Visualization of Turbot (Scophthalmus maximus) Primordial Germ Cells in vivo Using Fluorescent Protein Mediated by the 3′ Untranslated Region of nanos3 or vasa Gene
Primordial germ cells (PGCs) as the precursors of germ cells are responsible for transmitting genetic information to the next generation. Visualization of teleost PGCs in vivo is essential to research the origination and development of germ cells and facilitate further manipulation on PGCs isolation, cryopreservation, and surrogate breeding. In this study, artificially synthesized mRNAs constructed by fusing fluorescent protein coding region to the 3′ untranslated region (3′UTR) of nanos3 or vasa (mCherry-Smnanos3 3′UTR or mCherry-Smvasa 3′UTR mRNA) were injected into turbot (Scophthalmus maximus) fertilized eggs for tracing PGCs. The results demonstrated that the fluorescent PGCs differentiated from somatic cells and aligned on both sides of the trunk at the early segmentation period, then migrated and located at the dorsal part of the gut where the gonad would form. In the same way, we also found that the zebrafish (Danio rerio) vasa 3′UTR could trace turbot PGCs, while the vasa 3′UTR s of marine medaka (Oryzias melastigma) and red seabream (Pagrus major) failed, although they could label the marine medaka PGCs. In addition, through comparative analysis, we discovered that some potential sequence elements in the3 ′UTRs of nanos3 and vasa, such as GCACs, 62-bp U-rich regions and nucleotide 187–218 regions might be involved in PGCs stabilization. The results of this study provided an efficient, rapid, and specific non-transgenic approach for visualizing PGCs of economical marine fish in vivo.
KeywordsPrimordial germ cells nanos3 vasa Scophthalmus maximus Location
This study was conceived and designed by Li Zhou, Qinghua Liu, Xueying Wang, and Jun Li; Li Zhou and Xueying Wang contributed experimental work; Li Zhou and Qinghua Liu contributed the manuscript writing and revision, respectively; Shihong Xu, Haixia Zhao, Mingming Han, and Yunong Wang contributed experimental materials. All authors read and approve the final manuscript.
This research was funded by National Natural Science Foundation of China (31572602), National Key Research and Development Program (2018YFD0901205, 2018YFD0901204), the Marine S&T Fund of Shandong Province for Pilot National Laboratory for Marine Science and Technology(Qingdao)(2018SDKJ0302-4, 2018SDKJ0502-2),China Agriculture Research System (CARS-47), STS project (KFZD-SW-106, 2017T3017), Shandong Province Key Research and Invention Program (2017CXGC010K), the National Infrastructure of Fishery Germplasm Resource (2019DKA30470).
Compliance with Ethical Standards
All experiments were performed in accordance with the relevant national and international guidelines and approved by the Institutional Animal Care and Use Committee, Institute of Oceanology, Chinese Academy of Sciences.
Conflict of Interest
The authors declare that they have no conflict of interest.
- Bailey TL, Elkan C (1994) Fitting a mixture model by expectation maximization to discover motifs in biopolymers. Proc Int Conf Intell Syst Mol Biol 2:28–36Google Scholar
- Braat AK, Speksnijder JE, Zivkovic D (1999) Germ line development in fishes. Int J Dev Biol 43:745–760Google Scholar
- Curtis D, Treiber DK, Tao F, Zamore PD, Williamson JR, Lehmann R (1997) A CCHC metal-binding domain in Nanos is essential for translational regulation. EMBO J 16:834–843Google Scholar
- Kawakami Y, Ishihara M, Saito T, Fujimoto T, Yamaha E (2012) Cryopreservation of green fluorescent protein (GFP)-labeled primordial germ cells with GFP fused to the 3′UTR of the nanos gene by vitrification of Japanese eel (Anguilla japonica) somite-stage embryos. J Anim Sci 90:4256–4265CrossRefGoogle Scholar
- Koprunner M, Thisse C, Thisse B, Raz E (2001) A zebrafish nanos-related gene is essential for the development of primordial germ cells. Genes Dev 15:2877–2885Google Scholar
- Kuersten S, Goodwin EB (2003) The power of the 3′ UTR: translational control and development. Nat Rev Genet 4:626–637Google Scholar
- Lei JL (2003) The development direction of turbot farming industry in China (in Chinese). Sci Fish Farm 7:26–28Google Scholar
- Lindeman RE, Pelegri F (2010) Vertebrate maternal-effect genes: insights into fertilization, early cleavage divisions, and germ cell determinant localization from studies in the zebrafish. Mol Reprod Dev 77:299–313Google Scholar
- Saitou N, Nei M (1987) The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4:406–425Google Scholar
- Slanchev K, Stebler J, Goudarzi M, Cojocaru V, Weidinger G, Raz E (2009) Control of Dead end localization and activity-implications for the function of the protein in antagonizing miRNA function. Mech Dev 126:270–277Google Scholar
- Tanaka SS et al (2000) The mouse homolog of Drosophila Vasa is required for the development of male germ cells. Genes Dev 14:841–853Google Scholar
- Yoon C, Kawakami K, Hopkins N (1997) Zebrafish vasa homologue RNA is localized to the cleavage planes of 2- and 4-cell-stage embryos and is expressed in the primordial germ cells. Development 124:3157–3165Google Scholar