red fluorescence, respectively. These results suggest that O. dioica possesses a defence system against exogenous DNA and RNA, and that DNA fragment-induced gene silencing would be mediated through transcription blocking as well as mRNA degradation. This is the first report of DNAi in Metazoa.

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.

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.

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

BackgroundmiRNAs 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.ResultsWe 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.ConclusionOur 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.

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.

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.

DNA interference-mediated screening of maternal factors in the chordate Oikopleura dioica

The maternal contribution to the oocyte cytoplasm plays an important role during embryogenesis because it is involved in early cell fate specification and embryonic axis establishment. However, screening projects targeting maternal factors have only been conducted in a limited number of animal models, such as nematodes, fruit flies, and zebrafish, while few maternal genes have been analysed because of difficulties encountered in inhibiting gene products already expressed in the ovaries. Therefore, simple and efficient methods for large-scale maternal screening are necessary. The appendicularian Oikopleura dioica is a planktonic tunicate member of the chordates. Gonadal microinjection and a novel gene knockdown method, DNA interference (DNAi), have been developed for use in this animal with the aim of inhibiting gene functions during oogenesis within the gonad. In this study, we adapted these methods for large-scale maternal factor screening, and observed malformation phenotypes related to some maternal factors. Approximately 2000 (56.9%) ovary-enriched gene products were screened, of which the knockdown of seven encoding genes resulted in various abnormalities during embryonic development. Most of these were related to microtubules and cell adhesion-related proteins. We conclude that DNAi is a potentially powerful screening tool for the identification of novel maternal factors in chordates.