ホヤにおける新規母性因子特異的ノックダウン法によるmRNAの局在機構の解明

科学研究費助成事業 研究成果報告書：挑戦的萌芽研究2015-2017課題番号 : 15K1452

Transposon mediated transgenesis in a marine invertebrate chordate: Ciona intestinalis

Achievement of transposon mediated germline transgenesis in a basal chordate, Ciona intestinalis, is discussed. A Tc1/mariner superfamily transposon, Minos, has excision and transposition activities in Ciona. Minos enables the creation of stable transgenic lines, enhancer detection, and insertional mutagenesis

Study of genetic function of the ascidian based on the forward and reverse genetic approaches

課題番号：2068101

Monoaminergic modulation of photoreception in ascidian:evidence for a proto-hypothalamo-retinal territory

Background : The retina of craniates/vertebrates has been proposed to derive from a photoreceptor prosencephalic territory in ancestral chordates, but the evolutionary origin of the different cell types making the retina is disputed. Except for photoreceptors, the existence of homologs of retinal cells remains uncertain outside vertebrates. Methods : The expression of genes expressed in the sensory vesicle of the ascidian Ciona intestinalis including those encoding components of the monoaminergic neurotransmission systems, was analyzed by in situ hybridization or in vivo transfection of the corresponding regulatory elements driving fluorescent reporters. Modulation of photic responses by monoamines was studied by electrophysiology combined with pharmacological treatments. Results : We show that many molecular characteristics of dopamine-synthesizing cells located in the vicinity of photoreceptors in the sensory vesicle of the ascidian Ciona intestinalis are similar to those of amacrine dopamine cells of the vertebrate retina. The ascidian dopamine cells share with vertebrate amacrine cells the expression of the key-transcription factor Ptf1a, as well as that of dopamine-synthesizing enzymes. Surprisingly, the ascidian dopamine cells accumulate serotonin via a functional serotonin transporter, as some amacrine cells also do. Moreover, dopamine cells located in the vicinity of the photoreceptors modulate the light-off induced swimming behavior of ascidian larvae by acting on alpha2-like receptors, instead of dopamine receptors, supporting a role in the modulation of the photic response. These cells are located in a territory of the ascidian sensory vesicle expressing genes found both in the retina and the hypothalamus of vertebrates (six3/6, Rx, meis, pax6, visual cycle proteins). Conclusion : We propose that the dopamine cells of the ascidian larva derive from an ancestral multifunctional cell population located in the periventricular, photoreceptive field of the anterior neural tube of chordates, which also gives rise to both anterior hypothalamus and the retina in craniates/vertebrates. It also shows that the existence of multiple cell types associated with photic responses predates the formation of the vertebrate retina

Refining the Ciona intestinalis Model of Central Nervous System Regeneration

BACKGROUND: New, practical models of central nervous system regeneration are required and should provide molecular tools and resources. We focus here on the tunicate Ciona intestinalis, which has the capacity to regenerate nerves and a complete adult central nervous system, a capacity unusual in the chordate phylum. We investigated the timing and sequence of events during nervous system regeneration in this organism. METHODOLOGY/PRINCIPAL FINDINGS: We developed techniques for reproducible ablations and for imaging live cellular events in tissue explants. Based on live observations of more than 100 regenerating animals, we subdivided the regeneration process into four stages. Regeneration was functional, as shown by the sequential recovery of reflexes that established new criteria for defining regeneration rates. We used transgenic animals and labeled nucleotide analogs to describe in detail the early cellular events at the tip of the regenerating nerves and the first appearance of the new adult ganglion anlage. CONCLUSIONS/SIGNIFICANCE: The rate of regeneration was found to be negatively correlated with adult size. New neural structures were derived from the anterior and posterior nerve endings. A blastemal structure was implicated in the formation of new neural cells. This work demonstrates that Ciona intestinalis is as a useful system for studies on regeneration of the brain, brain-associated organs and nerves

piRNA-like small RNAs are responsible for the maternal-specific knockdown in the ascidian Ciona intestinalis Type A

The mRNAs stored in eggs are crucial for embryogenesis. To address functions of maternal mRNAs, we recently reported the novel method MASK (maternal mRNA-specific knockdown), which we used to specifically knockdown maternal transcripts in the ascidian Ciona intestinalis Type A. In MASK, the cis element of a maternal gene is fused with eGFP or Kaede reporter gene, and the cassette is introduced into Ciona genome by transposon-mediated transgenesis. In eggs of the transgenic lines, the maternal expression of the gene whose cis element is used for driving the reporter gene is suppressed. The zygotic expression of the gene is not suppressed, suggesting that the MASK method can distinguish between maternal and zygotic functions of a gene. Here we investigated the cis and trans factors responsible for MASK results. In the ovaries in which knockdown of a maternal gene occurs, a number of antisense small RNAs are expressed that are complementary to the sequence of the knocked-down genes. We suspect that these antisense small RNAs are the factor responsible for MASK results. The antisense small RNAs have several features that are seen in PIWI-interacting RNAs (piRNAs), suggesting that MASK is likely to use a piRNA-mediated mechanism to knock down maternal mRNAs

The nervous system of the adult ascidian Ciona intestinalis Type A (Ciona robusta): Insights from transgenic animal models

The nervous system of ascidians is an excellent model system to provide insights into the evolutionary process of the chordate nervous system due to their phylogenetic positions as the sister group of vertebrates. However, the entire nervous system of adult ascidians has yet to be functionally and anatomically investigated. In this study, we have revealed the whole dorsal and siphon nervous system of the transgenic adult ascidian of Ciona intestinalis Type A (Ciona robusta) in which a Kaede reporter gene is expressed in a pan-neuronal fashion. The fluorescent signal of Kaede revealed the innervation patterns and distribution of neurons in the nervous system of Ciona. Precise microscopic observation demonstrated the clear innervation of the anterior and posterior main nerves to eight and six lobes of the oral and atrial siphons, respectively. Moreover, visceral nerves, previously identified as unpaired nerves, were found to be paired; one nerve was derived from the posterior end of the cerebral ganglion and the other from the right posterior nerve. This study further revealed the full trajectory of the dorsal strand plexus and paired visceral nerves on either side from the cerebral ganglion to the ovary, and precise innervation between the cerebral ganglion and the peripheral organs including the gonoduct, cupular organ, rectum and ovary. The differential innervation patterns of visceral nerves and the dorsal strand plexus indicate that the peripheral organs including the ovary undergo various neural regulations. Collectively, the present anatomical analysis revealed the major innervation of the dorsal and siphon nervous systems of adult Ciona

Regeneration of oral siphon pigment organs in the ascidian Ciona intestinalis

Author Posting. © The Author(s), 2009. This is the author's version of the work. It is posted here by permission of Elsevier B.V. for personal use, not for redistribution. The definitive version was published in Developmental Biology 339 (2010): 374-389, doi:10.1016/j.ydbio.2009.12.040.Ascidians have powerful capacities for regeneration but the underlying mechanisms are poorly understood. Here we examine oral siphon regeneration in the solitary ascidian Ciona intestinalis. Following amputation, the oral siphon rapidly reforms oral pigment organs (OPO) at its distal margin prior to slower regeneration of proximal siphon parts. The early stages of oral siphon reformation include cell proliferation and re-growth of the siphon nerves, although the neural complex (adult brain and associated organs) is not required for regeneration. Young animals reform OPO more rapidly after amputation than old animals indicating that regeneration is age dependent. UV irradiation, microcautery, and cultured siphon explant experiments indicate that OPOs are replaced as independent units based on local differentiation of progenitor cells within the siphon, rather than by cell migration from a distant source in the body. The typical pattern of eight OPOs and siphon lobes is restored with fidelity after distal amputation of the oral siphon, but as many as sixteen OPOs and lobes can be reformed following proximal amputation near the siphon base. Thus, the pattern of OPO regeneration is determined by cues positioned along the proximal distal axis of the oral siphon. A model is presented in which columns of siphon tissue along the proximal-distal axis below pre-existing OPO are responsible for reproducing the normal OPO pattern during regeneration. This study reveals previously unknown principles of oral siphon and OPO regeneration that will be important for developing Ciona as a regeneration model in urochordates, which may be the closest living relatives of vertebrates.This research was supported by PhD fellowships from MRT and ARC to HA, Grants-in-Aid from MEXT, Japan, and the NIJ Cooperative Program (2008-B02) to YS, INRA, CNRS, the ANR Grant Choregnet, and the Marine Genomics Center of Excellence to J-SJ, Laura and Arthur Colwin and Frederick Bang Fellowships from the Marine Biological Laboratory, Woods Hole, MA to WRJ., and NSF grant (IBN-0611529) to WRJ