The International Journal of Developmental Biology

Int. J. Dev. Biol. 59: 465 - 470 (2015)

Vol 59, Issue 10-11-12

Visualization of primordial germ cells in the fertilized pelagic eggs of the barfin flounder Verasper moseri

Technical Article | Published: 15 January 2016

Rie Goto*,1, Taiju Saito1, Yutaka Kawakami1, Tomoe Kitauchi1, Misae Takagi1, Takashi Todo2, Katsutoshi Arai2 and Etsuro Yamaha1

1Nanae Fresh Water Laboratory, Field Science Center for Northern Biosphere, Hokkaido University, Nanae, Kameda, Hokkaido 041-1105 and 2Faculty of Fisheries Sciences, Hokkaido University, Hakodate, Hokkaido 041-8611, Japan


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 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.


teleost, PGC, flounder, pelagic egg, microinjection

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