Fig. 4

Labeling with DBCO-SRB does not alter the subcellular localization of injected mRNA and allows resolving the dynamic positioning of the transcript. (A) 1-cell stage embryos were injected with in vitro synthesized mRNA containing a non-translatable mcherry sequence upstream of nos 3′ UTR (STOP mcherry-nos mRNA). The injected mRNA was either not labeled (upper panels) or labeled (lower panels) with SRB (emission 610 nm). After 10 hpf, the embryos were fixed and hybridized with RNAscope probes, targeting the mcherry sequence (labeling the injected mRNA, emission filter 509 nm) and that of nanos open reading frame (labeling the endogenous nanos mRNA, emission filter 647 nm). An overlay of the three detection channels and DAPI (labeling the nuclei, emission filter 470) is presented in the right panels. (B) SRB-labeled gfp-nos mRNA was injected into 1-cell stage zebrafish embryos and tracked over time in PGCs after 1 day of development. The panels show maximum intensity projections spanning 9 μm, with time intervals of 3.5 seconds between frames. Lines highlight changes in distance (μm) between the edges of two RNA-containing structures over time. Changes in the shape of an RNA-containing structure (marked by an asterisk) are presented in the insets on the bottom left of each plane. Scale bar = 5 μm.