Characterization of TRPA channels in the starfish Patiria pectinifera: involvement of thermally activated TRPA1 in thermotaxis in marine planktonic larvae.

The vast majority of marine invertebrates spend their larval period as pelagic plankton and are exposed to various environmental cues. Here we investigated the thermotaxis behaviors of the bipinnaria larvae of the starfish, Patiria pectinifera, in association with TRPA ion channels that serve as thermal receptors in various animal species. Using a newly developed thermotaxis assay system, we observed that P. pectinifera larvae displayed positive thermotaxis toward high temperatures, including toward temperatures high enough to cause death. In parallel, we identified two TRPA genes, termed PpTRPA1 and PpTRPA basal, from this species. We examined the phylogenetic position, spatial expression, and channel properties of each PpTRPA. Our results revealed the following: (1) The two genes diverged early in animal evolution; (2) PpTRPA1 and PpTRPA basal are expressed in the ciliary band and posterior digestive tract of the larval body, respectively; and (3) PpTRPA1 is activated by heat stimulation as well as by known TRPA1 agonists. Moreover, knockdown and rescue experiments demonstrated that PpTRPA1 is involved in positive thermotaxis in P. pectinifera larvae. This is the first report to reveal that TRPA1 channels regulate the behavioral response of a marine invertebrate to temperature changes during its planktonic larval period.

Identification of morphogenetic capability limitations via a single starfish embryo/larva reconstruction method.

Reconstruction of a starfish embryo provides unique morphogenesis during the developmental process that is not observed in normal development. Here, we established a novel method for reconstruction from single embryos/larvae. By using this method, we investigated the morphogenetic capabilities in critical steps during the reconstruction process as showed by the reconstructed embryos generated from embryos/larvae at the six developmental stages, or from segregated ectodermal and/or endomesodermal cells. Additionally, the novel method addressed several problems found in prior methods related to reproducibly generating reconstructed embryos. In the reconstructions from the various stage embryos/larvae, the morphogenetic capabilities were substantively reduced in the reconstructed embryos generated from 3-day bipinnaria (3dBp). The combination experiments using ectodermal or endomesodermal cells segregated from 2dBp or 3dBp showed a reduction of the morphogenetic capabilities in both cells types in 3dBp. The reconstructed embryos generated from ectodermal or endomesodermal cells segregated from 2dBp possessed partial morphological features, such as formation of the epithelium or blastopore, but all failed to develop into bipinnariae. These results indicate two limitations of the morphogenetic capabilities during the reconstruction process. Firstly, the morphogenetic capabilities to reconstruct an embryo are considerably reduced between 2dBp and 3dBp. Secondly, cells specified as ectoderm or endomesoderm possess limited morphogenetic capabilities to reconstruct bipinnaria. Furthermore, our results demonstrate that the interaction between these specified cell types is required for reconstruction.

Wnt, Frizzled, and sFRP gene expression patterns during gastrulation in the starfish Patiria (Asterina) pectinifera.

By the initial phase of gastrulation, Wnt pathway regulation mediates endomesoderm specification and establishes the animal-vegetal axis, thereby leading to proper gastrulation in starfish. To provide insight into the ancestral mechanism regulating deuterostome gastrulation, we identified the gene expression patterns of Wnt, Frizzled (Fz), and secreted frizzled-related protein (sFRP) family genes, which play a role in the initial stage of the Wnt pathway, in starfish Patiria (Asterina) pectinifera embryos using whole mount in situ hybridization. We identified ten Wnt, four Fz, and two sFRP paralogues. From the hatching blastula to the late gastrula stage, the majority of the Wnt genes and both Fz5/8 and sFRP1/5 were expressed in the posterior and anterior half of the embryo, respectively. Wnt8, Fz1, and Fz4 showed restricted expression in the lateral ectoderm. On the other hand, several genes were expressed de novo in the restricted domain of the archenteron at the late gastrula stage. These results suggest that the canonical and/or non-canonical Wnt pathway might implicate endomesoderm specification, anterior-posterior axis establishment, anterior-posterior patterning, and archenteron morphogenesis in the developmental context of starfish embryos. From comparison with the expression patterns observed in Patria miniata, we consider that the Wnt pathway is conserved among starfishes.

Hox10-regulated endodermal cell migration is essential for development of the ascidian intestine.

Hox cluster genes play crucial roles in development of the metazoan antero-posterior axis. Functions of Hox genes in patterning the central nervous system and limb buds are well known. They are also expressed in chordate endodermal tissues, where their roles in endodermal development are still poorly understood. In the invertebrate chordate, Ciona intestinalis, endodermal tissues are in a premature state during the larval stage, and they differentiate into the digestive tract during metamorphosis. In this study, we showed that disruption of a Hox gene, Ci-Hox10, prevented intestinal formation. Ci-Hox10-knock-down larvae displayed defective migration of endodermal strand cells. Formation of a protrusion, which is important for cell migration, was disrupted in these cells. The collagen type IX gene is a downstream target of Ci-Hox10, and is negatively regulated by Ci-Hox10 and a matrix metalloproteinase ortholog, prior to endodermal cell migration. Inhibition of this regulation prevented cellular migration. These results suggest that Ci-Hox10 regulates endodermal strand cell migration by forming a protrusion and by reconstructing the extracellular matrix.

Tissue-specific and ubiquitous gene knockouts by TALEN electroporation provide new approaches to investigating gene function in Ciona.

Custom designed nucleases can simplify gene targeting experiments and have the potential to allow these techniques to be performed in a wide range of organisms. Transcriptional activator-like effector nucleases (TALENs) are starting to fulfill this potential with the advantages of low cost and fast construction times. Here, we report that TALENs are highly effective at inducing mutations in specific genomic loci in the ascidian chordate Ciona intestinalis. In Ciona there are well-established methods to introduce exogenous DNA by electroporation, and we show that this method can be used to introduce constructs that can express TALENs ubiquitously or in specific tissues. Our current protocols enable the rapid analysis of hundreds of TALEN-induced mutants. TALEN electroporations result in a high rate of mutations. These mutations can result in gene knockouts that recapitulate previously described functions of Fgf3 and Hox12. We show that TALENs can work efficiently to cause tissue-specific knockouts and demonstrate this by knocking out Hox12 in the epidermis and Fgf3 in neural tissues. We also use tissue-specific knockouts to reveal a new function of Fgf3 during ascidian larval metamorphosis.

Formation of the digestive tract in Ciona intestinalis includes two distinct morphogenic processes between its anterior and posterior parts.

BACKGROUND: In the ascidian Ciona intestinalis, the digestive tract, an essential system for animals, develops during metamorphosis from the two primordial tissues, the endoderm and endodermal strand, located in the larval trunk and tail, respectively. However, it has been largely unknown how the digestive tract develops from these primordial tissues. We examined the metamorphosing larvae for the tubular formation of the digestive tract, focusing on the epithelial organization of the endoderm, by combined confocal microscopy and computational rendering. RESULTS: The tubular structure of the esophagus to the stomach was formed through the folding and closure of the endodermal epithelia in the central-to-right posterior trunk. By contrast, the intestine was formed in the left posterior trunk through the accumulation and rearrangement of the cells originated from the endodermal strand. This was confirmed by the cell-tracing experiment using Kaede expression construct driven in the endodermal strand. Thus, the tubular formation of the digestive tract in C. intestinalis includes distinct morphogenetic processes and cell lineages between its anterior and posterior parts. CONCLUSION: This study provides the first detailed description of the digestive tract morphogenesis in C. intestinalis and serves as an important basis toward thorough understanding of its digestive tract development.

ß-Catenin-driven binary fate specification segregates germ layers in ascidian embryos.

ß-catenin is a transcriptional cofactor mediating the "canonical" Wnt signaling pathway, which activates target genes in a complex with TCF (LEF) transcription factors [1]. In many metazoans, embryos are first subdivided during early cleavage stages into nuclear ß-catenin-positive and -negative domains, with ß-catenin specifying endoderm or mesendoderm fate. This process has been demonstrated in a wide range of phyla including cnidarians, nemerteans, and invertebrate deuterostomes (echinoderms, hemichordates, and ascidians), implying that ß-catenin-dependent (mes)endoderm specification is evolutionarily ancient [2-10]. However, the mechanisms leading to the segregation of mesoderm and endoderm fates from a transient mesendodermal state are less well defined. We show that subdivision of the ascidian embryo into the three germ layers involves differential nuclear ß-catenin activity coupled with the first two animal-vegetal (A-V)-oriented cell divisions. We reveal that each of these A-V divisions operates as a binary fate choice: the first between ectoderm and mesendoderm and the second between margin (notochord and neural) and endoderm, such that a ß-catenin activation sequence of ON-to-ON specifies endoderm, OFF-to-OFF ectoderm, and ON-to-OFF margin.

Efficient TALEN construction and evaluation methods for human cell and animal applications.

Transcription activator-like effector nucleases (TALENs) have recently arisen as effective tools for targeted genome engineering. Here, we report streamlined methods for the construction and evaluation of TALENs based on the 'Golden Gate TALEN and TAL Effector Kit' (Addgene). We diminished array vector requirements and increased assembly rates using six-module concatemerization. We altered the architecture of the native TALEN protein to increase nuclease activity and replaced the final destination vector with a mammalian expression/in vitro transcription vector bearing both CMV and T7 promoters. Using our methods, the whole process, from initiating construction to completing evaluation directly in mammalian cells, requires only 1 week. Furthermore, TALENs constructed in this manner may be directly applied to transfection of cultured cells or mRNA synthesis for use in animals and embryos. In this article, we show genomic modification of HEK293T cells, human induced pluripotent stem cells, Drosophila melanogaster, Danio rerio and Xenopus laevis, using custom-made TALENs constructed and evaluated with our protocol. Our methods are more time efficient compared with conventional yeast-based evaluation methods and provide a more accessible and effective protocol for the application of TALENs in various model organisms.

Efficient targeted mutagenesis of the chordate Ciona intestinalis genome with zinc-finger nucleases.

Zinc-finger nucleases (ZFNs) are engineered nucleases that induce DNA double-strand breaks (DSBs) at target sequences. They have been used as tools for generating targeted mutations in the genomes of multiple organisms in both animals and plants. The DSB induced by ZFNs is repaired by non-homologous end joining (NHEJ) or by homologous recombination (HR) mechanisms. Non-homologous end joining induces some errors because it is independent of a reference DNA sequence. Through the NHEJ mechanism, ZFNs generate insertional or deletional mutations at the target sequence. We examined the usability, specificity and toxicity of ZFNs in the basal chordate Ciona intestinalis. As the target of ZFNs, we chose an enhanced green fluorescent protein (EGFP) gene artificially inserted in the C. intestinalis genome because this locus is neutral for the development and growth of C. intestinalis, and the efficiency of mutagenesis with ZFNs can thus be determined without any bias. We introduced EGFP -ZFN mRNAs into the embryos of an EGFP -transgenic line and observed the mutation frequency in the target site of EGFP . We also examined the effects of the EGFP -ZFNs at off-target sites resembling the EGFP target sequence in the C. intestinalis genome in order to examine the specificity of ZFNs. We further investigated the influence of ZFNs on embryogenesis, and showed that adequate amounts of ZFNs, which do not disrupt embryogenesis, can efficiently induce mutations on the on-target site with less effect on the off-target sites. This suggests that target mutagenesis with ZFNs will be a powerful technique in C. intestinalis.

Retinoic acid-driven Hox1 is required in the epidermis for forming the otic/atrial placodes during ascidian metamorphosis.

Retinoic acid (RA)-mediated expression of the homeobox gene Hox1 is a hallmark of the chordate central nervous system (CNS). It has been suggested that the RA-Hox1 network also functions in the epidermal ectoderm of chordates. Here, we show that in the urochordate ascidian Ciona intestinalis, RA-Hox1 in the epidermal ectoderm is necessary for formation of the atrial siphon placode (ASP), a structure homologous to the vertebrate otic placode. Loss of Hox1 function resulted in loss of the ASP, which could be rescued by expressing Hox1 in the epidermis. As previous studies showed that RA directly upregulates Hox1 in the epidermis of Ciona larvae, we also examined the role of RA in ASP formation. We showed that abolishment of RA resulted in loss of the ASP, which could be rescued by forced expression of Hox1 in the epidermis. Our results suggest that RA-Hox1 in the epidermal ectoderm played a key role in the acquisition of the otic placode during chordate evolution.

Large-scale infection of the ascidian Ciona intestinalis by the gregarine Lankesteria ascidiae in an inland culture system.

An important way to keep transgenic and mutant lines of the ascidian Ciona intestinalis, a model system for e.g. genetic functions, in laboratories is via culturing systems. Here we report a disease of C. intestinalis observed in an inland culturing system. The disease, called 'long feces syndrome,' is expressed in affected animals by the following characteristic symptoms of the digestive system: (1) excretion of long and thin feces, (2) pale color of the stomach, and (3) congestion of the digestive tube by digested material. Severely diseased animals usually die within a week after the first symptoms occur, implying a high risk of this disease for ascidian culturing systems. The digestive tubes of the diseased animals are occupied by the gregarine apicomplexan parasite Lankesteria ascidiae, suggesting that large-scale infection by this parasite is the cause of long feces syndrome.

Macho-1 regulates unequal cell divisions independently of its function as a muscle determinant.

The anterior-posterior (A-P) axis in ascidian embryos is established through the posteriorizing activities of a localized egg region known as the posterior vegetal cortex/cytoplasm (PVC). Here we describe a novel function of macho-1, a maternally-localized muscle determinant, in establishment of the A-P axis in the Halocynthia roretzi embryo. Macho-1, in addition to its known function in the formation of posterior tissue such as muscle and mesenchyme, and suppression of the anterior-derived notochord fate, acts independently of its transcriptional activity as a regulator of posterior-specific unequal cell divisions, in cooperation with beta-catenin. Our results suggest that macho-1 and beta-catenin regulate the formation of a microtubule bundle that shortens and pulls the centrosome toward a sub-cellular cortical structure known as centrosome-attracting body (CAB), which is located at the posterior pole of the embryo during unequal cell divisions, and act upstream of PEM, a recently-identified regulator of unequal cell divisions. We also present data that suggest that PEM localization to the CAB may not be required for unequal cleavage regulation. The present study provides an important and novel insight into the role of the zinc-finger-containing transcription factor and indicates that it constitutes a major part of the PVC activity.

Efficient transposition of a single Minos transposon copy in the genome of the ascidian Ciona intestinalis with a transgenic line expressing transposase in eggs.

Transgenesis with transposons is an important technique for studying genetic functions. In the ascidian Ciona intestinalis, methods for germline transformation with the Tc1/mariner transposon Minos have been established. A system to remobilize a single Minos copy in the genome is needed to refine this transgenic technique. In this study, such an experimental system was established with a transgenic line expressing Minos transposase in eggs. In the eggs of a double transgenic animal from a cross between the egg transposase line and a transgenic line having a single Minos insertion, the transposon was transposed into new positions of the Ciona genome, thus creating new insertions. Some of the new insertions caused enhancer detection. The majority of the new insertion sites were mapped on different chromosomes from that of the transposon donor. This characteristic of Minos is in contrast to that of the Sleeping Beauty transposon, which causes frequent intrachromosomal transposition.

Localized PEM mRNA and protein are involved in cleavage-plane orientation and unequal cell divisions in ascidians.

BACKGROUND: Orientation and positioning of the cell division plane are essential for generation of invariant cleavage patterns and for unequal cell divisions during development. Precise control of the division plane is important for appropriate partitioning of localized factors, spatial arrangement of cells for proper intercellular interactions, and size control of daughter cells. Ascidian embryos show complex but invariant cleavage patterns mainly due to three rounds of unequal cleavage at the posterior pole. RESULTS: The ascidian embryo is an emerging model for studies of developmental and cellular processes. The maternal Posterior End Mark (PEM) mRNA is localized within the egg and embryo to the posterior region. PEM is a novel protein that has no known domain. Immunostaining showed that the protein is also present in the posterior cortex and the in centrosome-attracting body (CAB) and that the localization is extraction-resistant. Here we show that PEM of Halocynthia roretzi is required for correct orientation of early-cleavage planes and subsequent unequal cell divisions because it repeatedly pulls a centrosome toward the posterior cortex and the CAB, respectively, where PEM mRNA and protein are localized. When PEM activity is suppressed, formation of the microtubule bundle linking the centrosome and the posterior cortex did not occur. PEM possibly plays a role in anchoring microtubule ends to the cortex. In our model of orientation of the early-cleavage planes, we also amend the allocation of the conventional animal-vegetal axis in ascidian embryos, and discuss how the newly proposed A-V axis provides the rationale for various developmental events and the fate map of this animal. CONCLUSIONS: The complex cleavage pattern in ascidian embryos can be explained by a simple rule of centrosome attraction mediated by localized PEM activity. PEM is the first gene identified in ascidians that is required for multiple spindle-positioning events.

Nuclear accumulation of beta-catenin and transcription of downstream genes are regulated by zygotic Wnt5alpha and maternal Dsh in ascidian embryos.

Nuclear beta-catenin plays crucial roles in the establishment of the embryonic axis and formation of mesendoderm tissues in ascidians and other animals. However, the cue responsible for nuclear accumulation of beta-catenin in the vegetal hemisphere is still unknown in ascidians. Here, we investigated the roles of Wnt5alpha and Dsh in the nuclear accumulation of beta-catenin and activation of its downstream genes in the ascidian Halocynthia roretzi. Wnt5alpha knockdown embryos lost nuclear accumulation of beta-catenin at the 64-cell stage but not at the 32-cell stage, and expression of Hr-lim, one of the targets of beta-catenin, was impaired in the anterior region of the embryo. Zygotic Wnt5alpha expression in the anterior-vegetal blastomeres was primarily responsible for these defects. Dsh knockdown showed no effect on nuclear localization of beta-catenin, but inhibited Hr-lim expression in the posterior region. These results suggest that maintenance of nuclear Hr-beta-catenin after the 64-cell stage is regulated by zygotic Hr-Wnt5alpha, and that expression of its target genes is modulated by both Hr-Wnt5alpha and Hr-Dsh. Our results also highlight the importance of nuclear accumulation of beta-catenin up to the 32-cell stage through a still unclarified mechanism.

Regulation of NF-kappaB/Rel by IkappaB is essential for ascidian notochord formation.

We previously reported that two NF-kappaB/Rel family members are involved in notochord formation of the ascidian Halocynthia roretzi. Here, we present evidence that the NF-kappaB/Rel signaling pathway plays important roles in the notochord formation in another ascidian, Ciona intestinalis. We first found that two NF-kappaB/Rel family members of C. intestinalis, Ci-rel1 and Ci-rel2, are splice variants: Ci-rel1 is a typical member, while Ci-rel2 is a C-terminally truncated short one. Ectopic expression of GFP-fusion proteins in the C. intestinalis notochord revealed that Ci-rel1 transiently moved into the nucleus in the initial tailbud stage, when concomitant expression of Ci-IkappaB, a C. intestinalis IkappaB homologue, was observed, indicating that Ci-rel1 is transiently activated in this stage. Ci-rel1, as well as Ci-rel2, is capable of binding to the kappaB sequence present upstream of Ci-IkappaB, suggesting that Ci-IkappaB is a target gene of Ci-rel1. Reporter gene assay suggests that the expression of Ci-IkappaB in the notochord is controlled by its kappaB sequence. Gene silencing of Ci-IkappaB by injection of the corresponding antisense morpholino oligonucleotide resulted in impairment of notochord formation in C. intestinalis, particularly in a defect in intercalation of notochord cells. Taken together, the results suggest that the regulation of Ci-rel1 by Ci-IkappaB, whose transcription is regulated by Ci-rel1, in the tailbud stage is essential for notochord formation in C. intestinalis.

Regulation of ascidian Rel by its alternative splice variant.

The Rel/NF-kappaB family of transcription factors play key roles in morphogenesis and immune responses. We reported previously that As-rel1 and As-rel2 of the ascidian Halocynthia roretzi are involved in notochord formation. The As-rel1 protein is a typical Rel/NF-kappaB family member, whereas the As-rel2 protein is a novel truncated product of As-rel1 that lacks a nuclear localization signal and the unique C-terminal region. Here, we present conclusive evidence that As-rel1 and As-rel2 are generated from a single gene by alternative splicing. We analyzed the roles of As-rel2 using cells transfected with As-rel1 or As-rel2 or both. As-rel1 was localized in the nucleus and As-rel2 in the cytoplasm when they were transfected individually. In contrast, when they were transfected simultaneously, both were localized in the nucleus because of the association of As-rel2 with As-rel1. In this case, the transcriptional activity of As-rel1 was suppressed by As-rel2. Ascidian IkappaB was found to sequester As-rel1 in the cytoplasm and suppress its transcriptional activity when As-rel1 and IkappaB were transfected simultaneously. In contrast, when As-rel1 and IkappaB were transfected together with As-rel2, As-rel1 was transported into the nucleus and its transcriptional activity was rescued from inhibition by IkappaB, whereas As-rel2 remained localized in the cytoplasm, suggesting IkappaB sequestration in the cytoplasm by As-rel2. From these findings, we conclude that the alternative splice variant, As-rel2, regulates the nuclear localization and transcriptional activity of As-rel1.