Visualization of gonadal development and sex differentiation in medaka using in vivo imaging

本项目旨在利用新近开发的BAC转基因技术，对发育性腺中特异性表达的基因进行转基因，描述参与角尾鱼性腺性别分化和发育的细胞系之间的细胞相互作用。我们成功构建了几种性腺特异性转录因子（sox9b ftz-f1和pod1）和medaka性别决定因子（dmy）的BAC转基因系；甾体生成酶p450c17，男性特异性体细胞标记基因dmrt1和女性特异性标记基因芳香化酶。对于每个转基因系，我们用全挂载免疫荧光法检测了转基因表达细胞的时空表达模式。通过使用sox9b-EGFP转基因细胞系，我们发现表达sox9b的细胞是睾丸和卵巢支持细胞的常见前体（Nakamura et al ., 2008）。我们还将BAC技术应用于突变系“真仔”的分析，该突变系表现出发育性种系耗竭现象。利用候选基因“赞仔”转基因系和表达“赞仔”- egfp融合基因的挽救系，研究了表达“赞仔”的细胞的发育动态。此外，通过将sox9b-EGFP系或olvasegfp系与zenzai突变系杂交，我们发现生殖细胞分裂模式与突变表型之间存在牢固的关系（Saito et al ., 2007）。另一个主要发现包括使用转基因表达细胞的延时成像技术分析成人卵巢表面的生殖细胞分裂。事实证明，双色转基因系在这方面特别有用。我们在成人卵巢组织中发现了假定的生殖系干细胞（Nakamura等人，未发表）。综上所述，我们发现了medaka在性腺发育过程中细胞相互作用的几个重要方面，这表明BAC转基因技术在发育和生殖生物学中的潜在用途。新产生的转基因品系将成为进一步研究的宝贵资源。少

This project aimed to depict the cellular interaction of cell lineages involved in the gonadal sex differentiation and development in the medakafish by applying recently developed BAC transgenic technology for genes specifically expressed in developing gonads. We successfully generated the BAC transgenic lines for several gonad-specific transcription factors such as sox9b ftz-f1, and pod1, and the medaka sex determination factor dmy; steroidgenesis enzyme p450c17, male-specific somatic marker gene dmrt1, and female-specific marker gene aromatase. For each transgenic line, we examined the temporal and spatial expression patterns of transgene-expressing cells by a whole-mount immunofluorescence method. Using sox9b-EGFP transgenic line, we found that sox9b-expressing cells are common precursors of both testicular and ovarian supporting cells (Nakamura et al, 2008). We also applied the BAC technology for the analysis of mutant line zenzai, which showed developmental germline depletion phen … More otype. Using the transgenic line for candidate gene zenzai and rescue line expressing zenzai-EGFP fusion gene, we examined the developmental dynamics of zenzai-expressing cells. Further, by crossing sox9b-EGFP line or olvas-EGFP line with zenzai mutant line, we found the firm relationship between pattern of germ cell division and mutant phenotype (Saito et al, 2007). Another major finding includes the analysis of germ cell division in adult ovarian surface using the time-lapse imaging of transgene-expressing cells. Double-color transgenic line turned out to be especially useful for such purposes. We found the putative germline stem cells in adult ovarian tissue (Nakamura et al, unpublished). Taken together, we found the several important aspects of cellular interaction during gonadal development in medaka, which showed the potential usefulness of BAC transgenic technology in developmental and reproductive biology. The newly generated transgenic lines will be valuable resources for further research. Less