Fig. 5

Late consequences of notochord infection. (A,B) Histological analysis at 5 dpi of larvae that had been injected in the notochord with PBS (A) or E. coli (B). Cross-sections (4 μm) stained with Masson’s Trichrome. In the infected larvae, collagen-enriched matrixes (blue) and supernumerary vacuoles (V) were seen. Cells inside the notochord (*) and ectopic matrix deposition (arrowhead) can be seen in the sections. (C,D) Neutrophil (green) and macrophage (red) infiltration in the notochord, multiscan confocal microscopy. Cross-sections (60 μm) of Tg(mpx:GFP; mpeg1:mCherryF) larvae at 5 days following injection of the notochord with PBS (C) or with E. coli (D). *Nonspecific staining. (E,F) Notochord of Tg(mpx:GFP) larvae at 3 dpi. (E) Transmitted-light image, overlaid in F with a maximal projection of confocal fluorescence images that show neutrophils (green) clustered at the damaged zone. (G–I) Sudan-black (SB) staining (black) and GFP expression (green) in Tg(mpx:GFP) larvae at 48 hours post-injection of either PBS (G) or E. coli (H,I) into the notochord. Arrowheads indicate neutrophils that have degranulated. (J) Graphic representation of the percentage of GFP⁺SB cells under the indicated conditions, mean±s.e.m., five or six fish per condition were analysed. *P<0.05 compared with the PBS control. (K) Infected larvae developed scoliosis and dysmorphic vertebrae. Larvae were injected with PBS or E. coli into the notochord at 48 hpf, grown to 60 dpf and fixed for analysis. Bones were stained with Alizarin red. Lateral and dorsal views of the anterior part of the vertebral column are shown. Arrowheads indicate mis-shaped and fused vertebral bodies. Scale bars: 20 μm (A-D,G-I); 50 μm (E,F).