gata2 is a bona fide target of miR-451. (A) Schematic diagram of the zebrafish gata2 3′UTR (blue, nts 128-782), which contains 2 predicted miR-451 binding sites (site a, orange; site b, yellow). Predicted seed complementarity sequences are boxed. Red nucleotides in “mut 3′UTR” indicate those mutated to destroy seed sequence binding. (B) Schema of a reporter assay to evaluate the gata2-3′UTR for interaction with miR-451. Microinjection of 1-cell embryos with a series of sensor mRNAs encoding GFP fused to various test 3′UTRs (as tabulated in panel D) was followed by separate injection of 50% of embryos with miR-451 (or mir-144 or control) duplex, tracing miRNA delivery by rhodamine, and the impact on GFP fluorescence intensity assessed at 24 to 28 hpf. (C) miR-451 negatively regulates the sensor GFP-3′UTR^gata2 mRNA. Top: compared with embryos receiving only sensor mRNA (top row in each panel), GFP-fluorescence brightness was reduced in those also injected with miR-451 (traced by rhodamine, red fluorescence in bottom row). Middle and bottom: results of embryos similarly arranged testing various GFP-gata2-3′UTR sensor RNA and miRNA combinations as labeled. Arrays of representative embryos were photographed together in a single image to ensure valid comparison of relative green fluorescence intensity between the 2 groups. (D) Summary of reporter assay outcomes using the series of GFP sensor RNA and miRNA combinations, using a comparative scale for GFP fluorescence (+ to ++++). The color-coding of the gata2-3′UTR variants in the sensor mRNA column refers to panel A; the red “X” indicates a mutated miR-451 site. Assays validating the bioactivity and specificity of the morpholino and duplex oligonucleotides used are presented in Figure S8.