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Fig. 4 Knockdown of miR-146a and miR-146b does not affect leukocyte development. Embryos were injected at the 1-cell stage with morpholinos targeting miR-146a (146aMO1 and 146aMO2) or miR-146b (146bMO1 and 146bMO2) or were injected with standard control morpholino (scMO). (A, B) Confirmation of morpholino knockdown. Embryos were injected with the indicated morpholinos and RNA was collected at 2 dpf. Knockdown of miR-146a (A) and miR-146b (B) was confirmed by TaqMan qPCR. This analysis also demonstrated the specificity of morpholinos 146aMO1, 146aMO2, and 146bMO2 for their respectively target miRNAs, whereas 146bMO1 could not discriminate between the a and b forms of miR-146. 146bMO2 positively affected miR-146a expression besides blocking miR-146b expression and caused developmental aberrations (J, K). (C) Quantification of L-plastin-positive leukocytes at 2 dpf. Embryos were injected with the control morpholino (scMO) or with a combination of 146aMO1 and 146bMO1 (146a/bMOs). L-plastin-labelled cells were counted manually on the left side of each embryo and the numbers present in the head, on the yolk sac, and in the tail were accumulated (n e 26 embryos per group). (D) Quantification of Mpx-positive neutrophils at 2dpf. Embryos were injected with the indicated morpholinos. Neutrophils stained for Mpx activity were counted manually as described for L-plastin immunostaining (n e 13 embryos per group). (E) Quantification of L-plastin-positive leukocytes at 26, 28, 30 and 32 hpf. Embryos were injected with the indicated morpholinos and L-plastin-labelled cells were counted as described above (n e 16 embryos per time point). (F-K) Example fluorescence images of the data shown in (E). The pattern of L-plastin-positive immune cells was comparable between embryos (32 hpf) injected with scMO (F), 146aMO1 (G), 146aMO2 (H), and 146bMO1 (I). 146bMO2 gave non-specific phenotypes and the number of immune cells was more variable dependent upon the severity of the phenotype (J, K).