(A) Expression of miR-206 analysed by qPCR at 1, 3, and 5 dpi. (B) Expression of miR-206 in uninfected and infected antagomir-injected embryos (miR-206 knockdown). (C) M. marinum burden in miR-206 knockdown embryos at 1 and 3 dpi. (D) Expression of miR-206 at 1 dpi following infection with either wild-type (WT) M. marinum, ΔESX1 M. marinum, or UPEC. (E) ΔESX1 M. marinum burden in miR-206 knockdown embryos at 1 and 3 dpi. (F) UPEC burden in antagomir-injected embryos at 6 hpi and 1 dpi. Each data point represents a single measurement, with the mean and SEM shown. For qPCR analysis, each data point represents 10 embryos, and contains 2 biological replicates. Bacterial burden analysis data points (WT M. marinum, ΔESX1 M. marinum, and UPEC) represent individual embryos (n = 40–50 embryos per group) and are representative of 2 biological replicates.

(A) Expression of candidate target genes measured by qPCR at 1 dpi in miR-206 knockdown embryos. (B-D) Expression of zebrafish CXCL12/CXCR4 pathway ortholog genes at 3 dpi. Each data point represents a single measurement of 10 pooled embryos and 2 biological replicates, with the mean and SEM shown.

(A) Representative images of infection phenotype at 3 dpi in control and miR-206 knockdown embryos. White arrows indicate bacterial foci. Neutrophils are red (lyzC:dsred) and M. marinum is green (wasabi); co-localisation is indicated by yellow fluorescence. (B) Measurement of whole-body neutrophil fluorescent area at 1 and 3 dpi in miR-206 knockdown embryos. (C) Measurement of neutrophil levels following trunk infection with M. marinum in miR-206 knockdown embryos. (D) Measurement of neutrophil retention at sites of infection following trunk infection with M. marinum in control and miR-206 knockdown embryos. (E) Measurement of neutrophil recruitment to a sterile tail fin wound in miR-206 knockdown embryos. Each data point represents a single measurement, with the mean and SEM shown. For time-lapse imaging, each data point represents the mean of 6 foci of infection from 6 separate embryos. Neutrophil retention was measured by selecting 10 random cells in 3 embryos per group and measuring the time spent at the granuloma. Bacterial burden analysis was performed on 15–20 embryos per treatment. Graphs are representative of 2 biological replicates. * P < 0.05, ** p < 0.01, *** p < 0.001.

(A-B) Representative images of bacterial granulomas in trunk-infected control and miR-206 knockdown embryos. (C) Quantification of M. marinum burden in trunk-infected control and miR-206 knockdown embryos. (D-E) Representative images of M. marinum-neutrophil interactions in trunk-infected control and miR-206 knockdown embryos. Neutrophils are red Tg(lyzC:dsred) and M. marinum is green (wasabi); co-localisation is indicated by yellow fluorescence. (F) Quantification of the proportion of the bacterial fluorescence overlapping with neutrophil fluorescence (co-localisation yellow fluorescence in D-E) in trunk-infected control and miR-206 knockdown embryos. Each data point represents the mean of 6 foci of infection from 6 separate embryos with SEM shown. Differences between groups was calculated using multiple t-tests and the Holm-Sidak method.

(A) Whole body neutrophil counts at 3 dpi of cxcr4b and double (cxcr4b and miR-206) knockdown embryos. (B) Measurement of neutrophil levels following trunk infection with M. marinum in double knockdown embryos. (C) Bacterial burden at 3 dpi in M. marinum-infected double knockdown embryos. (D) Whole body neutrophil counts at 3 dpi in miR-206 knockdown embryos treatment with AMD3100. (E) Measurement of neutrophil recruitment to M. marinum following trunk injection in miR-206 knockdown embryos treatment with AMD3100. (F) Bacterial burden at 3 dpi in miR-206 knockdown embryos treatment with AMD3100. Each data point represents a single measurement, with the mean and SEM shown. For time-lapse imaging, each data point represents the mean of 6 foci of infection from 6 separate embryos. Bacterial burden analysis was performed on 15–25 embryos per treatment. Graphs are representative of 2 biological replicates, except for AMD3100 data, which is a single biological replicate.

(A) Whole body neutrophil counts at 3 dpi of cxcl12a and double (cxcl12a and miR-206) knockdown embryos. (B) Measurement of neutrophil recruitment to M. marinum following trunk injection in double knockdown embryos. (C) Bacterial burden at 3 dpi in M. marinum-infected double knockdown embryos. Each data point represents a single measurement, with the mean and SEM shown. For time-lapse imaging, each data point represents the mean of 4 foci of infection from 4 separate embryos. Bacterial burden analysis was performed on 20–30 embryos per treatment. Graphs are representative of 2 biological replicates.