Journal: Development (Cambridge, England)
Article Title: Xenopus Nanos1 is required to prevent endoderm gene expression and apoptosis in primordial germ cells
Figure Lengend Snippet: Nanos1 represses VegT translation in PGCs. ( A ) RNA EMSA analysis shows that the Xenopus Pumilio1 RNA-binding domain (Xpum1 RBD) can bind biotin-labeled VegT PBE (1 ng) but not the mutant VegT PBE. This binding reaction was competed by unlabeled VegT PBE. ( B ) Experimental design for results shown in C. Venus-DEADSouth 3′UTR and DsRED-DEADSouth 3′UTR are germline lineage tracers. VegT PBE or its mutant was subcloned downstream of the DsRED open reading frame. Nanos1-MO was injected at stage 1. Germline lineage reporters were injected into four germ plasm-containing blastomeres at the 32-cell stage. Embryos were allowed to develop until stage 35 and the fluorescent images were taken from live embryos. ( C ) In vivo fluorescent reporter assay indicates that VegT PBE mediates translational repression of DsRED (red) in PGCs (green). Repression was lost in embryos bearing a PBE mutation or that were depleted of Nanos1. Reporters injected are noted across the top. This experiment was repeated three times. Arrows indicate DsRED signal detected in PGCs. Data from one experiment are presented beneath the images for each group. Scale bars: 50 μm.
Article Snippet: VegT 3′UTR (296 nt, 2336-2631) containing the PBE (UGUAAAUA) was subcloned downstream of the DsRED open reading frame to produce DsRED- VegT PBE-DS3′UTR (In-Fusion HD EcoDry Cloning System, Clontech).
Techniques: RNA Binding Assay, Labeling, Mutagenesis, Binding Assay, Injection, In Vivo, Reporter Assay