Fig. 3. Bioorthogonal labeling with DBCO-SRB reveals subcellular localization of mRNA injected into zebrafish embryos without interfering with transcript positioning and translation. (A) 1-cell stage zebrafish embryos were injected with mRNA containing the PGC-specific nos 3′ UTR synthesized in vitro, which was either not labeled or labeled with SRB (red circle). PGCs were imaged 10 hpf, focusing either on a cell cluster (as in B) or on single cells (as in C and D). (B) Injection of SRB-labeled-mRNA containing a non-translatable mcherry sequence upstream of nos 3′ UTR (STOP mcherry-nos) results in specific detection of the transcript in cells expressing the transgenic PGC membrane marker egfp-f′ nos (lower panels), while non-labeled mRNA is not visible (upper panels). An overlay of the two detection channels and DAPI (labeling the nuclei) is presented in the right panels. Scale bar = 10 μm. (C) Injection of gfp-nos mRNA results in GFP-expression in PGCs (as in ref. 69, upper panels). SRB-labeling of the mRNA does not interfere with PGC-specific GFP-expression and results in detection of granule-like mRNA structures (arrowheads, lower panels). An overlay of the detection channels is presented in the right panels. Scale bar = 5 μm. (D) Co-injection of SRB-labeled STOP mcherry-nos mRNA with mRNA encoding for the germ granule marker protein Vasa GFP (arrows, Fig. S7†), reveals the localization of labeled STOP mcherry-nos transcripts to germ granules in PGCs (arrowheads, lower panels). Non-labeled mRNA is not visible (upper panels). An overlay of the detection channels is presented in the right panels. Scale bar = 5 μm.