The Nanos3-3'UTR is required for germ cell specific NANOS3 expression in mouse embryos

Abstract

Background: The regulation of gene expression via a 3' untranslated region (UTR) plays essential roles in the discrimination of the germ cell lineage from somatic cells during embryogenesis. This is fundamental to the continuation of a species. Mouse NANOS3 is an essential protein required for the germ cell maintenance and is specifically expressed in these cells. However, the regulatory mechanisms that restrict the expression of this gene in the germ cells is largely unknown at present.

Methodology/principal findings: In our current study, we show that differences in the stability of Nanos3 mRNA between germ cells and somatic cells is brought about in a 3'UTR-dependent manner in mouse embryos. Although Nanos3 is transcribed in both cell lineages, it is efficiently translated only in the germ lineage. We also find that the translational suppression of NANOS3 in somatic cells is caused by a 3'UTR-mediated mRNA destabilizing mechanism. Surprisingly, even when under the control of the CAG promoter which induces strong ubiquitous transcription in both germ cells and somatic cells, the addition of the Nanos3-3'UTR sequence to the coding region of exogenous gene was effective in restricting protein expression in germ cells.

Conclusions/significance: Our current study thus suggests that Nanos3-3'UTR has an essential role in translational control in the mouse embryo.

Figure 1. Schematic representation of the transgenes used in this study.

The top line represents BAC RP24-325I12 which contains the Nanos3 gene, the second line is a larger scale schema of a portion of this construct. (A–D) Different modifications of the transgene. Blue lines denote sequences derived from the BAC RP24-325I12 construct and the red lines those of the CAG promoter. The meanings of each box is indicated. (E) Sequence of Nanos3-3′UTR we used.

Figure 2. Nanos3-mRFP is expressed dominantly in germ cells in BAC transgenic mouse lines.

Confocal images of embryos of the wild-type (A–D), BAC-Nanos3-mRFP(Nos3-3′UTR) (E-H) or BAC-Nanos3-mRFP(BghpA) (I–L). Panels (A–D) show images of immunostaining for anti-NANOS3. Panels (E–L) show images of mRFP fluorescence (not immunostaining) and (A′–L′) are merged images that include immunostaining for the germ cell marker anti-OCT3/4, anti-Stella/PGC7 or TRA98 (green signal). The developmental stage associated with each figure is indicated above each panel: E7.5 (A, E, and I), E8.5 (B), E9.5 (F and J), E11.5 (C, G and K), E13.5 male gonad (D, H and L). Asterisks represent non-specific signals by the secondary antibody. Scale bars, 50 µm.

Figure 3. Replacement of Nos3-3′UTR with BghpA results in the upregulation of NANOS3-mRFP protein in somatic tissues.

(A–D) Fluorescence images of male embryos derived from BAC-Nanos3-mRFP(Nos3-3′UTR) (A–B) and BAC-Nanos3-mRFP(BghpA) (C–D) transgenic embryos at E14.5. (A and C) The upper images are of the abdomens of embryos harboring the transgene (Tg⁺), whereas the lower images are of the same tissues from embryos with no transgene (Tg⁻). The broken gray lines indicate the gonads and broken yellow lines indicate the kidneys. (B and C) Whole body of Tg+ and Tg- embryos are shown. The image in the upper panel shows the mRFP fluorescence pattern, whilst the lower panels are the corresponding bright field images. (E and F) Developmental changes in the relative mRFP intensities in germ cells (red) and somatic cells (blue) derived from BAC-Nanos3-mRFP(Nos3-3′UTR) (E) and BAC-Nanos3-mRFP(BghpA) (F) transgenic embryos. Error bars represent the s.e.m.

Figure 4. The accumulation of somatic Nanos3 mRNA is suppressed by the Nanos3-3′UTR.

The levels of Nanos3 (A–C) or mRFP (D) mRNA were compared by quantitative RT-PCR using RNA samples derived from wild-type embryos (A–C) and BAC-Nanos3-mRFP(Nos3-3′UTR) (blue in D) and BAC-Nanos3-mRFP(BghpA) (green in D) transgenic embryos at E7.5, E9.5 and E13.5. Data were normalized by G3PDH in each sample. The relative mRNA levels in the E9.5A sample (an anterior part of E9.5 embryo) were assigned the reference value of 1.0. A, anterior part of the embryo; P, posterior part of the embryo; M–K or K, the male kidney; M–G or G, the male gonad; F–G, the female gonad; N, the posterior part of a Nanos3 knockout embryo at E9.5. Error bars represent the s.d.

Figure 5. Nanos3-3′UTR is sufficient to establish the germ cell-specific expression pattern in the mouse embryo.

(A–C) Fluorescence images of CAG-mRFP(Nos3-3′UTR) (A, male; B, female) and CAG-mRFP(BghpA) (C, male) transgenic embryos at E14.5. The upper images are of the abdomens of embryos harboring transgenes (Tg⁺), whereas the lower panels show corresponding images from embryos lacking a transgene (Tg⁻). Broken gray lines indicate gonads. (D–E) Quantitative RT-PCR analysis of mRFP in CAG-mRFP(Nos3-3′UTR) (D) or CAG-Lyn-mRFP(BghpA) (E) embryos at E14.5. The data were normalized using G3PDH. M–K, male kidney; M-Li, male limb; M–G, male gonad; F–G, female gonad; N, wild-type embryo. Error bars represent the s.d. Student t-test was used to calculate P values. *, P<0.05.

Figure 6. The Nanos3-3′UTR may function in both germ cells and somatic tissues after generation of the PGC.

Confocal images of CAG-Lyn-mRFP(BghpA) (A–D) and CAG-mRFP(Nos3-3′UTR) (E–J) transgenic embryos. (A–J) mRFP fluorescence; (A′–J′) merged images of mRFP fluorescence (red) and immunostaining signals (green) for NANOS3 (A′–D′ G′–J′), STELLA/PGC7 (E′–F′). Insets are high magnification of each panel. The embryonic stage for each sample is indicated. White arrowheads, germ cells; white open arrowheads, somatic cells that do not express mRFP; yellow arrowheads, somatic cells that express mRFP. Scale bars, 100 µm.