Pattern recognition receptors and effector mechanisms of the innate immune system. The localization of Tlrs on the cell surface or on endosomes is hypothetical and based on the known or proposed functions of their homologs in other fish or mammals. The ability of PRRs (depicted in green) to recognize PAMPs present on various types of microorganisms, like bacteria, viruses, and fungi, has been simplified here by depicting microorganisms as rod-like bacteria (in blue). PAMP recognition by PRRs leads to activation of transcription factors (TFs), which translocate to the nucleus and initiate transcription of cytokine genes, antimicrobial genes, and other immune-related genes. Defense mechanisms such as autophagy, ROS and NO production, and degranulation can be immediately activated upon microbial recognition, without de novo gene transcription.

In situ detection of autophagy by Lc3 accumulation. CMV::LC3-GFP transgenic [15] zebrafish embryos (28 hpf) were injected into the caudal vein with 200 colony-forming units (CFU) of M. marinum Mma20 expressing a pMST3::mCherry vector. Confocal images were taken of a tail region of the developing larva at 3 days after infection (3 dpi), a point at which the M. marinum infection (a) has been established. Low levels of Lc3-GFP signal (b) can be observed throughout the cells, whilst brighter regions (indicated by arrowheads) are only observed upon Lc3 accumulation and formation of autophagic membranes associated with bacteria (c). Scale bar: 10 μm.

In situ detection of reactive nitrogen species. Wild-type zebrafish embryos (Albino; 28 hpf) were injected into the caudal vein with 200 colony-forming units (CFU) of M. marinum Mma20 expressing a pMST3::mCherry vector. Confocal images were taken of a tail region of the developing larva at 3 days after infection (3 dpi), a point at which the M. marinum infection (a) has been established. Embryos were fixed in 4% paraformaldehyde at 3 dpi, and immunohistochemistry was performed, using an antinitrotyrosine antibody that detects tissue nitration (b) [21]. Colocalization (c) between bacteria and extensive tissue nitration can be observed at this time point. Scale bar: 10 μm.

Comparison of the zebrafish innate immune response to different bacterial pathogens. Gene expression profiles of zebrafish embryos and adults infected with E. tarda FL6-60 (Et), S. typhimuriumSL1027(St), and M. marinum Mma20 (Mm) are depicted in a heat map. Embryos were infected with 200 CFU of each pathogen into the caudal vein at 28 hpf and snap frozen individually at 8 hpi for E.  tarda and S. typhimurium, and at 8 hpi and 4 dpi for M. marinum. Triplicate samples for each infection condition were compared with samples from control embryos (injected with PBS) using a common reference microarray design. The raw data were deposited in the Gene Expression Omnibus database under accession number GSE35474. The data derived from embryonic infections were compared with data from a study in which adult zebrafish were infected intraperitoneally with M. marinum Mma20, after which RNA samples were taken at 1 dpi and 6 dpi [22]. The dose of the Mma20 strain used in the adult infection study was lethal within days after the final sampling point at 6 dpi. Only genes relevant to this paper were included in the heatmap. All selected genes are represented by a minimum of two probes that showed significant up or downregulation (significance cut-offs for the ratios of infected versus control groups were set at 2-fold with P < 10⁻⁵). Upregulation is indicated by increasingly bright shades of yellow, and downregulation is indicated by increasingly bright shades of blue. It should be noted that the genes listed in this figure are named according to sequence homology with mammalian counterparts and in most cases have not yet been confirmed functionally.