Fig. 1

Fig. 1

Macrophages are not involved in the clearance of bacteria injected within the notochord.

(A) Experimental scheme. One dpf tg(mpeg1:mCherry-F/mpx:GFP) or tg(mfap4:mCherry-F) or tg(mpeg1:mCherry-F) embryos were i.v. injected with Lipo-Clodronate (L-clo) or Lipo-PBS (L-PBS). Correctly depleted larvae were selected based on the loss of red fluorescent macrophages, and GFP or DsRed expressing E. coli were injected within their notochord at 2 dpf. The infection outcome was analyzed at 1 and 2 dpi using fluorescence microscopy. (B) Lipo-Clodronate efficiently depletes macrophages without affecting neutrophil population. Experiments were performed as described in (A) on tg(mpeg1:mCherry-F/mpx:GFP). GFP (neutrophils) and mCherry (macrophages) were analysed by fluorescence microscopy at 2 dpf. (C) qRT-PCR measurement of mpeg1 mRNA relative to ef1a in Lipo-PBS and Lipo-clodronate conditions in whole larvae at 3 dpf (pool of 10 larvae, mean values ± Standard Error of the Mean (SEM), three experiments, Mann Whitney test, one tailed, *P<0.05). (D) Tg(mfap4:mCherry-F) were treated with Lipo-Clodronate or Lipo-PBS as described in (A). mCherry (macrophages) was analysed by fluorescence microscopy at 2 dpf. Representative fluorescence overlaid with brightfield images show macrophage depletion in Lipo-Clodronate treated larvae. (E) Macrophage counts (mfap4⁺ cells) at 2 dpf in indicated conditions (horizontal lines indicate the mean ± SEM, Student test, one-tailed, ***p<0.001). (F) E. coli-GFP infections in the notochord of tg(mpeg1:mCherry-F) embryos are cleared in macrophage-depleted embryos. GFP (E. coli) and mCherry (macrophages) were imaged repeatedly in individual larvae using fluorescence microscopy at 6 hpi and 1 dpi. In both Lipo-PBS and Lipo-clodronate conditions, E. coli-GFP are present in the notochord at 6 hpi (white arrows) but are cleared at 1 dpi (N_L-PBS = 5 and N_L-clo = 9). Arrowhead shows the recruitment of macrophage in Lipo-PBS injected larvae. Asterisks show the auto-fluorescence of the yolk. (G) CFU counts at 1 dpi in notochord infected of Lipo-PBS and Lipo-Clodronate treated larvae (mean number of CFU per larva ± SEM, N_L-PBS = 9 and N_L-clo = 5, Mann Whitney test, two tailed, p>0.05, ns = not significant). (H) E. coli infections in the notochord of tg(mpx:GFP) embryos after macrophage depletion with Lipo-Clodronate. GFP (Neutrophils) was imaged in larvae using fluorescence microscopy at 2 dpi (N_L-PBS = 25 and N_L-clo = 24). Scale bars: 400 μm.