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 Lhomopropargylglycine 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.

Methods to label, isolate, and image sea urchin small micromeres, the primordial germ cells (PGCs)

“…Culture to a desired stage and fix the embryos with 4% PFA (ref method for fixation in Oulhen, Swartz, Laird, Mascaro, & Wessel, 2017). …”

Specification of the germline by Nanos-dependent down-regulation of the somatic synMuvB transcription factor LIN-15B

“…Paradoxically, in sea urchins, Nanos targets the mRNA coding for the CNOT6 deadenylase for degradation in PGCs, which indirectly stabilizes other maternal mRNAs (Swartz et al, 2014). In that system, Nanos was also found to silence eEF1A expression, leading to a transient period of translational quiescence in PGCs (Oulhen et al, 2017).…”

“…Nanos proteins are cytoplasmic and regulate gene expression post-transcriptionally by recruiting effector complexes that silence and degrade mRNAs in the cytoplasm. Six direct Nanos mRNA targets have been identified to date [Drosophila hunchback, cyclin B and hid(Asaoka-Taguchi et al, 1999;Dalby and Glover, 1993;Kadyrova et al, 2007;Murata and Wharton, 1995;Sato et al, 2007;Wreden et al, 1997), Xenopus VegT (Lai et al, 2012), and sea urchin CNOT6 and eEF1A (Oulhen et al, 2017;Swartz et al, 2014)], but none of these targets are sufficient to explain how Nanos activity might affect PGC chromatin. In this study, we characterize the gene expression defects of PGCs lacking nanos activity in C. elegans.…”

The painted sea urchin, Lytechinus pictus, as a genetically-enabled developmental model

“…The primary reasons for this have been: (1) a lack of molecular tools for generating mutants in marine organisms and (2) the use of echinoderm species with long generation times. The “molecular tools bottleneck” has recently been relaxed, with the demonstration of successful F0 genome editing using the CRISPR/Cas9 system in, among many other organisms, the purple sea urchin Strongylocentrotus purpuratus (Cui, Lin, & Su, 2017; Lin & Su, 2016; Mellott, Thisdelle, & Burke, 2017; Oulhen, Swartz, Laird, Mascaro, & Wessel, 2017; Oulhen & Wessel, 2016; Shevidi, Uchida, Schudrowitz, Wessel, & Yajima, 2017). As such, the next bottleneck to address is the shift to species with generation times suitable for maintaining stable germline transgenic lines in the lab.…”