Goto Rie, Saito Taiju, Kawakami Yutaka, Kitauchi Tomoe, Takagi Misae, Todo Takashi, Arai Katsutoshi, Yamaha Etsuro
Nanae Fresh Water Laboratory, Field Science Center for Northern Biosphere, Hokkaido University, Nanae, Kameda, Hokkaido 041-1105, Japan.
Int J Dev Biol. 2015;59(10-12):465-70. doi: 10.1387/ijdb.150008rg.
Primordial germ cells (PGCs) appear during early embryogenesis and differentiate into gametes through oogenesis or spermatogenesis. Teleost PGCs can be visualized by injecting RNA transcribed from the fusion product of a fluorescent protein gene attached to the 3' untranslated region (3'UTR) of zebrafish nanos3 (zf-nos3). Although this method has been widely applied to teleost PGCs, the visualization of PGCs in pelagic species that have eggs with a hard chorion is more problematic due to the technical difficulty of microinjection into their eggs. In this study, we developed a reliable method for microinjection of fertilized eggs in a pelagic species, the barfin flounder. Using a microneedle with a constriction "brake", we were able to introduce gfp-nos3 3'UTR mRNA into embryos and to determine the origin and migration route of PGCs. We also isolated the barfin flounder nos3 (bf-nos3) gene to compare its 3'UTR sequence with that of zebrafish. The 3'UTR of the bf-nos3 sequence was longer than that of zf-nos3. However, PGCs were also visualized after injection of gfp-bf-nos3 3'UTR mRNA both in zebrafish and barfin flounder. These results suggest that the function of nos3 is conserved between these species regardless of the sequence differences. The method developed here for labeling PGCs with gfp-nos3 mRNA will provide a means to study PGC development in the embryos of a wide range of marine fish species.
原始生殖细胞（PGCs）在胚胎发育早期出现，并通过卵子发生或精子发生分化为配子。硬骨鱼的PGCs可以通过注射由与斑马鱼nanos3（zf-nos3）的3'非翻译区（3'UTR）连接的荧光蛋白基因的融合产物转录的RNA来可视化。尽管这种方法已广泛应用于硬骨鱼的PGCs，但由于向具有硬卵壳的浮游物种的卵中显微注射技术难度较大，因此可视化这些物种中的PGCs更具问题。在本研究中，我们开发了一种可靠的方法用于对浮游物种条石鲷的受精卵进行显微注射。使用带有收缩“制动器”的微针，我们能够将gfp-nos3 3'UTR mRNA导入胚胎，并确定PGCs的起源和迁移途径。我们还分离了条石鲷nos3（bf-nos3）基因，以将其3'UTR序列与斑马鱼的进行比较。bf-nos3序列的3'UTR比zf-nos3的长。然而，在斑马鱼和条石鲷中注射gfp-bf-nos3 3'UTR mRNA后，PGCs也能被可视化。这些结果表明，无论序列差异如何，nos3在这些物种之间的功能是保守的。这里开发的用gfp-nos3 mRNA标记PGCs的方法将为研究多种海洋鱼类胚胎中的PGC发育提供一种手段。
