Zebrafish germline‐specific RNPs are homotypic and remain distinct during aggregation. Germ plasm RNAs as cortical pre‐aggregates (A–D) and furrow aggregate (E). Despite RNP‐RNP association, RNPs remain distinct and are homotypic, containing a single type of RNA. A–D:Pre‐aggregates at the cortex. Two different RNAs associate but do not fully colocalize (Pearson's coefficient: (A) vasa/dnd: r = 0.37, (B) nanos/vasa: r = 0.39, (C) dazl/vasa: r = 0.17, compared with a control experiment where the same RNA is targeted by identical probes that are labeled with two different fluorophores (D) nanos/nanos: r = 

Correlation as a measure of RNA localization within germ plasm aggregates. Diagram of whole embryo with insets indicating location of furrow aggregates, followed by micrographs (merge and channel separated) of magnified regions. Furrow aggregates labeled to detect two germ plasm RNAs as indicated. Pearson's coefficient for combinations of two different RNAs ranges from 0.20 to 0.33, in contrast to control labelings for the same RNA targeted by differently labeled probes (nanos/nanos: r = 0.80). For each double labeling combination, 10‐μm section line scans of fluorescence intensity of separate channels along each aggregate show that fluorescence in embryos labeled for two different RNAs exhibit divergent peak distribution, in contrast with the control labeled nanos/nanosaggregate, where fluorescence distributions run largely in parallel. Fluorescence intensity values (y‐axis) are min‐max normalized to a scale of 0–1. All images are confocal Z‐projections. Scale bar = 5 μm.

originally localized to the vegetal cortex, accumulates at the furrows (arrowheads) of the four cell‐staged blastocyst at the animal pole in wild‐type control (DMSO‐treated) embryos (A) but not in wild‐type embryos treated with F‐actin drugs cytochalasin‐D (B) and phalloidin (C), yet drug‐treated embryos exhibit germ plasm aggregate formation. D–E″:aura/mid1ip1Lmutant embryos show severely reduced dazl RNP accumulation at the furrow (Welch and Pelegri, 2015) (D), yet contain animal RNP aggregates (dnd/nanos, E, E″). F: Quantitation dnd RNP aggregates in the presence or absence of dazlRNPs: Panels A–E = confocal Z‐projections; Panels E′–E″ = SIM Z‐projections. Scale bars = 100 μm in A–D; 75 μm in E; 8 μm in E′–E″.

Germ plasm RNPs remain homotypic during asymmetric germ plasm segregation and dispersal. A–C″: The single‐type character of germ plasm RNPs appears to be maintained through aggregate formation at the furrows for the first several cell cycles (0–2 hpf [A,A′]), segregation in the blastula (2–4 hpf [B,B′]), and RNP dispersal at the end of the blastula period [4–6 hpf (C–C″]). Note in panels (C′,C″) RNPs are dispersed, largely occupying the cytoplasmic space, in contrast to aggregated, subcellularly localized germ plasm at earlier stages (A,B; see also Fig. 2 top left inset), Panel A′ = confocal Z‐projection; Panels B′–C″ = SIM Z‐projections. Scale bars = 5 μm in A′–C′; 1 μm in C″. Panels (A–C) adapted from Kimmel et al. (1995), with permission.

 

 

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