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Fig. 1
Injection of Escherichia coli into the notochord of zebrafish larvae. (A) Embryos at 48 hpf. Bacteria were injected (elongated arrowhead) into the notochord (red) dorsal to the urogenital opening. The dashed line with arrowheads shows the region where cross-sections have been performed for electron microscopy. (B,C) Transmission electron microscopy analysis of a larva 15 minutes after injection of bacteria into the notochord; C is a higher magnification of the boxed region in B. Arrowheads indicate bacteria. Nc, notochordal cell; v, vacuole; pbm, peri-notochordal basement membrane that comprises three layers – o, outer; m, medium (collagen); I, inner. (D-F) The fate and distribution of DsRed-expressing E. coli (red) after infection of muscle with 3000 CFU (D) or notochord with 2000 CFU (E) and 3000 CFU (F). Each larva was followed and imaged at 3 (top row) and 24 (bottom row) hpi. Arrowheads indicate clusters of bacteria. (G-I) Transmission electron microscopy analysis of the notochord of larvae that had been infected in the notochord (11 hpi); H,I show magnifications of regions of G. (G) Numerous bacteria (bracket) are found between the peri-notochordal basement membrane (pbm, arrowheads) and notochord cells (nc). (H) Collagen (col) disorganisation is observed next to bacteria. (I) Extracellularly replicating bacterium (arrowhead). (J) Survival was scored of uninfected larvae and those that were infected in the muscle or notochord (n>24 each). These results are representative of three independent experiments, P<0.0005 for notochord injection versus uninjected. (K) Whole embryo bacterial counts of E. coli when injected into the muscle or notochord. Results are expressed as the mean number of CFU per larvae±s.e.m. (n>5 larvae per timepoint). NS: not significant. Scale bars: 5 μm (B,G); 1 μm (C,H,I).