ホヤ胚初期発生過程における細胞分化メカニズムの解析

本研究では、細胞分化メカニズムを(1)卵内における決定因子の局在化と予定割球への分配、(2)組織予定割球における決定因子の核への移行、(3)分化のための遺伝子カスケードの進行、の3つの局面に区別して解析することで、統括的に細胞分化メカニズムを理解することを目指した。同時に、カタユウレイボヤとユウレイボヤの各組織分化マーカーを多数調製し、その分化メカニズムの研究基盤を整えることを目指した。 第3章前半では、遺伝子カスケードに注目し、モノクローナル抗体法を用いて細胞の違いを生み出す分子の検索を行った。その結果、一部の脊索細胞を特異的に認識するモノクローナル抗体CiNot-1を調製でき、形態的には均一な脊索細胞において発現している分子種に差異があることを示した。そして、CiNot-1を脊索における細胞系譜の分子マーカーとして確立した。 第3章後半では、決定因子の核への移行に注目し、卵割期の核タンパク質を認識するモノクローナル抗体を調製し、その中から分化の引き金を引く転写調節因子を見つけだそうとした。そのために免疫寛容法などを利用したが、期待されたほどの効果が得られず、転写調節因子は見つからなかった。そのかわり、卵割期の核を認識する5種類の抗体が得られた。そのエピトープは核と細胞質の両方に存在し、発生過程において独特の挙動を示す。この挙動は、特定の時期に核へ移行する転写調節因子の特性を知る上で興味深い。また、Nup-4エピトープをコードするcDNAを単離したところ、ポリユビキチン遺伝子であった。 第4章では、カスケードの終盤で働く組織特異的遺伝子に注目した。尾芽胚期で発現する遺伝子を高頻度に持つライブラリーを作製し、そこから組織特異的に発現し、分化マーカーとして有用な遺伝子を一度に多数単離した。それらのうち、表皮細胞特異的に発現する2クローンと筋肉細胞特異的に発現する2クローンについて詳細に解析を行った。そのなかでCsEpi-2は、ホヤzygotic遺伝子の中で最も早い8細胞期から発現が見られ、表皮細胞の分化が8細胞期ですでに始まっていることを明らかにした。CsMA-1はユウレイボヤではじめて単離された筋肉アクチンであった。また、CsEpi-2以外は、カタユウレイボヤ胚でもマーカーとして利用できることがわかった。 第5章では、卵細胞質再配置により決定因子が正確に局在することに注目し、筋肉細胞分化に関わることの知られているマイオプラズミンC1とp58の局在するメカニズムを細胞骨格繊維との関係を中心に解析した。その結果、局在に関与すると思われる新たな微小管の特異構造を発見した。また、2つの分子の局在はほぼ完全に一致し、その移動には細胞骨格繊維が関与していた。さらにin vitroでこの2つの分子が互いに結合できることも明らかにした。これらの結果は、決定因子の局在化と分配メカニズムを理解するうえで、重要な知見となった。以上の結果をもとに、決定因子が複合体として機能するという仮説をたて、ホヤ細胞分化メカニズムの全体像を考察し論議した。甲南大学平成10年度(1998年度

Atlantoaxial Stabilization Using C1 and C2 Laminar Screw Fixation

We describe the use of a C1 laminar screw in combination with a C2 laminar screw as a salvage technique to treat two patients, one with persistent first intersegmental artery and the other with vertebral artery occlusion after cervical spine fracture. The combined use of C1 and C2 laminar screws allows for good fixation of the atlantoaxial joint with a lower risk of vertebral artery injury; therefore, it can be an alternative surgical procedure for patients with congenital or traumatic anomalous vertebral artery

モンゴル コクサン キョウリュウ カセキ カラノ ユウキブツ ケンシュツ

P(論文)departmental bulletin pape

Isolation and characterization of cDNA clones for epidermis-specific and muscle-specific genes in Ciona savignyi embryos

Ascidian eggs and embryos have provided an appropriate experimental system to explore the cellular and molecular mechanisms involved in the embryonic cell specification and pattern formation of the embryo. In Japan, most of the studies of ascidian embryology have been carried out with the large eggs of Halocynthia roretzi. However, for future studies, Ciona species may provide a better experimental system, in particular with respect to the incorporation of genetic approaches. In order to establish Ciona as an experimental system, molecular markers with which to examine cellular differentiation are required. In the present study, we isolated and characterized cDNA clones for two epidermis-specific genes (CsEpi-1 and CsEpi-2) and for two muscle-specific genes (CsMA-1 and CsMu-1). CsEpi-1 encodes a polypeptide with three trefoil domains, while CsMA-1 encodes a muscle-type actin from C. savignyi Although CsEpi-2 and CsMu-1 transcripts seem to have a poly(A) tail at the 3' end, we could not find a distinct open reading frame in the sequences. Probes for CsEpi-1, CsMA-1 and CsMu-1 cross-reacted with C. intestinalis embryos. These cDNAs are useful as molecular markers for the specification of epidermis and muscle of Ciona embryos

Development of Ciona intestinalis juveniles (Through 2nd ascidian stage)

Following the reading of its draft genome sequence and the collection of a large quantity of cDNA information, Ciona intestinalis is now becoming a model organism for whole-genome analyses of the expression and function of developmentally relevant genes. Although most studies have focused on larval structures, the development of the adult form is also very interesting in relation to tissues and organs of vertebrate body. Here we conducted detailed observations of the development of tissues and organs in Ciona intestinalis larva and juveniles until so-called the 2nd ascidian stage. These observations included examination of the oral siphon, tentacle, oral pigments and atrial pigments, atrial siphon, ganglion and neural gland, longitudinal muscle, stigmata, transverse bar and languet, longitudinal bar and papilla, heart, digestive organ, gonad, endostyle, and stalk and villi. The findings from these observations make a new staging system for juvenile development possible. Based on the development of the internal organs, we propose here nine stages (stage 0 similar to stage 8) starting with swimming larvae and proceeding through juveniles until the 2nd ascidian stage. These descriptions and staging system provide a basis for studying cellular and molecular mechanisms underlying the development of adult organs and tissues of this basal chordate

brachyury null mutant-induced defects in juvenile ascidian endodermal organs

We report the isolation of a recessive ENU-induced short-tailed mutant in the ascidian Ciona intestinalis that is the product of a premature stop in the brachyury gene. Notochord differentiation and morphogenesis are severely disrupted in the mutant line. At the larval stage, variable degrees of ectopic endoderm staining were observed in the homozygous mutants, indicating that loss of brachyury results in stochastic fate transformation. In post-metamorphosis mutants, a uniform defect in tail resorption was observed, together with variable defects in digestive tract development. Some cells misdirected from the notochord lineage were found to be incorporated into definitive endodermal structures, such as stomach and intestine

Sustained heterozygosity across a self-incompatibility locus in an inbred ascidian.

Because self-incompatibility loci are maintained heterozygous and recombination within self-incompatibility loci would be disadvantageous, self-incompatibility loci are thought to contribute to structural and functional differentiation of chromosomes. Although the hermaphrodite chordate, Ciona intestinalis, has two self-incompatibility genes, this incompatibility system is incomplete and self-fertilization occurs under laboratory conditions. Here, we established an inbred strain of C. intestinalis by repeated self-fertilization. Decoding genome sequences of sibling animals of this strain identified a 2.4-Mbheterozygous region on chromosome 7. A self-incompatibility gene, Themis-B, was encoded within this region. This observation implied that this self-incompatibility locus and the linkage disequilibrium of its flanking region contribute to the formation of the 2.4-Mb heterozygous region, probably through recombination suppression. We showed that different individuals in natural populations had different numbers and different combinations of Themis-B variants, and that the rate of self-fertilization varied among these animals. Our result explains why self-fertilization occurs under laboratory conditions. It also supports the concept that the Themis-B locus is preferentially retained heterozygous in the inbred line and contributes to the formation of the 2.4-Mb heterozygous region. High structural variations might suppress recombination, and this long heterozygous region might represent a preliminary stage of structural differentiation of chromosomes