Nanos is expressed in multipotent cells, stem cells and primordial germ cells (PGCs) of organisms as diverse as jellyfish and humans. It functions together with Pumilio to translationally repress targeted mRNAs. Here we show by loss-of-function experiments that Xenopus Nanos1 is required to preserve PGC fate. Morpholino knockdown of maternal Nanos1 resulted in a striking decrease in PGCs and a loss of germ cells from the gonads. Lineage tracing and TUNEL staining reveal that Nanos1-deficient PGCs fail to migrate out of the endoderm. They appear to undergo apoptosis rather than convert to normal endoderm. Whereas normal PGCs do not become transcriptionally active until neurula, Nanos1-depleted PGCs prematurely exhibit a hyperphosphorylated RNA polymerase II C-terminal domain at the midblastula transition. Furthermore, they inappropriately express somatic genes characteristic of endoderm regulated by maternal VegT, including Xsox17α, Bix4, Mixer, GATA4 and Edd. We further demonstrate that Pumilio specifically binds VegT RNA in vitro and represses, along with Nanos1, VegT translation within PGCs. Repressed VegT RNA in wild-type PGCs is significantly less stable than VegT in Nanos1-depleted PGCs. Our data indicate that maternal VegT RNA is an authentic target of Nanos1/Pumilio translational repression. We propose that Nanos1 functions to translationally repress RNAs that normally specify endoderm and promote apoptosis, thus preserving the germline.

SUMMARYPrimordial germ cells (PGCs) in Xenopus are specified through the inheritance of germ plasm. During gastrulation, PGCs remain totipotent while surrounding cells in the vegetal mass become committed to endoderm through the action of the vegetal localized maternal transcription factor VegT. We find that although PGCs contain maternal VegT RNA, they do not express its downstream targets at the mid-blastula transition (MBT). Transcriptional repression in PGCs correlates with the failure to phosphorylate serine 2 in the carboxy-terminal domain (CTD) of the large subunit of RNA polymerase II (RNAPII). As serine 5 is phosphorylated, these results are consistent with a block after the initiation step but before the elongation step of RNAPII-based transcription. Repression of PGC gene expression occurs despite an apparently permissive chromatin environment. Phosphorylation of CTD-serine 2 and expression of zygotic mRNAs in PGCs are first detected at neurula, some 10 hours after MBT, indicating that transcription is significantly delayed in the germ cell lineage. Significantly, Oct-91, a POU subclass V transcription factor related to mammalian Oct3/4, is among the earliest zygotic transcripts detected in PGCs and is a likely mediator of pluripotency. Our findings suggest that PGCs are unable to respond to maternally inherited endoderm determinants because RNAPII activity is transiently blocked while these determinants are present. Our results in a vertebrate system further support the concept that one strategy used repeatedly during evolution for preserving the germline is RNAPII repression.

Background: It has not been possible to study the pumping and signaling functions of Na/K-ATPase independently in live cells. Results: Both cell-free and cell-based assays indicate that the A420P mutation abolishes the Src regulatory function of Na/K-ATPase. Conclusion: A420P mutant has normal pumping but not signaling function. Significance: Identification of Src regulation-null mutants is crucial for addressing physiological role of Na/K-ATPase.

Glioblastoma is an aggressive tumor that is associated with distinctive infiltrating microglia/macrophages populations. Previous studies demonstrated that chlorogenic acid (5-caffeoylquinic acid, CHA), a phenolic compound with low molecular weight, has an anti-tumor effect in multiple malignant tumors. In the present study, we focused on the macrophage polarization to investigate the molecular mechanisms behind the anti-glioma response of CHA in vitro and in vivo. We found that CHA treatment increased the expression of M1 markers induced by LPS/IFNγ, including iNOS, MHC II (I-A/I-E subregions) and CD11c, and reduced the expression of M2 markers Arg and CD206 induced by IL-4, resulting in promoting the production of apoptotic-like cancer cells and inhibiting the growth of tumor cells by co-culture experiments. The activations of STAT1 and STAT6, which are two crucial signaling events in M1 and M2-polarization, were significantly promoted and suppressed by CHA in macrophages, respectively. Furthermore, In G422 xenograft mice, CHA increased the proportion of CD11c-positive M1 macrophages and decreased the distribution of CD206-positive M2 macrophages in tumor tissue, consistent with the reduction of tumor weight observed in CHA-treated mice. Overall these findings indicated CHA as a potential therapeutic approach to reduce glioma growth through promoting M1-polarized macrophage and inhibiting M2 phenotypic macrophage.

Nanos family members have been shown to act as translational repressors in the Drosophila and Caenorhabditis elegans germline, but direct evidence is missing for a similar function in vertebrates. Using a tethered function assay, we show that Xenopus Nanos1 is a translational repressor and that association with the RNA is required for this repression. We identified a 14 amino acid region within the N-terminal domain of Nanos1 that is conserved in organisms as diverse as sponge and Human. The region is found in all vertebrates but notably lacking in Drosophila and C. elegans. Deletion and substitution analysis revealed that this conserved region was required for Nanos1 repressive activity. Consistent with this observation, deletion of this region was sufficient to prevent abnormal development that results from ectopic expression of Nanos1 in oocytes. Although Nanos1 can repress capped and polyadenylated RNAs, Nanos1 mediated repression did not require the targeted RNA to have a cap or to be polyadenylated. These results suggest that Nanos1 is capable of repressing translation by several different mechanisms. We found that Nanos1, like Drosophila Nanos, associates with cyclin B1 RNA in vivo indicating that some Nanos targets may be evolutionarily conserved. Nanos1 protein was detected and thus available to repress mRNAs while PGCs were in the endoderm, but was not observed in PGCs after this stage.