F.GENETIC STRAINS RESEARCH CENTER
F-d. Model Fish Genomics Resource - Noriyoshi Sakai Group

RESEARCH ACTIVITIES

(1) Gene targeting system with RNA interference (RNAi) in zebrafish

Minori Shinya, Kimiko Saka and Noriyoshi Sakai

--Gene silencing via short interfering RNAs (siRNAs) has proved to be a useful tool in studying gene function in plants, invertebrates and mammalian systems. To date, gene silencing effects of siRNAs have confirmed in the zebrafish, which is an emerging model for developmental and diseases analysis. However, the effects were temporal (only in early developmental stages) and sometimes mosaic in an embryo because of the method injecting siRNAs into the one-cell stage of embryos. We recently succeeded in the production of transgenic zebrafish from in vitro-cultured sperm¹⁾. The advantage of this technique is that the mosaicism inherent in other conventional transgenic methods is avoided. Our aim in the present study is the establishment of a rapid system with cultured sperm to generate transgenic zebrafish for gene silencing by siRNA. To achieve this, we first selected the targeted genes that we have already known the silenced phenotype and also the phenotype can be easily recognized. Then, we established the zebrafish cultured cells expressing the targeted genes, in order to determine the best sequence of siRNA for specific suppression of the gene by transfecting several possible designed siRNAs. After finding out the best siRNA, we will construct the retroviral vector of DNA vector-based siRNA constructs and then infect to the cultured spermatogonia with the virus. By in vitro insemination with the sperm derived from the infected spermatogonia, we can obtain the transgenic zebrafish producing siRNA in all the cells. Once the above system is developed, the inducible siRNA in the specific cells or organs will be easily conducted, and thus, the technique leads to a high-throughput system for genome-wide loss-of-function studies.

(2) Analysis of functionally distinctive testicular cell lines of zebrafish to support male germ cell development

Kayoko Kurita, Kataaki Okubo¹, Masaru Matsuda¹, Yoshitaka Nagahama¹ and Noriyoshi Sakai ( ¹Laboratory of Reproductive Biology, National Institute for Basic Biology)

--Sertoli cells are important to germ cells in everything from male sex-determination to spermatogenesis. In spermatogenesis, Sertoli cells interact directly with germ cells in the testis to induce the complex process required for the production of functional sperm. These cells mediate the production of many molecules as well as cell junctions and adhesion. The function of many of these molecules and the regulation of gene expression remain unclear. We recently established two testicular cell lines of zebrafish with distinct functions to support the development of male germ cells²⁾. Twelve cell lines were established by single-colony isolation from tumor-like testis-derived ZtA6 cells. Multiple features characteristic of Sertoli cells such as phagocytic activity and transcription specific genes (sox9a and Wilms' tumor suppressor WT1) were analyzed in the lines. The lines, ZtA6-2 and ZtA6-12, showed almost the same characteristics as Sertoli cells, but exhibited distinctive features when male germ cells were co-cultured with each line as feeders. The in vitro fertilization by the culture of germ cells with ZtA6-12 produced more embryos than that with ZtA6-2. In contrast, ZtA6-2 gave rise to significantly larger clumps of germ cells after a 12-day culture compared to the ZtA6-12 cell line. Expression of vas, strongly expressed in spermatogonia and premeiotic spermatocytes, was prolonged in the culture using ZtA6-2 feeders, while it was reduced with the germ cells on the ZtA6-12 feeders. Compared with the previous results obtained on the original ZtA6 cells, these results suggested that the function of the ZtA6-2 cells was directed to stimulate the proliferation of spermatogonia, and ZtA6-12 to stimulate the differentiation into sperm.
--These cell lines will facilitate investigation of Sertoli cell molecules that contribute to the proliferation and differentiation of spermatogonia. We are currently working on the molecular cloning of genes expressed specifically in each line. 205 clones and 256 clones were isolated by the subtraction experiments with ZtA6-2 cDNA minus ZtA6-12 cDNA and vice versa. Several have already been confirmed by screening with dot blot hybridization and virtual Northern hybridization to cDNA of each line. In situ hybridization for sections of a testis showed that some of the cDNAs were expressed in Sertoli cells. Analysis of other cDNAs is under investigation.

(3) Culture condition for zebrafish spermatogonial stem cells

Kenji Saito and Noriyoshi Sakai

--Spermatogonial stem cells (SSCs) maintain spermatogenesis by continuous production of the daughter cells and cells that differentiate into spermatozoa. Sertoli cells are the only type of somatic cells that closely interact with SSCs to create a favorable environment inter testis. However, the regulatory mechanisms are still unclear because of the difficulty of identifying and manipulating an individual SSC and the surroundings.
--To establish in vitro system in which SSCs proliferate continuously, we performed the isolation procedure of A type spermatogonia and determined a culture condition to proliferate A type spermatogonia on a Sertoli cell feeder layer. When zebrafish were treated with busulfan reagent, we found the decline of differentiating germ cells, such as spermatocytes and spermatids, while A type spermatogonia increased after 4 days of treatment. Then, on day 7 germ cells of the testis were consisted of only A type spermatogonia and sperm. In teleosts, a single spermatogonium that enclosed within germinal cysts of Sertoli cells is defined as A type. Furthermore, we observed that the A type spermatogonia proliferate rapidly after the damage with busulfan reagent. The definition and the observation suggest that A type spermatogonia are SSCs of a teleost. When enzymatically dissociated testicular cells containing A type spermatogonia were co-cultured on ZtA6-6 Sertoli cell line, proliferation of A type spermatogonia was observed without differentiation by a BrdU incorporation experiment after 14 days of culture. These results indicate that A type spermatogonia self-renew in the culture condition that might represent testicular microenvironments to maintain SSCs.

(4) Analysis of the ability of cultured embryonic cells derived from different developmental stages to induce the anterior-posterior axis

Megumi Hashiguchi and Noriyoshi Sakai

--One of the most fascinating phenomena in primary embryonic induction is the regional specificity of the neural structures that are produced. Primary embryonic induction can be divided into three major components; head specific, trunk specific and tail specific induction.
--We established the condition to culture zebrafish embryonic cells continuously without any artificial immortalization treatment. When cultured cells were transplanted into a blastulae embryo, the cells from different developmental stages had different abilities to induce specific second axes. Cells derived from earlier stage embryos (gastrula stage) induce complete anterior structure. Cells from the pharyngula stage (embryos segmented) induce anterior structures either with cyclopia or without an eye. Cells from later stages (early larva) induce posterior structure with otic vesicles and a heart. Interestingly, cells from later stages had low induction efficiency, but the efficiency increased when proliferation of the cells was arrested with mitomycin C. Whole-mount in situ hybridization for emx-1 (telencephalon marker), krox 20 (rhombomere marker), and shh (notochord marker) indicated similar patterns of gene expression to the specific induction. Cultured cells derived from embryos at various developmental stages, therefore, change their properties to induce the second axis from an anterior to a posterior orientation according to their developmental stage. In addition, cells may secrete more inducer(s) when proliferation is stopped, particularly in later stages. These cultured cells can be used to find cellular factors involved in anterior-posterior specific induction.

PUBLICATIONS

Papers
1. Kurita, K., Burgess, S.M. and Sakai, N. (2004). Transgenic zebrafish produced by retroviral infection of in vitro-cultured sperm. Proc. Natl. Acad. Sci. USA 101, 1263-1267.
2. Kurita, K. and Sakai, N. (2004). Functionally distinctive testicular cell lines of zebrafish to support male germ cell development. Mol. Reprod. Dev. 67, 430-438.
3. Matsumoto, T., Yukawa, W., Nozaki, Y., Nakashige, R., Shinya, M., Makino, S., Yagura, M., Ikuta, T., Imanishi, T., Inoko, H., Tamiya, G. and Gojobori, T. (2004). Novel algorithm for automated genotyping of microsatellites. Nucleic Acids Res 32, 6069-6077.

Reviews
4. Sakai, N. (2004). Genetically modified sperm in fish. ISB News Report April, 1-3.

ORAL PRESENTATIONS

1. Sakai, N., Kurita, K., Saito, K. and Burgess, S. M. Transgenic zebrafish produced by in vitro-cultured sperm. 6th International Conference on Zebrafish Development & Genetics, Madison-Wisconsin, USA, July, 2004.
2. 斉藤憲二、長濱嘉孝、酒井則良「ゼブラフィッシュA型精原細胞の単離法とその培養系の開発」日本動物学会平成16年度中部支部大会、静岡市、2004年7月
3. 橋口恵、酒井則良「ゼブラフィッシュ初期胚由来培養細胞の二次胚誘導能の解析」日本動物学会平成16年度中部支部大会、静岡市、2004年7月

POSTER PRESENTATIONS

1. Kurita, K., Okubo, K., Matsuda, M., Nagahama, Y. and Sakai, N. Functionally distinctive testicular cell lines to support male germ cell development. 6th International Conference on Zebrafish Development & Genetics, Madison-Wisconsin, USA, July, 2004.
2. 新屋みのり、木村哲晃、吉田恵子、島田敦子、三谷啓志、成瀬清、武田洋幸、猪子英俊、田宮元「メダカを用いた顔貌形成に関する量的形質遺伝子座解析」日本発生生物学会第37回大会、名古屋市、2004年6月

EDUCATION

他大学/機関での講義やセミナー
1. Dr. N. Sakai gave a seminar on “Genetically modified sperm in fish" at Shizuoka University, March, 2004 (in Japanese).
2. Dr. N. Sakai gave a course of lectures at the Department of Marine Bioscience of Fukui Prefectural University, April-August, 2004 (in Japanese).
3. Dr. N. Sakai was invited to give a seminar on “Genetically modified sperm in fish" at National Institute of Child Health and Human Development of NIH, Bethesda, USA, July, 2004.