Abstract
In embryonic male germ cells, the RNA-binding protein NANOS2 recruits its target RNAs to processing bodies (P-bodies), where they are repressed. This process is necessary to promote male-type germ cell differentiation. However, it remains unclear whether all NANOS2 functions depend on P-bodies. To address this question, we established ES cell lines containing a germ cell-specific inducible Cre and reporter together with the floxed Ddx6 allele. We deleted the Ddx6 gene by administering tamoxifen to chimeric embryos containing germ cells derived from recombinant ES cells. DDX6-null germ cells exhibited both similar and distinct defects from those observed in NANOS2-null germ cells. These results demonstrate that NANOS2 function is carried out via both P-body-dependent and -independent mechanisms. RNA-seq analyses further supported the phenotypic differences between DDX6-null and NANOS2-null germ cells, and indicated distinct molecular cascades involved in NANOS2-mediated gene regulation.
Highlights
Germ cells are specialized cells required for transmitting genetic information to the generation
We introduced a method of chimera analysis to facilitate germ-cell-specific gene knockout studies
We consider this unlikely because we established a Ddx6-conditional KO (cKO) mouse line using the same ES cells and confirmed all phenotypes observed in the chimera analyses
Summary
Germ cells are specialized cells required for transmitting genetic information to the generation. Many NANOS2-null germ cells ectopically express STRA8 and initiate meiosis even in the male gonad. NANOS2-null germ cells fail to express DNMT3L, one of the epigenetic regulators important for DNA methylation, including genomic imprinting[9,10,11] These NANOS2-null phenotypes may be due to the upregulation of NANOS2 target genes. ES-derived chimeric mice are used to generate cKO mouse lines This line has to be crossed with a germ cell-specific Cre line to establish a male mouse with Cre. the male floxed-Ddx6/ Cre mouse can be crossed with a homozygous floxed female mouse. We demonstrate here that the chimera method is highly effective Using this method, we generated chimeric embryos that contain DDX6-null germ cells. NANOS2 may work in a DDX6- and P-bodyindependent manner
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