Transient translational quiescence in primordial germ cells

Abstract

Stem cells in animals often exhibit a slow cell cycle and/or low transcriptional activity referred to as quiescence. Here, we report that the translational activity in the primordial germ cells (PGCs) of the sea urchin embryo (Strongylocentrotus purpuratus) is quiescent. We measured new protein synthesis with O-propargyl-puromycin and L-homopropargylglycine Click-iT technologies, and determined that these cells synthesize protein at only 6% the level of their adjacent somatic cells. Knockdown of translation of the RNA-binding protein Nanos2 by morpholino antisense oligonucleotides, or knockout of the Nanos2 gene by CRISPR/Cas9 resulted in a significant, but partial, increase (47%) in general translation specifically in the PGCs. We found that the mRNA of the translation factor eEF1A is excluded from the PGCs in a Nanos2-dependent manner, a consequence of a Nanos/Pumilio response element (PRE) in its 3'UTR. In addition to eEF1A, the cytoplasmic pH of the PGCs appears to repress translation and simply increasing the pH also significantly restores translation selectively in the PGCs. We conclude that the PGCs of this sea urchin institute parallel pathways to quiesce translation thoroughly but transiently.

Keywords: Metabolism; Primordial germ cells; Sea urchin; Translation.

Fig. 1.

Translation is transiently reduced in the PGCs at blastula stage. At different time points after fertilization: 5.5 h post fertilization at cleavage stage (A-C), 18 h (blastula stage) (D-F) or 3 days (larva stage) (G-I). Embryos were treated with OPP. Protein synthesis is represented in red and Vasa antibody (green) is used as a marker to localize the PGCs. Arrows indicate PGCs and transient quiescence. Approximately 100 embryos were visualized and representative embryos are presented. Scale bar: 20 µm.

Fig. 2.

Nanos is essential to maintain a translational quiescence in the PGCs. (A-F) Fertilized eggs were injected with either a control morpholino or Nanos morpholino, and treated with OPP at blastula stage (18 h post-fertilization) to visualize protein synthesis (red). Vasa immunofluorescence (green) indicates the location of the PGCs (arrows). Approximately 100 embryos were visualized and representative embryos are presented. Scale bar: 20 µm. (G) For each morpholino, the intensity of OPP was measured in the animal pole, the vegetal pole and the PGCs; the results are presented as percentages compared with the animal pole. Thirty-five blastulae were quantified for the control morpholino and 29 for the Nanos morpholino. Significance was assessed for each area of the blastula between control and Nanos morpholino using Student's t-test (*P<0.001).

Fig. 3.

The CRISPR approach supports the function of Nanos in the TTQ. Fertilized eggs were injected with either the Cas9 mRNA alone (control) or the Cas9 mRNA mixed with all four gRNAs directed against Nanos (Nanos CRISPR). These injected embryos were used to test the expression of Vasa protein in the PGCs (arrows, red) during gastrulation (A-D). DNA was labeled with Hoechst (blue). Sp Nanos2 gene was sequenced after genomic DNA extraction (E). Embryos injected with the Nanos CRISPR (Na and Nb) show several representative examples of mutations and deletions, compared with the control (C). One of the embryos even had a 117 nucleotide excision (not shown). Only a partial sequence of Sp Nanos2 is shown here, corresponding to the region targeted by the gRNAs (represented in red). (F-I) Finally, some injected embryos were also treated with HPG at blastula stage (18 h post-fertilization) to visualize protein synthesis (red). Vasa immunofluorescence (green) indicates the location of the PGCs (arrows). (J) For each embryo, the intensity of the HPG was measured in the animal pole, the vegetal pole and the PGCs; the results are presented as percentages compared with the animal pole. Ten blastulae were quantified for the control and 10 for the Nanos CRISPR. Significance was assessed for each area of the blastula between control and Nanos CRISPR with the use of Student's t-test (*P<0.001).(A-D,F-I) Approximately 100 embryos were visualized and representative embryos are presented. Scale bars: 20 µm.

Fig. 4.

eEF1A mRNA depletion in the PGCs requires Nanos. (A-P) Fertilized eggs were injected with either a control morpholino or Nanos morpholino, and fixed at blastula stage for eEF1A in situ hybridization (red) followed by an immunofluorescence using Vasa antibody (green). DNA was stained with Hoechst (blue). Images are magnified in the area surrounding the PGCs (E-H for the control morpholino; M-P for Nanos morpholino). (Q) The ratio of eEF1A mRNA present in the PGCs versus the somatic cells was quantified using 13 blastulae for the control morpholino and 11 for the Nanos morpholino, and are presented as percentages. The significance of the differences between the control and the Nanos morpholino was assessed using Student's t-test (*P<0.001). (A-P) Arrows indicate the PGCs. Approximately 100 embryos were visualized and representative embryos are presented. Scale bars: 20 µm.

Fig. 5.

eEF1A mRNA depletion in the PGCs depends on its PRE. (A-D) Fertilized eggs were injected with either a control morpholino or a morpholino targeting the eEF1A PRE. Embryos were fixed at blastula stage for eEF1A in situ hybridization (red) followed by an immunofluorescence using Vasa antibody (green). Approximately 100 embryos were visualized and representative embryos are presented. Arrows indicate the PGCs. Scale bar: 20 µm. (E) The ratio of eEF1A mRNA present in the PGCs versus the somatic cells was quantified using 10 blastulae for the control morpholino and 12 for the PRE morpholino, and presented as percentages. Significance was assessed between the control and the PRE morpholino using Student's t-test (*P<0.005).

Fig. 6.

Overexpression of eEF1A protein specifically increases protein synthesis in the PGCs. (A-L) Fertilized eggs were injected with mRNA coding for either GFP or GFP eEF1A, and treated with OPP at blastula stage before fixation. OPP represents the protein synthesis (red) and Vasa antibody is used to localize the PGCs (blue). Approximately 100 embryos were visualized and representative embryos are presented. Arrows indicate the PGCs. Scale bar: 20 µm. (M) The protein synthesis in the PGCs and in the somatic cells was quantified using 11 blastulae for GFP, and 16 for GFP eEF1A. Significance was assessed between the GFP and the GFP eEF1A using Student's t-test (*P<0.05).

Fig. 7.

TTQ starts before the exclusion of eEF1A protein from the PGCs. Early blastula were treated with either HPG (A-D) or OPP (E-H). After the Click-iT reaction (red), the DNA was stained with Hoechst (blue) and eEF1A protein was followed by immunofluorescence (green). A′ to H′ are magnifications of the corresponding blastulae (A-H). Approximately 100 embryos were visualized and representative embryos are presented. Arrows indicate the PGCs. Scale bars: 20 µm.

Fig. 8.

Mitochondrial activity is reduced in the PGCs. Fluorescent in situ hybridization shows that the RNA coding for the ADP/ATP translocase 1 (A-C) is depleted from the PGCs during gastrulation. The embryos were co-labeled using an antibody against Vasa protein (A′-C′). (D-F′) Embryos were labeled with Mitotracker red to observe the mitochondrial activity (red) and with Edu (green) to stain the PGCs. Approximately 100 embryos were visualized and representative embryos are presented. Arrows indicate the PGCs. Scale bars: 20 µm.