The zebrafish lysozyme C promoter drives myeloid-specific expression in transgenic fish
© Hall et al; licensee BioMed Central Ltd. 2007
Received: 08 November 2006
Accepted: 04 May 2007
Published: 04 May 2007
How different immune cell compartments contribute to a successful immune response is central to fully understanding the mechanisms behind normal processes such as tissue repair and the pathology of inflammatory diseases. However, the ability to observe and characterize such interactions, in real-time, within a living vertebrate has proved elusive. Recently, the zebrafish has been exploited to model aspects of human disease and to study specific immune cell compartments using fluorescent reporter transgenic lines. A number of blood-specific lines have provided a means to exploit the exquisite optical clarity that this vertebrate system offers and provide a level of insight into dynamic inflammatory processes previously unavailable.
We used regulatory regions of the zebrafish lysozyme C (lysC) gene to drive enhanced green fluorescent protein (EGFP) and DsRED2 expression in a manner that completely recapitulated the endogenous expression profile of lysC. Labeled cells were shown by co-expression studies and FACS analysis to represent a subset of macrophages and likely also granulocytes. Functional assays within transgenic larvae proved that these marked cells possess hallmark traits of myelomonocytic cells, including the ability to migrate to inflammatory sources and phagocytose bacteria.
These reporter lines will have utility in dissecting the genetic determinants of commitment to the myeloid lineage and in further defining how lysozyme-expressing cells participate during inflammation.
A major challenge faced by researchers in various fields of immunology is understanding the cellular interactions that govern a successful immunological response. A typical vertebrate immune response depends upon the highly orchestrated migration and motility of various hematopoietic compartments and their subsequent interactions that ultimately control the magnitude of the response [1, 2]. Current techniques used to study the cellular dynamics of such processes include intravital microscopy and tissue explant assays [3, 4]. Although extremely informative, both methods rely upon invasive surgical procedures and do not represent a true whole animal setting. The zebrafish is emerging as an extremely promising model system to study specific aspects of disease mechanisms [5–11]. Not only is the early zebrafish embryo optically clear, it is also highly amenable to genetic manipulation and small chemical screens [11, 12]. These attributes have been exploited to study aspects of immune system development [13–16].
Zebrafish, like mammals experience two separate waves of hematopoiesis. The first 'primitive wave' generates embryonic erythrocytes as well as early myeloid-derived macrophages and neutrophils from two distinct intraembryonic compartments, the caudally-located intermediate cell mass (ICM) and a rostral blood-forming region derived from cephalic mesoderm [17–19]. Whether these early myeloid cell populations are specifically embryonic or persist to contribute to more adult immune cell populations is unclear. The second, 'definitive' wave, believed to initiate within the ventral wall of the dorsal aorta eventually gives rise to all adult blood lineages, including erythrocytes, macrophages, several types of granulocytes, lymphocytes and thrombocytes [18, 20, 21]. Zebrafish also possess a B and T cell repertoire as part of a functional adaptive immune system; a rag-dependent system that emerged with the evolution of the jawed vertebrates [14, 22].
Macrophages represent the main differentiated cell of the monocyte phagocyte system and an essential primary innate barrier to infection [23, 24]. Their primary function is phagocytosis and they are widely distributed throughout the organism, displaying variations in behavior and morphology . Despite these variations, most macrophages can be defined by the expression of certain enzymes, such as lysozyme and peroxidase . In addition to phagocytosis of infectious materials, macrophages also function during host tissue repair, regulation of inflammatory events (through secretion of cytokines), regulation of immune responses (through antigen presentation to lymphocytes) and are believed to influence certain aspects of neovascularization [23, 25, 26].
In zebrafish, embryonic macrophage precursors originate from the rostral blood compartment and derive from lateral plate mesoderm . During early somitogenesis, anterior lateral plate mesoderm converges to the midline and eventually lies just beneath the paraxial mesoderm. From this region, macrophage precursors expressing the early myeloid marker pu.1 migrate to initially populate the anterior yolk surface and then more posterior yolk regions [17, 27–31]. The transcription factor pu.1 has been demonstrated, by whole mount in situ hybridization, to mark the myeloid lineage up to 30 hours post fertilization (hpf) [17, 32]. Once on the yolk, these primitive macrophages begin to express genes encoding the actin-binding protein L-plastin and lysozyme C [17, 28, 29, 33]. Upon initiation of embryonic circulation, a subset of macrophages enter the circulation and are distributed throughout the embryonic tissues. This is in contrast to neutrophils, where appearance on the yolk is preceded by that within the posterior ICM, as revealed by expression of the myeloperoxidase (mpo) [17, 19], suggesting a degree of anatomical separation during embryonic monocytopoiesis and granulopoiesis.
To generate a macrophage reporter line, and facilitate direct observation of macrophage events in situ in real-time, we have generated novel transgenic lines in which the zebrafish lysC promoter was used to drive macrophage expression of EGFP and DsRED2. Lysozyme is a cationic antibacterial enzyme capable of hydrolyzing specific linkages within the bacterial cell wall . In humans, lysozyme is synthesized within both granulocytic and monocytic cells [34, 35]. Regulatory regions of the mouse lysozyme M gene, one of two lysozyme genes found in mice , have been demonstrated to be sufficient to drive myelomonocytic-specific expression of the EGFP fluorescent reporter [37, 38]. In zebrafish, lysC expression has been reported to specifically mark the macrophage compartment, based upon initial expression within cells on the yolk and co-localised expression with L-plastin . We used the highly efficient Tol2 transposon system to generate germline lysC::EGFP and lysC::DsRED2 transgenic founders . Approximately 6.35 kb of promoter sequence was sufficient to drive reporter expression in a manner that recapitulated the endogenous expression profile of lysC and was consistent with the known ontogeny of the macrophage lineage. Inflammation, bacterial infection and phagocytic assays demonstrated that these labeled cells possessed hallmark traits of the macrophage lineage while time-lapse confocal microscopy revealed their highly dynamic morphology, proliferative potential and ability to roll along vascular endothelia. Within transgenic embryos, labeled cells expressed markers of the macrophage lineage, including L-plastin. Some fluorescent cells also contained transcripts for myeloperoxidase (mpo), raising questions regarding the macrophage-specificity of lysC previously reported , but consistent with the presence of lysozyme within both macrophages and granulocytes of mammals. In summary, we present the lysC::EGFP and lysC::DsRED2 reporter lines as having marked myelomonocytic compartments that will prove useful in defining the determinants of lineage commitment during myelopoiesis and in examining the contribution of innate immune cells during inflammatory events.
Results and Discussion
Strategy to generate a zebrafish lysozyme Creporter line
The lysC promoter region was selected for generation of a reporter line, as expression of the lysC gene had been described as being specific to the zebrafish macrophage lineage . To facilitate comparison with the lysC transgenic reporter lines generated in this work, we have included a detailed expression analysis of lysC during embryonic/larval development that extends previous studies (see Additional file 1: lysC expression during early embryonic/larval development). A zebrafish genomic BAC clone was initially identified in silico as containing sequence of the Ensembl-predicted lysC gene. This BAC clone (I.D. zC250A24) was confirmed via amplification and sequence verification as containing the first coding exon of lysC. Fragments corresponding to sequence upstream of the initiation codon were cloned from this BAC (for details see Methods section) into EGFP- and DsRED2-encoding pT2KXIGΔin Tol2-containing vectors . This generated the pT2K/lysC::EGFP and pT2K/lysC::DsRED2 expression vectors. To verify that these constructs could drive expression of the fluorescent reporters to simulate endogenous lysC, we injected 1-cell stage zebrafish embryos with 30 pg of either construct (injection of higher doses resulted in toxicity). EGFP and DsRED2 expression was first observed exclusively on the yolk surface with EGFP first detectable around 24 hpf and DsRED2 approximately 4 hours later. This is most likely due to the longer time required for proper folding of the DsRED2 tetrameric protein (data not shown). A subset of these cells entered the circulation and could be seen moving over the yolk ventrally towards the embryonic heart. This expression pattern is consistent with the endogenous expression pattern of lysC, as detected by whole mount in situ hybridization as shown here and by others  and the ontogeny of early macrophage development .
These constructs were then injected into 1-cell stage embryos along with capped transposase transcript to induce transposition and generate germline transgenic founders. Six germline transgenic founders were identified for both the lysC::EGFP and lysC::DsRED2 lines. Both lines displayed correct Mendelian inheritance ratios in subsequent generations and identical reporter expression profiles of comparable intensities (data not shown).
Next we describe, in detail, reporter expression within stable lysC::EGFP transgenic animals. It is of note that the lysC::DsRED2 line possessed an identical expression profile throughout development (see Additional file 2: Co-localized expression of EGFP and DsRED2 within lysC::EGFP/lysC::DsRED2 compound transgenic larvae), albeit commencing slightly later.
EGFP expression within lysC::EGFP transgenic embryos recapitulates the endogenous expression profile of lysCthat traces the early development of the macrophage lineage
In addition to the domains described above, EGFP-labeled cells were observed within the developing brain and retina (see Additional file 5: Labeled cells are located within the developing brain and retina). However, no colonization of the optic tectum by EGFP-expressing cells was seen within the lysC::EGFP line throughout embryonic/larval development. Previous studies have demonstrated specific colonization of the optic tectum by L-plastin-expressing macrophages . Analysis of our lysC::EGFP reporter line and the expression of lysC (see Additional file 1, lysC expression during early embryonic/larval development) suggest that these cells that mark the optic tectum represent a subset of macrophages that are lysozyme C-deficient. It is of note that within lysC::EGFP animals, fewer labeled cells were detected within the developing head when compared with those revealed by L-plastin and fms expression .
EGFP-expressing cells within lysC::EGFPadult fish mark the head kidney and display myelomonocytic morphology
EGFP-expressing cells are of the myeloid lineage
To confirm that these EGFP-expressing cells within the lysC::EGFP line were derived from hematopoietic progenitors, and more specifically were of myeloid descent, transgenic embryos were depleted of Scl and Gata1 by morpholino injection. Depletion of Scl, a transcription factor absolutely required in mammals for both primitive and definitive hematopoiesis [45–47], resulted in an almost complete ablation of EGFP-expressing cells (see Additional file 6: Labeled cells are restricted to the myeloid lineage). This was in contrast to Gata1-depleted embryos which demonstrated marked expansion of the EGFP-marked compartment (see Additional file 6: Labeled cells are restricted to the myeloid lineage) consistent with previous studies in which Gata1-deficient embryos possess an expanded myeloid and diminished erythroid compartment .
Recently a medaka transgenic line has been described in which a fugu mpo promoter fragment is reported to drive macrophage-specific expression  illustrating an interesting difference with zebrafish where the same gene is believed to be largely neutrophil-specific . It seems that within the embryonic myeloid compartment of zebrafish, myeloid-restricted genes are subject to dynamic temporal regulation. The expression profile of the labeled cells within lysC::EGFP animals described here, in concert with their initial appearance on the yolk surface suggests that populations of macrophages and also some neutrophils are fluorescently marked.
Labeled cells within transgenic larvae demonstrate a robust response to inflammation and bacterial infection
During bacterial infections macrophages function to clear invading pathogens and also act as key modulators of both the innate and adaptive immune systems . In zebrafish, macrophages exhibit a robust response to infection [27, 57, 59]. To examine the response of the marked cell population within the lysC reporter lines, we infected lysC::DsRED2 zebrafish larvae with GFP-expressing Salmonella enterica serovar Typhimurium. Following infection, a robust response was detected within the posterior extremities of the intestine (see Additional file 11: Infection with GFP-expressing Salmonella results in a robust inflammatory response within the posterior intestine). Labeled cells within this region were detected containing fluorescent bacteria (Fig. 7D) confirming their contribution to the bacterial inflammatory response.
Together, these results demonstrate that the lysC::EGFP/DsRED2 reporter lines mark populations of macrophages and likely also neutrophils. These lines should prove valuable in further dissecting the genetic determinants of lineage commitment during myelopoiesis. They will also have utility in studying the relative contributions to innate immune responses of the leukocyte compartments.
Zebrafish (Danio rerio) embryos were obtained from natural spawning between lysC::EGFP/DsRED2, fli1::EGFP , I-FABP::RFP  and wild type (obtained from the Zebrafish International Resource Center) adult fish. Embryos were raised at 28°C in Embryo Medium (E3)  and developmentally staged as described . Research was conducted with approval from The University of Auckland Animal Ethics Committee (AEC/04/2005/R370).
Whole mount in situ hybridization and immunohistological analysis
Whole mount in situ hybridizations were performed essentially as described . Pigmentation in embryos older than 24 hpf was inhibited either using PTU (I-phenyl-2-thiourea; Sigma) as described  or by bleaching in a 0.5 × SSC, 5% formamide, 10% H2O2 solution. For experiments where EGFP was detected by immunohistochemistry following whole mount in situ hybridization, the following modifications were used. Following Fast Red (Roche) staining, larvae were washed several times in PBS then transferred to blocking solution before binding of a rabbit anti-GFP-Alexa488 conjugated antibody (Molecular Probes) diluted 1:200 in blocking solution for 4 hours at room temperature. Larvae were then washed several times in PBS, fixed in 4% (w/v) PFA in PBS, mounted in 1% (w/v) low melting point agarose (Sigma) and images captured using a TCS SP2 confocal laser scanning microscope (Leica) through FITC (for Alexa488) and TRITC (for Fast Red stain) channels.
Cloning of zebrafish lysCpromoter and construction of pT2K/lysC::EGFP/DsRED2 constructs
A lysC-containing BAC clone (zC250A24) obtained from the RZPD (German Resource Center for Genome Research) was digested with XhoI and EcoRI which cut approximately 11,040 bp and 330 bp from the lysC initiation codon, respectively. This generated a 10.7 kb genomic fragment encompassing regulatory elements of the lysC gene starting 330 bp from the initiation codon. The remaining 3' sequence was generated by amplification using the following primer set: LysPA, 5'-CACCGGTGTCACTAATAACACGATCG-3' (which corresponded to sequence starting 30 nucleotides upstream of the EcoRI site); LysPB, 5'-TATACCCGGGAGGTGTATGGTGGAG-3' (which corresponded to sequence starting 39 nucleotides upstream of the initiation codon and contained a SmaI restriction site). This 330 bp PCR fragment was then cloned into the pGEM-T Easy Vector (Promega), sequence verified then digested with EcoRI and SmaI. This restriction fragment was cloned, together with the 10.7 kb XhoI/EcoRI fragment, into XhoI/SmaI-linearized pBS/KS to generate 11kblysCP/pBSKS. The 11 kb lysC promoter fragment was then liberated from 11kblysCP/pBSKS using XhoI and SmaI and ligated into XhoI/BamHI(blunted)-linearized pT2KXIGΔin to generate pT2K/lysC::EGFP. The pT2K/lysC::DsRED2 construct was generated by first cloning the XhoI/SmaI-linearized 11 kb lysC promoter fragment from 11kblysCP/pBSKS together with a BamHI (blunted)/KpnI DsRED2-encoding construct into XhoI/KpnI-linearized pBS/KS to create 11kblysCP/DsRED2/pBSKS. This construct was then digested with SacII and XhoI to release 6.35 kb of lysC promoter sequence upstream of DsRED2 which was cloned into SacII/XhoI-linearized pCS2+. Subsequent digestion with ApaI/ClaI facilitated the cloning of this 6.35 kb lysC promoter/DsRED2-encoding fragment into similarly linearized pT2KXIGΔin.
To confirm the efficiency with which the cloned lysC::EGFP/DsRED2 constructs could drive EGFP/DsRED2 expression and recapitulate the endogenous expression pattern of lysC, the transient expression profile of injected embryos was examined under fluorescence microscopy.
Generation of transgenic lysC::EGFP/DsRED2lines
Capped transposase transcript was generated using the mMESSAGE mMACHINE SP6 kit (Ambion Inc.) from the pCS-TP expression vector as previously described . Zebrafish embryos were microinjected with the p2TK/lysC::EGFP/DsRED2 (30 pg/embryo) construct combined with transposase transcript (50 pg/embryo) at the 1-cell stage of development. These potential founders were then screened for reporter expression under fluorescence microscopy following 2 days of development. Only fluorescent embryos were selected to form the founder population. Once at sexual maturity, founders were intercrossed and F1 progeny screened for reporter expression. Pairs that generated a positive clutch were then individually outcrossed to wild type animals to identify the germline transgenic founder.
Images of whole transgenic embryos/larvae were generated using a DC200 digital camera and supporting software (Leica) connected to an MZFLIII fluorescence stereomicroscope equipped with GFP and DsRED filter sets (Leica).
Microangiography was performed as described . In brief, red fluorescent microspheres of 0.02 μm in diameter (Molecular Probes) were diluted 1:1 with a 2% BSA (Sigma) solution then sonicated. LysC::EGFP larvae to be injected were anaesthetized in tricaine as described  before being mounted in 1% (w/v) low melting point agarose (Sigma) in E3 medium ventral side up. The microsphere suspension was then injected either into the sinus venosus (for 2 dpf larvae) or directly into the heart (for 6 dpf larvae) using a FemtoJet pressure injection system (Eppendorf). The success of injection was monitored under an MZFLIII stereo microscope equipped with a DsRED filter set (Leica).
FACS analysis, sorting and cytospin preparations
Cell collection, FACS analysis and sorting was performed essentially as described . In brief, lysC::EGFP adult fish were anaesthetized in tricaine before dissection of kidneys which were dissociated by teasing through a 40 μm cell strainer (BD Falcon) in ice-cold 0.9 × PBS supplemented with 5% FBS. Cells were washed several times and resuspended in 0.9 × PBS/5% FBS before addition of propidium iodide to a final concentration of 1 μg/ml for exclusion of dead cells. FACS analysis was based on forward and side scatter characteristics, propidium iodide exclusion and GFP fluorescence using a FACS Vantage flow cytometer (Beckton Dickenson). Cytospin preparations were made using a Cytofuge 2 cytocentrifuge (StatSpin) followed by Leishman's staining for morphological analysis.
Adult animals were anaesthetized in 4% paraformaldehyde, decalcified in 0.5 M EDTA (pH 7.8) for several days, dehydrated through an ethanol series, cleared in xylol overnight before infiltration and embedding in paraffin. Five-micrometer tissue sections were deparaffinized, rehydrated and immunohistochemical detection of EGFP performed as previously described  using a rabbit anti-GFP (Torrey Pines Biolabs Inc.) as a primary antibody and a HRP-conjugated anti-rabbit antibody (Sigma) as a secondary. Sections were counter stained with eosin.
Injection of morpholino oligonuleotides
Morpholino oligonucleotides (Gene Tools, Philomath, OR) used in this study and their sequence are as follows: gata1ATGMO, 5'-CTGCAAGTGTAGTATTGAAGATGTC-3'  and sclspliceMO, 5'-AATGCTCTTACCATCGTTGATTTCA-3' . MOs were injected essentially as described . Effective doses for each morpholino were as described [48, 68, 69].
The inflammation assay was conducted essentially as described . In brief, 7 dpf lysC::EGFP larvae were anaesthetized in E3 medium supplemented with tricaine before the posterior portion of the developing caudal fin was transected with a sterile scalpel. Larvae were then left to recover in E3 medium before analysis under fluorescence microscopy using an MZFLIII stereo microscope equipped with a GFP filter set (Leica) during the progression of inflammation. The high resolution inflammation assay was performed by making a small incision in the ventral fin of anaesthetized 6 dpf lysC::DsRED2/fli1::EGFP compound transgenic larvae using a sterile scalpel followed by mounting in 1% low melting point agarose (Sigma) in E3 medium and time-lapse confocal microscopy using an Olympus FV1000 confocal microscope equipped with a heated chamber which was kept at 29°C and water-immersion lenses. Z-series were collected at 1 minute intervals and were no greater than 4 μm between each section as to ensure detection of all DsRED2-labeled cells. Projections of summed Z stacks, time-lapse animations and cell tracking were generated using ImageJ .
Red fluorescent microspheres of 2 μm diameter (Molecular Probes) were selected to monitor the phagocytic ability of EGFP-expressing cells within 4 dpf lysC::EGFP larvae. Larvae to be injected were anaesthetized in tricaine then mounted in 1% (w/v) low melting point agarose (Sigma) in E3 medium. The microspheres were diluted 3:1 in sterile distilled water then microinjected into the embryonic tissue of 4 dpf transgenic larvae using a FemtoJet pressure injection system (Eppendorf). Injections were typically 0.5 μl in volume and targeted just posterior to the swim bladder within the somitic tissues. The success of injections was confirmed through fluorescence microscopy using an MZFLIII stereo fluorescence microscope equipped with a DsRED filter set (Leica). Phagocytosis of the individual microspheres was detected using a TCS SP2 confocal laser-scanning microscope (Leica) using GFP and TRITC channels. Projections of summed Z stacks and time-lapse animations were generated using ImageJ .
Bacterial infection assay
GFP-labeled Salmonella enterica serovar Typhimurium were used to infect zebrafish. To label Salmonella, the strain SM022  was recreated by P22 transduction  of the rpsM::gfp fusion and linked kanamycin resistance genes from SM022 into the original parental Salmonella strain SL1344 . Five dpf lysC::DsRED2 larvae (raised in E3 medium supplemented with PTU to inhibit pigmentation, as described ) were immersed in 2.93 × 109/ml CFU GFP-labeled Salmonella strain SL1344. Following a 24 hour infection period surviving larvae were washed several times in PTU-supplemented E3. Uptake of GFP-expressing Salmonella was monitored through fluorescence microscopy. Successfully infected transgenic larvae were then anaesthetized, mounted in 1% (w/v) low melting point agarose (Sigma) and imaged using an Olympus FV1000 confocal microscope equipped with a heated chamber which was kept at 29°C.
We thank the Biomedical Imaging Research Unit, School of Medical Sciences, The University of Auckland, for their expert assistance in fluorescence and confocal microscopy, Annie Chien and Lisa Pullin for their excellent technical assistance, Stephen Edgar for FACS analysis support and Simon Swift for facilitating and assisting with bacterial infection work. This work would not have been possible without the expert management of our zebrafish facility by Alhad Mahagaonkar. We are grateful to K. Guillemin and D. Monack for generously gifting the Salmonella enterica bacteria and GFP-bearing P22 phage. We also thank K. Kawakami, G. Lieschke and L. Zon for generously supplying the Tol2/transposase expression vectors, mpo and L-plastin/fms probe templates, respectively and Jen-Leih Wu for the I-FABP::RFP transgenic zebrafish line. Funding for this work was provided by a grant awarded to PC from the New Economy Research Fund, Foundation for Research Science and Technology, New Zealand.
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