Visualization of Turbot (Scophthalmus maximus) Primordial Germ Cells in vivo Using Fluorescent Protein Mediated by the 3' Untranslated Region of nanos3 or vasa Gene

Mar Biotechnol (NY). 2019 Oct;21(5):671-682. doi: 10.1007/s10126-019-09911-z. Epub 2019 Sep 9.

Abstract

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.

Keywords: Location; Primordial germ cells; Scophthalmus maximus; nanos3; vasa.

MeSH terms

  • 3' Untranslated Regions
  • Animals
  • Base Sequence
  • Cell Tracking / methods*
  • DEAD-box RNA Helicases / genetics
  • DEAD-box RNA Helicases / metabolism
  • Fish Proteins / genetics*
  • Fish Proteins / metabolism
  • Flatfishes / genetics*
  • Flatfishes / growth & development
  • Flatfishes / metabolism
  • Genes, Reporter
  • Germ Cells / cytology
  • Germ Cells / growth & development
  • Germ Cells / metabolism*
  • Luminescent Proteins / genetics
  • Luminescent Proteins / metabolism
  • Microinjections
  • Nucleic Acid Conformation
  • Oryzias / genetics
  • Oryzias / growth & development
  • Oryzias / metabolism
  • RNA-Binding Proteins / genetics
  • RNA-Binding Proteins / metabolism
  • Recombinant Fusion Proteins / genetics*
  • Recombinant Fusion Proteins / metabolism
  • Red Fluorescent Protein
  • Sequence Alignment
  • Sequence Homology, Nucleic Acid
  • Species Specificity
  • Zebrafish / genetics*
  • Zebrafish / growth & development
  • Zebrafish / metabolism
  • Zebrafish Proteins / genetics
  • Zebrafish Proteins / metabolism
  • Zygote

Substances

  • 3' Untranslated Regions
  • Fish Proteins
  • Luminescent Proteins
  • RNA-Binding Proteins
  • Recombinant Fusion Proteins
  • Zebrafish Proteins
  • ddx4 protein, zebrafish
  • DEAD-box RNA Helicases