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

Abstract

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.

Migration behavior of PGCs and asymmetrical gonad formation in pond smelt Hypomesus nipponensis

“…In pond smelt, PGCs were clearly visualized using GFP nos3 3' UTR mRNA, as is the case in other fish species (Saito et al, 2006(Saito et al, , 2011(Saito et al, , 2014Nagasawa et al, 2013;Goto et al, 2015). However, PGCs were detected earlier in embryonic development in pond smelt than zebrafish and other teleost species, because of…”

“…PGCs can be visualized in real time by microinjection of GFP-conjugated zebrafish nos3 3'UTR mRNA, an artificially synthesized mRNA, into fertilized eggs (Saito et al, 2006). This technology has been applied to a wide range of species, from archaic fish species such as sturgeon (Saito et al, 2014) to more highly evolved species such as flounder (Goto et al, 2015). With this new approach, the migratory behavior of PGCs has been characterized in a variety of fish species.…”

Targeted mutagenesis of the ryanodine receptor by Platinum TALENs causes slow swimming behaviour in Pacific bluefin tuna (Thunnus orientalis)

“…The microinjection of ryr1b -TALENs was performed using a needle (G-1, Narishige, Tokyo, Japan) with an added constriction (3‒5 μm inner diameter) in the upper part of the tip in order to control the pressure required for injecting the RNA solution into the embryo and to prevent backflow of cytoplasm from the embryo as described previously 39–41 . Embryos in the 1‒4 cell stage were placed on a 1% agar-coated Petri dish filled with 50% sterilised seawater, and then microinjected with TALEN mRNA.…”

The Regulatory Region of Muscle-Specific Alpha Actin 1 Drives Fluorescent Protein Expression in Olive Flounder Paralichthys olivaceus

“…The purified DNA solution (25 ng/uL) was injected into one-cell-stage zebrafish embryos (n=100) using an air pressure microinjector (PicoPump820, World Precision Instruments, Sarasota, FL, USA), as described previously (Kong et al, 2013). For the microinjection into olive flounder, the method of preparing microinjection needle modified to that described in Goto et al (2015). Glass microinjection needles were produced by pulling of a borosilicate glass capillary tubes (World Precision Instruments, Inc., Sarasota, FL, USA) using micropipette puller device (Narishige, Tokyo, Japan).…”