Journal: bioRxiv

Article Title: Spatial organization and function of RNA molecules within phase-separated condensates are controlled by Dnd1

doi: 10.1101/2023.07.09.548244

Figure Lengend Snippet: (A) 1-cell stage embryos that lack the chemokine guidance cue Cxcl12a were injected with a morpholino antisense oligonucleotide (anti-Nos Morpholino), inhibiting the translation of nanos3 mRNA. The germ cells were then evaluated for somatic cell morphologies at 24 hpf. Images show an example of a Nanos3-deficient germ cell exhibiting elongated muscle precursor-like appearance. (B) Evaluation of the proportion of wildtype and Nanos3-deficient germ cells with soma-like morphologies. Cells were only counted as soma-like when they displayed clear morphologies of neurons, muscle or notochord cells. (C) Scheme depicting early germ cell development. Germ cells are specified at 4 hpf, become migratory and form two bilateral clusters at 10 hpf. The black box shows a representative image of perinuclear germ granules marked by the Vasa-GFP protein, with enrichment of the endogenous germline mRNAs tdrd7 and nanos3 (detected by RNAscope probes). (D) Distribution of nanos3 mRNA relative to tdrd7 mRNA (upper panels) and Dnd1 protein (lower panels) within germ granules marked by Vasa-GFP (upper panels) or endogenous Vasa protein (lower panels). One confocal plane is presented. (E) 3D analysis of the distribution of germ granule components across concentric layers of the condensate. (F) Heat map images of germ granule single confocal planes showing the distribution of nanos3 mRNA, tdrd7 mRNA or Dnd1 protein in different layers of the condensate. White arrows point at distinct clusters of these molecules, primarily detected in peripheral layers (layer d, e) of the condensate. (G) Proportion of bright clusters of nanos3 mRNA, tdrd7 mRNA, Dnd-GFP protein or Vasa protein detected in the core (dark grey) or peripheral (light grey) regions of the granule. The area occupied by bright clusters in the core or periphery was normalized to the size of the respective region. The peripheral region also includes the immediate area outside of the granule. Each dot in the graph represents the value derived from a single germ granule. For each granule, the brightest 5% of pixels were defined as bright clusters. Single confocal planes were used for the analysis. n (nanos, tdrd7) = 36, n (Vasa) = 35, n (Dnd-GFP) = 31. N = 3. See also .

Article Snippet: RNAscope probes Probe Diluent (ACD Bio 300041), Dr-nanos-C2 (ACD Bio 404521-C2), Dr-tdrd7-C2 (ACD Bio 536551-C2), EGFP-C1 (ACD Bio 400281-C1) and Dr-nanos3-CDS-C3 (ACD Bio 431191-C3) were hybridized overnight at 40°C.

Techniques: Injection, Protein Enrichment, RNAscope, Derivative Assay