C. DEPARTMENT OF DEVELOPMENTAL GENETICS
C-c. Division of Molecular and Developmental Biology - Koichi Kawakami Group

RESEARCH ACTIVITIES

(1) Gene trap and enhancer trap approaches in zebrafish

Koichi Kawakami, Yasuyuki Kishimoto, Kazuhide Asakawa, Tomoya Kotani, Saori Nagayoshi and Akihiro Urasaki

--In order to understand the genetic basis for developmental processes in vertebrate, we have been using a small tropical fish, the zebrafish, as a model animal. Because it is practically possible to breed and maintain very large numbers of fish in the lab, and because zebrafish embryos develop in water and are transparent, forward genetic approaches (i.e., collecting a large number of mutations affecting developmental processes and analyzing genes responsible for the mutant phenotypes) are feasible in the fish. The Tol2 element is a transposable element identified from the genome of the medaka fish. Previously we found that the Tol2 element encodes a fully functional transposase and showed that the Tol2 element can transpose into the zebrafish genome in the germ lineage. To date, the Tol2 element is the only natural transposon in vertebrate for which an autonomous element has been identified. We have been interested in developing novel genetic methodologies in zebrafish using the Tol2 element. Using this Tol2 transposon system, we have constructed various gene trap and enhancer trap vectors, that contain either a splice acceptor or a basal promoter and a promoterless fluorescent reporter gene (i.e., GFP, RFP, etc). The plasmid DNAs containing these transposon vectors were coinjected in zebrafish fertilized eggs with the transposase mRNA, and offspring from the injected fish were analyzed for expression of the reporter gene in specific tissues and organs. The integration site of the transposon vector and the gene trapped by the vector insertion were cloned rapidly by PCR-based techniques such as inverse PCR and 5'RACE. These approaches should facilitate our understanding of vertebrate morphogenesis and organogenesis.

(2) Genetic analysis of zebrafish maternal-effect mutations affecting early embryogenesis

Yasuyuki Kishimoto, Sumito Koshida1, Makoto Furutani-Seiki2, Atsushi Kawakami3, Hisato Kondoh2, 4 and Koichi Kawakami (1National Institutes of Natural Sciences, 2Kondoh Differentiation Signaling Project, JST, 3University of Tokyo, 4Osaka University)

--Maternal-effect genes play essential roles in early embryogenesis in many animals. We have carried out a genetic screen for mutations affecting such maternal-effect genes employing an F3 screen strategy, identifying six recessive mutations out of 60 mutagenized genomes (ref. 6). Among them, four mutations were kept and analyzed. Three of the mutations (acytokinesis mutations: ackkt5, ackkt62 and ackkt119) prohibited the cell cleavage in the embryos from homozygous females without affecting nuclear divisions. These embryos are defective in generating contractile rings; the ackkt62 mutation abolished the organization of cortical F-actin, while other mutations caused abortive contractile ring-like structures at ectopic sites. Defects in contractile ring formation leads to the absence of microtubule arrays at the prospective cleavage plane. Thus, these mutations reveal the sequence of events associated with cytokinesis. It is remarkable that in all acytokinetic embryos, daughter nuclei after mitosis are arranged in spatially normal positions, and maternal vasa mRNAs accumulate in the prospective planes of the first and the second cell cleavages despite complete loss of cytokinesis. This indicates that the basic cell architectures of early embryos are largely established by the autonomous activities of the mitotic apparatus, without any dependence on the cell cleavage machinery. The fourth mutation, bobtail (btl), caused a strong reduction of the tailbud outgrowth. The expression of myoD in somites and eve1 in tailbud is reduced in the mutant embryos, whereas that of ntl in notochord and pax2.1 in pronephros is comparable between wild-type and mutant embryos, suggesting that the btl gene product may regulate gene expression involved in myogenesis and posterior mesodermal development. We also found that the third ventricle in the tectal region is inhibited in the btl mutant embryo. In btl mutant embryos, expression of MHB genes such as wnt1, fgf8 and pax2.1 are once activated at the 8-somite stage, but eliminated from the MHB region afterward. Thus, the btl gene product is important for maintenance of the MHB gene expression. btl has been mapped on two BAC and PAC clones on chromosome 17. We are currently focusing on identification of the btl gene from these genomic clones.

(3) Transposition of the Tol2 element in mouse embryonic stem cells

Koichi Kawakami and Tetsuo Noda1 (1Tohoku University)

--It has not been known whether Tol2 can transpose in vertebrates other than fish. We investigated transposition of Tol2 in mouse embryonic stem (ES) cells. We constructed a transposon donor plasmid containing a nonautonomous Tol2 element with the neomycin resistance gene and a helper plasmid capable of expressing the transposase, and introduced the donor plasmid with various amounts of the helper plasmid by electroporation into mouse ES cells. The number of G418-resistant ES colonies increased as the amount of helper plasmid was increased, in a dose-dependent manner, indicating that the transposase activity elevated the integration efficiency. These G418-resistant ES colonies were cloned and the structure of the junction of the integrated Tol2 element and the genomic DNA was analyzed by inverse PCR. In those clones, Tol2 was surrounded by mouse genomic sequences and an 8-bp direct repeat was created adjacent to the both ends of Tol2, indicating that Tol2 was integrated in the genome through transposition. The Tol2 transposon system is thus active in mouse as well as in fish. We propose that it should be used as a genetic tool to develop novel gene transfer, transgenesis and mutagenesis methods in mammals (ref. 1).

(4) A transposon-mediated gene trap approach identifies developmentally regulated genes in zebrafish

Koichi Kawakami, Hisashi Takeda1, Noriko Kawakami, Makoto Kobayashi2, Naoto Matsuda1 and Masayoshi Mishina1 (1University of Tokyo, and 2Tsukuba University)

--We developed a novel gene trap method in zebrafish using the Tol2 transposon system. First, we established a highly efficient transgenesis method in which a plasmid DNA containing the Tol2 transposon vector and the transposase mRNA synthesized in vitro were coinjected into one-cell stage embryos. The transposon vector inserted in the genome could be transmitted to the F1 progeny at high frequencies, and regulated gene expression by a specific promoter could be recapitulated in transgenic fish. Then we constructed a transposon-based gene trap vector containing a splice acceptor and the GFP gene, performed a pilot screen for gene trapping, and obtained fish expressing GFP in temporally and spatially restricted patterns. We confirmed the endogenous transcripts were indeed trapped by the insertions, and the insertion could interfere with expression of the trapped gene. We propose our gene trap approach should facilitate studies of vertebrate development and organogenesis (ref. 2, 5).

(5) Excision of the Tol2 transposable element of the medaka fish Oryzias latipes in Xenopus laevis and Xenopus tropicalis

Koichi Kawakami, Kosuke Imanaka1, Mari Itoh1 and Masanori Taira1 (1University of Tokyo)

--We demonstrated transposase-dependent excision of the Tol2 element in Xenopus laevis and Xenopus (Silurana) tropicalis embryos. We coinjected a plasmid DNA containing a nonautonomous Tol2 element and the transposase mRNA synthesized in vitro into two-cell-stage embryos, and analyzed DNA extracted from the injected embryos by polymerase chain reaction (PCR). We demonstrated that the Tol2 element could be excised from the plasmid DNA in both X. laevis and X. tropicalis only when it was coinjected with the transposase mRNA. In most cases, a complete loss of the Tol2 sequence was accompanied by addition of a short DNA sequence to the target sequence, indicating that transposase-dependent excision occurred. While these footprints were characteristic to those created upon excision of transposons of the hAT family, the additional bases found in Xenopus were longer and their structures were more complicated than those detected upon excision in zebrafish. This may reflect differences in the activities of host factors involved in either transposition, repair, or both between fish and frog. Our present study suggests that the Tol2 transposon system should be used as a novel genetic tool to develop transgenesis and mutagenesis methods in Xenopus (ref. 3).

(6) The status, quality, and expansion of the NIH full-length cDNA project: the Mammalian Gene Collection (MGC)

Gerhard D. S. and 113 others (The MGC project team).

--The National Institutes of Health's Mammalian Gene Collection (MGC) project was designed to generate and sequence a publicly accessible cDNA resource containing a complete open reading frame (ORF) for every human and mouse gene. The project initially used a random strategy to select clones from a large number of cDNA libraries from diverse tissues. Candidate clones were chosen based on 5'-EST sequences, and then fully sequenced to high accuracy and analyzed by algorithms developed for this project. Currently, more than 11,000 human and 10,000 mouse genes are represented in MGC by at least one clone with a full ORF. The random selection approach is now reaching a saturation point, and a transition to protocols targeted at the missing transcripts is now required to complete the mouse and human collections. Comparison of the sequence of the MGC clones to reference genome sequences reveals that most cDNA clones are of very high sequence quality, although it is likely that some cDNAs may carry missense variants as a consequence of experimental artifact, such as PCR, cloning, or reverse transcriptase errors. Recently, a rat cDNA component was added to the project, and ongoing frog (Xenopus) and zebrafish (Danio) cDNA projects were expanded to take advantage of the high-throughput MGC pipeline (ref. 4). We have made a contribution to this project by constructing the zebrafish full-length cDNA library.

PUBLICATIONS

Papers
1. Kawakami, K. and Noda, T. (2004). Transposition of the Tol2 element, an Ac-like element from the Japanese medaka fish Oryzias latipes, in mouse embryonic stem cells. Genetics 166, 895-899.
2. Kawakami, K., Takeda, H., Kawakami, N., Kobayashi, M., Matsuda, N. and Mishina, M. (2004). A transposon-mediated gene trap approach identifies developmentally regulated genes in zebrafish. Developmental Cell 7, 133-144.
3. Kawakami, K., Imanaka, K., Itoh, M. and Taira, M. (2004). Excision of the Tol2 transposable element of the medaka fish Oryzias latipes in Xenopus laevis and Xenopus tropicalis. Gene 338, 93-98.
4. Gerhard, D. S. and 113 others (The MGC project team). (2004). The status, quality, and expansion of the NIH full-length cDNA project: The mammalian gene collection (MGC). Genome Research 14, 2121-2127.
5. Kawakami, K. (2004). Transgenesis and gene trap methods in zebrafish by using the Tol2 transposable element. Methods in Cell Biology 77, 201-222.
6. Kishimoto, Y., Koshida, S., Furutani-Seiki, M. and Kondoh, H. (2004). Zebrafish maternal-effect mutations causing cytokinesis defect without affecting mitosis or equatorial vasa deposition. Mech Dev. 121, 79-89.

Reviews
7. 川上浩一（2004）ゼブラフィッシュのファンクショナルゲノミクス.「ゲノミクス・プロテオミクスの新展開」今中忠行監修（エヌ・ティー・エス）,pp362-372.
8. 小谷友也,川上浩一（2004）ゼブラフィッシュの時空間特異的な遺伝子発現.蛋白質核酸酵素49, 2111-2116.

ORAL PRESENTATIONS

1. Kawakami, K. The Tol2 transposable element and its application in gene trapping in zebrafish. EMBO Workshop: Molecular Mechanisms of Transposition, Its Regulation and Evolution, Roscoff, France, June, 2004.
2. Kawakami, K., Ito, A., Kotani, T., Morvan, G., Nagayoshi, S., Sasaki, T., Urasaki, A., and Kishimoto, Y. Transgenesis and gene trap approaches in zebrafish using the Tol2 transposon system. The 6th International Conference on Zebrafish Development and Genetics, Madison, U.S.A., July, 2004.
3. Kawakami, K. A transposon-mediated gene trap approach in zebrafish. The 11th CDB Meeting: Asia-Oceania Fish Meeting, Kobe, Japan, November, 2004.
4. 三嶋雄一郎、小坂恭子、橋本祥子、藤原俊伸、川上浩一、安田国雄、坂本博、井上邦夫.ゼブラフィッシュ生殖細胞形成過程に働くRNA-蛋白質複合体.日本分子生物学会第27会大会、神戸、2004年12月.

POSTER PRESENTATIONS

1. 小坂恭子、三嶋雄一郎、川上浩一、坂本博、井上邦夫（2004）ゼブラフィッシュにおける母性mRNA局在化機構の解析.日本発生生物学会第37会大会、名古屋、2004年6月.
2. Inoue, F., Nagayoshi, S., Odaira, Y., Kawakami, K. and Yamasu, K. Transcription of fgf8 is regulated by multiple cis regions during development of the zebrafish. The 6th International Conference on Zebrafish Development and Genetics, Madison, U.S.A., July, 2004.
3. Kotani, T. and Kawakami, K. Trapping of maternal genes in zebrafish. The 6th International Conference on Zebrafish Development and Genetics, Madison, U.S.A., July, 2004.
4. Nagayoshi, S. and Kawakami, K. Characterization of the zebrafish lines constructed by the transposon-mediated gene trap method, which express GFP in the central nervous system. The 6th International Conference on Zebrafish Development and Genetics, Madison, U.S.A., July, 2004.
5. Kishimoto, Y., Koshida, S., Furutani-Seiki, M., Kawakami, K., and Kondoh, H. Isolation and characterization of the maternal-effect bobtail mutation affecting tailbud outgrowth and brain morphogenesis. The 6th International Conference on Zebrafish Development and Genetics, Madison, U.S.A., July, 2004.
6. Asakawa, K., Ito, A., Urasaki, A., Morvan, G., Kotani, T., Sasaki, T., Nagayoshi, S., Kishimoto, Y., Hibi, M. and Kawakami, K. Development of Gal4-enhancer trap system using the Tol2 transposon in zebrafish. The 11th CDB Meeting: Asia-Oceania Fish Meeting, Kobe, Japan, November, 2004.
7. Urasaki, A., Kotani, T., Nagayoshi, S. and Kawakami, K. Integration and remobilization of a gene trap transposon construct in zebrafish. The 11th CDB Meeting: Asia-Oceania Fish Meeting, Kobe, Japan, November, 2004.
8. 永吉さおり、川上浩一.ゼブラフィッシュの遺伝子トラップ法による脊椎動物中枢神経系で発現する遺伝子の探索.日本分子生物学会第27会大会、2004年12月.
9. 浅川和秀、伊藤安希、浦崎明宏、Ghislaine Morvan、小谷友也、佐々木剛、永吉さおり、岸本康之、日比正彦、川上浩一.ゼブラフィッシュにおけるトランスポゾンを用いたGal4エンハンサートラップ法の構築.日本分子生物学会第27会大会、2004年12月.
10. 浦崎明宏、川上浩一.ゼブラフィッシュにおけるトランスポゾンTol2を用いた新しい発生遺伝学的方法論の開発.日本分子生物学会第27会大会、2004年12月.
11. 小谷友也、川上浩一.Tol2転移システムを用いたゼブラフィッシュの母性遺伝子トラップ.日本分子生物学会第27会大会、2004年12月.
12. 今中康介、伊藤万里、川上浩一、平良真規.Xenopus laevisとtropicalisにおけるトランスポゾンを介したトランスジェニック法の確立.日本分子生物学会第27会大会、2004年12月.
13. 岸本康之、越田澄人、川上厚志、古谷-清木誠、近藤寿人、川上浩一.尾芽伸長および頭部形態形成に異常をきたすゼブラフィッシュ母性効果変異bobtailの解析.日本分子生物学会第27会大会、2004年12月.

SOCIAL CONTRIBUTIONS AND OTHERS

1. K. Kawakami served as a Chair of the 6th International Conference on Zebrafish Development and Genetics held at Madison, U.S.A. on July, 2004.