Infection of Zebrafish Embryos with Intracellular Bacterial Pathogens

Zebrafish (Danio rerio) embryos are increasingly used as a model for studying the function of the vertebrate innate immune system in host-pathogen interactions 1. The major cell types of the innate immune system, macrophages and neutrophils, develop during the first days of embryogenesis prior to the maturation of lymphocytes that are required for adaptive immune responses. The ease of obtaining large numbers of embryos, their accessibility due to external development, the optical transparency of embryonic and larval stages, a wide range of genetic tools, extensive mutant resources and collections of transgenic reporter lines, all add to the versatility of the zebrafish model. Salmonella enterica serovar Typhimurium (S. typhimurium) and Mycobacterium marinum can reside intracellularly in macrophages and are frequently used to study host-pathogen interactions in zebrafish embryos. The infection processes of these two bacterial pathogens are interesting to compare because S. typhimurium infection is acute and lethal within one day, whereas M. marinum infection is chronic and can be imaged up to the larval stage 2, 3. The site of micro-injection of bacteria into the embryo (Figure 1) determines whether the infection will rapidly become systemic or will initially remain localized. A rapid systemic infection can be established by micro-injecting bacteria directly into the blood circulation via the caudal vein at the posterior blood island or via the Duct of Cuvier, a wide circulation channel on the yolk sac connecting the heart to the trunk vasculature. At 1 dpf, when embryos at this stage have phagocytically active macrophages but neutrophils have not yet matured, injecting into the blood island is preferred. For injections at 2-3 dpf, when embryos also have developed functional (myeloperoxidase-producing) neutrophils, the Duct of Cuvier is preferred as the injection site. To study directed migration of myeloid cells towards local infections, bacteria can be injected into the tail muscle, otic vesicle, or hindbrain ventricle 4-6. In addition, the notochord, a structure that appears to be normally inaccessible to myeloid cells, is highly susceptible to local infection 7. A useful alternative for high-throughput applications is the injection of bacteria into the yolk of embryos within the first hours after fertilization 8. Combining fluorescent bacteria and transgenic zebrafish lines with fluorescent macrophages or neutrophils creates ideal circumstances for multi-color imaging of host-pathogen interactions. This video article will describe detailed protocols for intravenous and local infection of zebrafish embryos with S. typhimurium or M. marinum bacteria and for subsequent fluorescence imaging of the interaction with cells of the innate immune system.

. The site of micro-injection of bacteria into the embryo (Figure 1) determines whether the infection will rapidly become systemic or will initially remain localized. A rapid systemic infection can be established by micro-injecting bacteria directly into the blood circulation via the caudal vein at the posterior blood island or via the Duct of Cuvier, a wide circulation channel on the yolk sac connecting the heart to the trunk vasculature. At 1 dpf, when embryos at this stage have phagocytically active macrophages but neutrophils have not yet matured, injecting into the blood island is preferred. For injections at 2-3 dpf, when embryos also have developed functional (myeloperoxidase-producing) neutrophils, the Duct of Cuvier is preferred as the injection site. To study directed migration of myeloid cells towards local infections, bacteria can be injected into the tail muscle, otic vesicle, or hindbrain ventricle [4][5][6] . In addition, the notochord, a structure that appears to be normally inaccessible to myeloid cells, is highly susceptible to local infection 7 . A useful alternative for high-throughput applications is the injection of bacteria into the yolk of embryos within the first hours after fertilization 8 . Combining fluorescent bacteria and transgenic zebrafish lines with fluorescent macrophages or neutrophils creates ideal circumstances for multi-color imaging of host-pathogen interactions. This video article will describe detailed protocols for intravenous and local infection of zebrafish embryos with S. typhimurium or M. marinum bacteria and for subsequent fluorescence imaging of the interaction with cells of the innate immune system.

Video Link
The video component of this article can be found at http://www.jove.com/video/3781/ Protocol 1. Prepare Injection Needles 1. Prepare borosilicate glass microcapillary injection needles (Harvard Apparatus, 300038, 1 mm O.D. × 0.78 mm I.D.) using a micropipette puller device (Sutter Instruments Inc., Flaming/Brown p-97 with the following settings for a longer tip: air pressure 400; heat 610; pull 40; velocity 50; time 30 and with the following settings for a shorter tip: air pressure 500; heat 510; pull 100; velocity 200; time 60). 2. Break off the needle tip with fine tweezers to obtain a tip opening diameter of 5-10 μm. It is advisable to bevel the needle tip opening at a 45 degrees angle with a microgrinder (Narishige Inc., EG-400) to yield a sharper tip. This will facilitate puncturing the epidermal layer and thus result in more reproducible injections with less tissue damage than with blunt needles. Longer tipped needles are preferred for caudal vein (step 5) and Duct of Cuvier (step 6.1) injections and shorter tipped needles are preferred for the other injection protocols (steps 6.2-6.6).

Prepare Zebrafish Embryos for Injections
1. Set up zebrafish breeding pairs and collect embryos as shown in another video article 10 . Keep embryos in a Petri dish filled with egg water (60 μg/mL Sea salts; ca. 60 embryos/dish) and incubate at 28.5 °C. 2. If required, add 0.003% N-Phenylthiourea (PTU, Sigma-Aldrich) to the egg water when the embryos are approximately 12 hpf to prevent melanization. 3. Around 24 hours post fertilization (hpf), dechorionate the embryos with fine tweezers (Fine Science Tools Inc., Dumont #5 Forceps -Inox Biology. 4. Keep the embryos in a Petri dish filled with egg water and with a layer of 1% agarose on the bottom to prevent embryos from sticking to the plastic surface. 5. Anesthetize the embryos with 200 μg/mL buffered 3-aminobenzoic acid (Tricaine, Sigma-Aldrich) approximately 10 min prior to injections.

Representative Results
Injection of Salmonella typhimurium or Mycobacterium marinum bacteria into the blood island of embryos at 1 dpf results in the rapid phagocytosis by macrophages. The mpeg1 gene has recently been identified as a faithful marker of embryonic macrophages, colocalizing with the well-established macrophage marker csf1r (fms) and not overlapping with neutrophil markers such as mpx (mpo) and lyz 4,14 . For live imaging of bacterial phagocytosis we used transgenic lines in which the mpeg1 promoter drives fluorescent protein expression in macrophages 14 . These transgenic lines either have the mpeg1 promoter fused directly to the gfp gene, or employ a two-component system where the mpeg1 promoter drives expression of the yeast Gal4 transcription factor that activates a second transgene with the Gal4 recognition sequence (UAS, upstream activating sequence) fused to the kaede gene. When the blood island injection (protocol 5; Figure 1A) is performed correctly, the bacteria will immediately flow through the blood circulation and spread throughout the embryo. Dissemination of the relatively large and brightly  (Figure 3A), and confocal imaging at 2 hpi shows that many bacteria are phagocytosed by fluorescent macrophages (Figure 3B-C). The injection of as little as 25 cfu of wild type S. typhimurium bacteria will result in a lethal infection, while a similar dose of bacteria of an avirulent strain, such as Ra, can be cleared by the embryonic immune system 3 . An injection dose of 250 cfu was used to determine transcriptional responses to S. typhimurium infection in the zebrafish embryo and demonstrated the induction of a strong pro-inflammatory gene expression response 15 . In contrast, the intravenous injection of M. marinum bacteria does not elicit a strong pro-inflammatory response, but leads to a persistent infection where infected macrophages form tight aggregates that are considered as the initial stages of granulomas, which are the hallmark of tuberculosis 2 . Confocal imaging of such a granuloma-like aggregate in the Tg(mpeg1:EGFP)gl22 line 14 at 5 dpi shows the intracellular growth of mCherry-labeled M.
Other routes of infection are useful for different purposes. Bacteria can be injected into the hindbrain ventricle at 32 hpf (Figure 1C), which is a compartment devoid of macrophages. Injection of 20-100 mCherry-labeled M. marinum bacteria into this compartment leads to the rapid infiltration by macrophages that phagocytose the bacteria (Figure 4A). Another method to study the directed migration of innate immune cells is injection of bacteria into the tail muscle ( Figure 1D). However, tail muscle injections also cause tissue damage that by itself elicits some attraction of leukocytes. Such a wounding response can be avoided when carefully injecting a small volume (0.5-1 nL) into the otic vesicle ( Figure 1E). As shown here by using the Tg(mpx:EGFP)i114 line 16 , injection of approximately 20 cfu of S. typhimurium into the otic vesicle leads to the attraction of neutrophils at 3 hpi (Figure 4D), while this response is not observed in PBS control injections (Figure 4E). The notochord, which appears to be resistant to infiltration by leukocytes, is a permissive compartment for the growth of M. marinum mutants that are strongly attenuated when injected in other tissues 7 ; (Figure 4F). Finally, early injection of M. marinum into the yolk of embryos provides an alternative method to achieve a systemic infection. We generally perform these injections around the 16 cell stage (Figure 1G), but bacterial injections into the yolk can also be performed at later stages (up to the 1000 cell stage), or earlier (1 to 8 cell stage) for co-injections with morpholinos 8 .
Following yolk injection of a dose of 20-40 cfu, M. marinum bacteria spread over several days into the embryonic tissues and form granulomalike aggregates similar to those observed upon the conventional intravenous injection method 8 ; (Figure 4G). The yolk injection method is not suitable for S. typhimurium infection, because its rapid growth in the yolk causes early lethality. The yolk infection method for M. marinum infection will be useful for high-throughput applications since it can be automated using an injection robot 8 .

Discussion
The infection methods described in this article are frequently used to study the function of host innate immunity genes or bacterial virulence genes 1 . The intravenous micro-injection methods (protocols 5 and 6.1) are used most often for such studies. The caudal vein at the posterior blood island is the most convenient location for intravenous injection of 1-day-old embryos. As the caudal vein becomes more difficult to penetrate at later stages, we prefer the Duct of Cuvier as intravenous injection site for embryos at 2-3 dpf. In all cases it is critical to inject the embryos with consistent numbers of bacteria, which should be checked by plating injection inocula for cfu counting. The following aspects must be considered to achieve reproducible injections. Firstly, it is essential to have high-quality glass microcapillary injection needles. If the needle tip is too large, the injection will create a puncture hole large enough for bleeding to occur and the injected bacteria will flow out of the blood circulation. Secondly, bacteria must be injected directly into the blood circulation. If the needle tip is not inside the vein or if injection fluid spreads into the yolk sac extension, the embryo must be discarded from the experiment. Thirdly, using PVP40 as a carrier for injecting M. marinum will assist in preventing the bacteria from sinking in the glass needle. PVP40 improves the homogeneity of the suspension, resulting in more reproducible inocula throughout the duration of injections. PVP40 may also be used for S. typhimurium injections, but here it is less important because the larger size and bright fluorescence of the bacteria allows a visual control over the injection inoculum during experiments. For practising the injections we recommend the use of phenol red dye (1% v/v) or 1 μm fluorescent spheres available in different colors (Invitrogen). Once the technique is mastered, it takes approximately 30 minutes to inject 50-100 embryos, including the time required for aligning the embryos on agarose plates and adjusting the bacterial injection volume.
While intravenous injections into the blood island (protocol 5) or into the duct of Cuvier (protocol 6.1) are most commonly used, the alternative routes of infection described in this video article have proved useful to study macrophage and neutrophil chemotaxis (protocols 6.2, 6.3, and 6.4), to study growth of attenuated bacterial strains (protocol 6.5), and to adapt the zebrafish infection model for high-throughput applications (protocol 6.6) [4][5][6][7][8] . The described micro-injection methods can also be applied for injections of viruses 18,19 , fungal spores 14,20 or protozoan parasites (Maria Forlenza, personal communication). A useful addition to the injection procedures described in this video article is a recently described procedure for subcutaneous injection of bacteria into zebrafish embryos 21 . This procedure was applied to study phagocytosis of bacteria by neutrophils, which were found to efficiently engulf bacteria on tissue surfaces but, in contrast to macrophages, were virtually unable to phagocytose bacteria upon injection into the blood or into fluid-filled body cavities. For subcutaneous injections embryos are positioned similar as for the tail muscle injections described in this video article, but the needle is inserted just under the skin to inject the bacteria over a somite.
It is important to consider that micro-injection is essentially an artificial route of infection. However, early embryos are highly resistant to external exposure to bacterial pathogens. In fact, immersion assays, where embryos are bathed in a bacterial suspension, are not reproducible in our hands 22 . While some embryos may become infected upon immersion, we have observed a large variation in mortality rates and in the expression of inflammatory marker genes between individual embryos in such assays. The micro-injection methods described in this article achieve reproducible infections and are particularly useful to study bacterial interactions with host innate immune cells. An efficient approach to quantify bacterial infection in individual embryos is to analyze the fluorescent images of infected embryos with custom-made, dedicated pixel quantification software 17,23 . This approach has been shown to correlate well with results of cfu counts after plating of infected embryos. Similarly, relative changes in leukocyte numbers per embryo have been quantified by digital image analysis in transgenic leukocyte fluorophore reporter lines; this approach would be applicable to quantifying the host leukopoietic response to infection 24 .
The function of host innate immune genes can be investigated efficiently in vivo by combining the described infection models with morpholino knockdown. Morpholinos are synthetic antisense oligonucleotides that target specific RNAs and reduce the expression of the gene products when injected into 1-cell stage zebrafish embryos as shown in other video articles in this journal 10,25 . In morpholino knockdown studies, it is of great importance that all embryo groups to be compared are staged correctly prior to bacterial injections. This is especially important because embryos have a developing immune system that becomes increasingly competent over time.
There are several transgenic reporter lines currently available to distinguish the major innate immune subsets, macrophages and neutrophils, in zebrafish embryos. The mpx and lyz promoters have been used to generate neutrophil reporter lines 16,26,27 , and the csf1r (fms) and mpeg1 promoters were used to produce macrophage reporters 14,28 . While csf1r (fms) is additionally expressed in xanthophores, mpeg1 expression is exclusively in macrophages. As shown in this video article, the recently developed mpeg1 reporter lines are highly useful for imaging bacterial phagocytosis by macrophages.

Disclosures
The authors have nothing to disclose.