Name: Live Imaging of Chemokine Receptors in Zebrafish Neutrophils During Wound Responses (Video) | JoVE Journal
Uploaded: 2020-12-04T13:30:00.000+00:00
Duration: 6 min 48 s
Description: Learn live imaging of chemokine receptor dynamics in zebrafish neutrophils during wound responses. This protocol enables visualization of CXCR1 and CXCR2 trafficking to study immune cell behavior in vivo.

ai_agent_IP

ai_agent

maintenance

instantTrialDurationMinutes

isBiopharma

enableNewMyJoveSectionDesign

educationFeatures

seoMetaApi

ai_copilot_institution_blacklist

ai_chat_institution_blacklist

freeAccessWindow

ssoRoleCapture

scheduledMaintenanceBanner

joveLabsFeatures

disable_autoplay_for_bots

extended_trial_duration

5 minutes

We thank Christine Holt and Bill Harris for help with confocal microscopy. We thank Darren Gilmour for the fluorescent timer backbone constructs and Anna Huttenlocher for the Tol2-Lyz backbone vector. We thank Steve Renshaw for the Tg(mpx:GFP)i114 line. This work was supported by the MRC (MR/L019523/1), the Wellcome Trust [204845/Z/16/Z]; Isaac Newton Trust [12.21(a)i] and an Isaac Newton Trust grant 19.23(n). C.C. was supported by an MRC DTP studentship and partly by the Wellcome Trust [204845/Z/16/Z]; Isaac Newton Trust [12.21(a)i] grant. H.W. was supported by an MRC DTP Studentship. H.P. was supported by a Wellcome Trust PhD grant (105391/Z/14/Z) and partly by a Wellcome Trust [204845/Z/16/Z]; Isaac Newton Trust [12.21(a)i] grant and the MRC (MR/L019523/1).

<ol><li>Rot, A., von Andrian, U. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Chemokines+in+innate+and+adaptive+host+defense%3A+basic+chemokinese+grammar+for+immune+cells%5BTitle%5D)+AND+%22Annual+Review+of+Immunology%22%5BJournal%5D)">Chemokines in innate and adaptive host defense: basic chemokinese grammar for immune cells.</a> Annual Review of Immunology. 22, 891-928 (2004).</li>
<li>Cotton, M., Claing, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((G+protein-coupled+receptors+stimulation+and+the+control+of+cell+migration%5BTitle%5D)+AND+%22Cellular+Signalling%22%5BJournal%5D)">G protein-coupled receptors stimulation and the control of cell migration.</a> Cellular Signalling. 21 (7), 1045-1053 (2009).</li>
<li>Liu, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Bidirectional+regulation+of+neutrophil+migration+by+mitogen-activated+protein+kinases%5BTitle%5D)+AND+%22Nature+Immunology%22%5BJournal%5D)">Bidirectional regulation of neutrophil migration by mitogen-activated protein kinases.</a> Nature Immunology. 13 (5), 457-464 (2012).</li>
<li>Wen, X., Jin, T., Xu, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Imaging+G+Protein-coupled+Receptor-mediated+Chemotaxis+and+its+Signaling+Events+in+Neutrophil-like+HL60+Cells%5BTitle%5D)+AND+%22Journal+of+Visualized+Experiments%22%5BJournal%5D)">Imaging G Protein-coupled Receptor-mediated Chemotaxis and its Signaling Events in Neutrophil-like HL60 Cells.</a> Journal of Visualized Experiments. (115), e54511(2016).</li>
<li>Arnon, T. I., et al. GRK2-dependent S1PR1 desensitization is required for lymphocytes to overcome their attraction to blood. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aArnon%2C+TI+%22Science%22">Science</a>. 333 (6051), 1898-1903 (2011).</li>
<li>Jung, H., Mithal, D. S., Park, J. E., Miller, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Localized+CCR2+Activation+in+the+Bone+Marrow+Niche+Mobilizes+Monocytes+by+Desensitizing+CXCR4%5BTitle%5D)+AND+%22PloS+One%22%5BJournal%5D)">Localized CCR2 Activation in the Bone Marrow Niche Mobilizes Monocytes by Desensitizing CXCR4.</a> PloS One. 10 (6), 0128387(2015).</li>
<li>Kwan, K. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+Tol2kit%3A+a+multisite+gateway-based+construction+kit+for+Tol2+transposon+transgenesis+constructs%5BTitle%5D)+AND+%22Developmental+Dynamics%3A+An+Official+Publication+of+the+American+Association+of+Anatomists%22%5BJournal%5D)">The Tol2kit: a multisite gateway-based construction kit for Tol2 transposon transgenesis constructs.</a> Developmental Dynamics: An Official Publication of the American Association of Anatomists. 236 (11), 3088-3099 (2007).</li>
<li>Donà, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Directional+tissue+migration+through+a+self-generated+chemokine+gradient%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Directional tissue migration through a self-generated chemokine gradient.</a> Nature. 503 (7475), 285-289 (2013).</li>
<li>Venkiteswaran, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Generation+and+dynamics+of+an+endogenous%2C+self-generated+signaling+gradient+across+a+migrating+tissue%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">Generation and dynamics of an endogenous, self-generated signaling gradient across a migrating tissue.</a> Cell. 155 (3), 674-687 (2013).</li>
<li>Minina, S., Reichman-Fried, M., Raz, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Control+of+receptor+internalization%2C+signaling+level%2C+and+precise+arrival+at+the+target+in+guided+cell+migration%5BTitle%5D)+AND+%22Current+Biology%22%5BJournal%5D)">Control of receptor internalization, signaling level, and precise arrival at the target in guided cell migration.</a> Current Biology. 17 (13), 1164-1172 (2007).</li>
<li>Yoo, S. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+role+of+microtubules+in+neutrophil+polarity+and+migration+in+live+zebrafish%5BTitle%5D)+AND+%22Journal+of+Cell+Science%22%5BJournal%5D)">The role of microtubules in neutrophil polarity and migration in live zebrafish.</a> Journal of Cell Science. 125, Pt 23 5702-5710 (2012).</li>
<li>Yoo, S. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Differential+regulation+of+protrusion+and+polarity+by+PI3K+during+neutrophil+motility+in+live+zebrafish%5BTitle%5D)+AND+%22Developmental+Cell%22%5BJournal%5D)">Differential regulation of protrusion and polarity by PI3K during neutrophil motility in live zebrafish.</a> Developmental Cell. 18 (2), 226-236 (2010).</li>
<li>Buchan, K. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+transgenic+zebrafish+line+for+in+vivo+visualisation+of+neutrophil+myeloperoxidase%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">A transgenic zebrafish line for in vivo visualisation of neutrophil myeloperoxidase.</a> PLoS One. 14 (4), 0215592(2019).</li>
<li>Coombs, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Chemokine+receptor+trafficking+coordinates+neutrophil+clustering+and+dispersal+at+wounds+in+zebrafish%5BTitle%5D)+AND+%22Nature+Communications%22%5BJournal%5D)">Chemokine receptor trafficking coordinates neutrophil clustering and dispersal at wounds in zebrafish.</a> Nature Communications. 10 (1), 5166(2019).</li>
<li>Hall, C., Flores, M. V., Storm, T., Crosier, K., Crosier, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+zebrafish+lysozyme+C+promoter+drives+myeloid-specific+expression+in+transgenic+fish%5BTitle%5D)+AND+%22BMC+Developmental+Biology%22%5BJournal%5D)">The zebrafish lysozyme C promoter drives myeloid-specific expression in transgenic fish.</a> BMC Developmental Biology. 7, 42(2007).</li>
<li>Renshaw, S. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+transgenic+zebrafish+model+of+neutrophilic+inflammation%5BTitle%5D)+AND+%22Blood%22%5BJournal%5D)">A transgenic zebrafish model of neutrophilic inflammation.</a> Blood. 108 (13), 3976-3978 (2006).</li>
<li>Khmelinskii, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Tandem+fluorescent+protein+timers+for+in+vivo+analysis+of+protein+dynamics%5BTitle%5D)+AND+%22Nature+Biotechnology%22%5BJournal%5D)">Tandem fluorescent protein timers for in vivo analysis of protein dynamics.</a> Nature Biotechnology. 30 (7), 708-714 (2012).</li>
<li>Westerfield, M. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aWesterfield%2C+M+%22The+zebrafish+book.+A+guide+for+the+laboratory+use+of+zebrafish+%28Brachydanio+rerio%29%22">The zebrafish book. A guide for the laboratory use of zebrafish (Brachydanio rerio)</a>. , University of Oregon Press. Eugene, Oregon. (2007).</li>
<li>Rosen, J. N., Sweeney, M. F., Mably, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Microinjection+of+Zebrafish+Embryos+to+Analyze+Gene+Function%5BTitle%5D)+AND+%22Journal+of+Visualized+Experiments%22%5BJournal%5D)">Microinjection of Zebrafish Embryos to Analyze Gene Function.</a> Journal of Visualized Experiments. (25), 1115(2009).</li>
<li>Detrich, H. W., Zon, L. I., Westerfield, M. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aDetrich%2C+HW+%22The+Zebrafish%3A+Genetics%2C+Genomics+and+Informatics%22">The Zebrafish: Genetics, Genomics and Informatics</a>. , Elsevier. Burlington. (2004).</li>
<li>Le Guyader, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Origins+and+unconventional+behavior+of+neutrophils+in+developing+zebrafish%5BTitle%5D)+AND+%22Blood%22%5BJournal%5D)">Origins and unconventional behavior of neutrophils in developing zebrafish.</a> Blood. 111 (1), 132-141 (2008).</li>
<li>Chan, T. F., Vese, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Active+contours+without+edges%5BTitle%5D)+AND+%22IEEE+Transactions+on+Image+Processing%22%5BJournal%5D)">Active contours without edges.</a> IEEE Transactions on Image Processing. 10 (2), 266-277 (2001).</li>
<li>Haralick, R. M., Shanmugam, K., Dinstein, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Textural+features+for+image+Classification%5BTitle%5D)+AND+%22IEEE+Transactions+on+Systems%2C+Man%2C+and+Cybernetics%22%5BJournal%5D)">Textural features for image Classification.</a> IEEE Transactions on Systems, Man, and Cybernetics. SMC-3 (6), 610-621 (1973).</li>
<li>Deng, Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Localized+bacterial+infection+induces+systemic+activation+of+neutrophils+through+Cxcr2+signaling+in+zebrafish%5BTitle%5D)+AND+%22Journal+of+Leukocyte+Biology%22%5BJournal%5D)">Localized bacterial infection induces systemic activation of neutrophils through Cxcr2 signaling in zebrafish.</a> Journal of Leukocyte Biology. 93 (5), 761-769 (2013).</li>
<li>Torraca, V., Mostowy, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Zebrafish+infection%3A+From+pathogenesis+to+cell+biology%5BTitle%5D)+AND+%22Trends+in+Cell+Biology%22%5BJournal%5D)">Zebrafish infection: From pathogenesis to cell biology.</a> Trends in Cell Biology. 28 (2), 143-156 (2018).</li>
<li>Feng, Y., Santoriello, C., Mione, M., Hurlstone, A., Martin, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Live+imaging+of+innate+immune+cell+sensing+of+transformed+cells+in+zebrafish+larvae%3A+parallels+between+tumor+initiation+and+wound+inflammation%5BTitle%5D)+AND+%22PLoS+Biology%22%5BJournal%5D)">Live imaging of innate immune cell sensing of transformed cells in zebrafish larvae: parallels between tumor initiation and wound inflammation.</a> PLoS Biology. 8 (12), 1000562(2010).</li>
<li>Poplimont, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Neutrophil+swarming+in+damaged+tissue+is+orchestrated+by+connexins+and+cooperative+calcium+alarm+signals%5BTitle%5D)+AND+%22Current+Biology%22%5BJournal%5D)">Neutrophil swarming in damaged tissue is orchestrated by connexins and cooperative calcium alarm signals.</a> Current Biology. 30 (14), 2761-2776.e7 (2020).</li>
<li>Cornet, C., Di Donato, V., Terriente, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Combining+Zebrafish+and+CRISPR%2FCas9%3A+Toward+a+more+efficient+drug+discovery+pipeline%5BTitle%5D)+AND+%22Frontiers+in+Pharmacology%22%5BJournal%5D)">Combining Zebrafish and CRISPR/Cas9: Toward a more efficient drug discovery pipeline.</a> Frontiers in Pharmacology. 9, 703(2018).</li>
<li>Kienle, K., Lämmermann, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Neutrophil+swarming%3A+an+essential+process+of+the+neutrophil+tissue+response%5BTitle%5D)+AND+%22Immunological+Reviews%22%5BJournal%5D)">Neutrophil swarming: an essential process of the neutrophil tissue response.</a> Immunological Reviews. 273 (1), 76-93 (2016).</li>
<li>Lisse, T. S., Brochu, E. A., Rieger, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Capturing+tissue+repair+in+zebrafish+larvae+with+time-lapse+brightfield+stereomicroscopy%5BTitle%5D)+AND+%22Journal+of+Visualized+Experiments%22%5BJournal%5D)">Capturing tissue repair in zebrafish larvae with time-lapse brightfield stereomicroscopy.</a> Journal of Visualized Experiments. (95), e52654(2015).</li>
</ol>

The authors declare no conflict of interest

The method described allows live imaging of receptor dynamics in response to endogenous ligands in situ during an inflammatory response to tissue damage. The use of Cxcr1/Cxcr2 neutrophil reporters could be expanded to other physiological settings, such as infection, tumor models or other types of tissue damage14,25,26,27. In addition, transgenic rescue lines, in which the endogenous receptor is suppressed and rescued by an exogenous mutant receptor, could provide useful tools to dissect the importance of specific neutrophil migration patterns in immune responses. For example, Cxcr1 receptor mutants that have impaired desensitization cause more prominent neutrophil clustering at inflammatory sites14. This gain of function phenotype could be used to understand the role of neutrophil congregation in different physiological processes, e.g., wound repair, infectious disease, or tumor evolution, and complement receptor knockdown/knockout experiments. The methodology also provides a basis to expand the range of available reporters. The choice of fluorescent reporter is important to consider and depends on the biological question. We found that the constitutive turnover of these chemokine receptors in neutrophils was high, in comparison to epithelial cells, and that reporters with fast maturation (e.g., sfGFP) were required to report membrane levels at steady state and resolve differences upon neutrophil stimulation8,14. Thus, membrane ratios of sfGFP/tagRFP are not applicable for measuring ligand-induced internalization in this cell type, but the pattern of tagRFP allows tracking of the intracellular fates of the receptor, which could be useful in some studies. We also found that the more concentrated intracellular signal of tagRFP is useful for screening individual larvae. An alternative approach for measuring receptor levels at the plasma membrane would be to co-express a fluorescent membrane marker in neutrophils either in the same transgene9 or in an independent transgene14. In the former scenario the transgene would provide an additional means for screening the fish and expression levels would be comparable between the marker and the receptor. The latter approach would be more modular, in that a zebrafish line with a receptor reporter could be combined with different reporter lines. In either case, it is worth noting that membrane quantification of the receptor levels is challenging in clustered neutrophils (see below). Finally, we note that a possible extension of this protocol would be to follow up the live imaging by immunohistochemistry for more detailed localization analyses.
The Tol2 transgenesis system is well established7 and the lysozyme C promoter has been used extensively for neutrophil expression11,15. The transgenesis approach is, therefore, relatively straightforward and the expression level achieved with this promoter is high enough to provide sufficient contrast for analysis of receptor dynamics. A possible limitation is that the expression level does not recapitulate endogenous receptor expression levels. New CRISPR technologies could be deployed to establish knock-in lines for receptors of particular interest28. These technologies are still cumbersome and may not guarantee the required expression levels for subcellular imaging, but their successful development would be an important breakthrough for understanding endogenous signaling dynamics. Functional validations are important for interpreting data with transgenic receptor constructs. For example, ligand recognition assays can be used to establish that the fluorescent fusion protein is functional and rescue of knockout phenotypes could be used to establish that the transgenic neutrophil expression levels are compatible with functionality14. Finally, a more direct way to validate the receptor fusion would be to utilize an in vitro functional assay with labelled receptor alongside non-labeled versions14.
The quantification approach addresses specific difficulties in accurate membrane segmentation in neutrophils in vivo. In cells of epithelial nature, quantification of receptor levels can be executed automatically by normalizing membrane receptor levels to a control marker, which can be expressed in tandem or separately9. Indeed, we have applied such an approach, when using the ligand-recognition assay in gastrulating embryos14. However, neutrophils undergo complex, rapid changes in cell shape in vivo, making the membrane segmentation difficult both in 2D and 3D14. This is even more challenging when neutrophils cluster, which occurs in many physiological settings29. The contrast metric overcomes this limitation as it does not require membrane segmentation but instead reflects the overall state of receptor distribution in the cell (membranous/smooth vs vesicular/dotty). It is important to note that contrast metric can be affected by the overall contrast of the image, so normalization of individual cell values to an internal reference is required to account for variability of signal in different embryos/samples. For example, we used the mean cell contrast value of non-responsive neutrophils in the CHT (i.e., neutrophils that remain stationary and do not migrate into the ventral fin)14. An additional possibility would be to normalize with contrast values of a control marker in the same cell. This would provide a solution when an internal reference of non-responding cells is not available and may likely resolve finer quantitative differences in receptor dynamics between different conditions.
The location of imaging is another variable to consider. The reason for choosing the ventral fin wound here, as opposed to the more commonly used tail fin wound model16,30, is because the site of wounding is nearby the site of neutrophil residence/migratory origin. This accelerates the timeline of the assay, as it takes relatively little time for neutrophils to arrive. Additionally, it provides the opportunity to capture cell behavior both at the migration origin (CHT) and the target location of interest (wound). This is relevant here, because the spatial and temporal resolution required for subcellular imaging is difficult to combine with a large field of view or multi-position scanning. Thus, the ventral fin wound assay permits tracking of the evolution of the migratory response from the migration origin and simultaneous capturing of unspecific receptor fluctuations in cells that do not respond. As mentioned above, the latter is useful for quantification purposes as it provides an internal reference for unspecific dynamics. In other systems, it may not be possible to have such an internal reference, in which case the contrast values of a co-expressed membrane marker would provide an alternative control.
In summary, we anticipate that the methodology is applicable to other systems and can be deployed for a variety of purposes. For example, the same reporters could be utilized in other inflammatory settings, such as infection settings or other disease models. The repertoire of zebrafish receptor reporter lines could be expanded to other signaling receptors, to understand signaling mechanisms or report ligand dynamics in vivo. The approach can be combined with knockdown/knockout techniques to interrogate the mechanistic basis of observed dynamics. For example, perturbation of ligand expression can indicate the ligand dependency for observed receptor dynamics. In the future, we envisage that the system could be further refined to incorporate knock-in insertion of reporters. Ultimately, findings using this methodology would provide novel insights valuable beyond the zebrafish community, given the conservation of these signaling receptors in mammals and the relative challenge of conducting these studies in larger organisms.

Leukocyte migration is of paramount importance for immune responses. Immune cells are prototypical migratory cells, which are remarkably capable of traversing tissues and blood vessels and sensing a range of chemical guidance cues to migrate directionally towards microbes or other host cells of importance. Correct guidance relies on the recognition of chemoattractants, among which chemokines represent the most prominent category1. Chemokines are recognized by highly specific seven-transmembrane G protein coupled receptors. Upon chemokine binding, chemokine receptors change conformation leading to the activation of associated trimeric G proteins and their dissociation into functional signaling subunits that promote cytoskeletal changes and directed migration1. Secondarily, chemokine receptors are phosphorylated, and this modification leads to desensitization to attractant, which can be followed by rapid re-sensitization/recycling or intracellular degradation and down-regulation from the cell surface2. These receptor dynamics influence the duration and dose of signaling received by the cells but how they affect leukocyte migration behavior has been difficult to elucidate in vivo.
Tracking receptor dynamics in live leukocytes in traditional mammalian systems faces several challenges. For live studies, receptor fusions with fluorescent proteins must be expressed in the cells. This is challenging in primary leukocytes, particularly in neutrophils, and studies so far have used surrogate neutrophil cell lines to express chemokine receptors3,4. Generation of transgenic mouse models, in which leukocytes express a fluorescent receptor or mutant receptors with informative trafficking defects5,6, entails considerable investment of time and resources. Even in these instances, the imaging resolution and contrast for imaging receptor dynamics in the live animal can be limited and studies have used immunohistochemistry on fixed tissue sections5. Given these technical challenges, our understanding of how chemoattractant receptors dynamics affect cell behavior in a live tissue setting is currently limited.
Here we provide a methodology to monitor receptor trafficking in zebrafish neutrophils. Zebrafish are genetically tractable, like mice, but transgenesis is relatively more straightforward through the use of efficient transposon systems and direct zygote manipulation7. The transparent larva is ideally amenable to imaging. The chemokine receptor dynamics have been visualized in primordial germ cells and the lateral line primordium by expression of corresponding fusions with fluorescent reporters8,9,10. Zebrafish larvae are equipped with mature neutrophils that have highly conserved genetic and cellular properties with respect to mammalian neutrophils. Subcellular signaling dynamics such as cytoskeletal dynamics and polarity regulators have been visualized in these cells by the generation of corresponding transgenic lines11,12,13. Recently, we visualized and functionally analyzed chemokine receptor dynamics in neutrophils during the course of inflammatory responses to tissue damage14. Here, we describe the generation of transgenic reporter lines for chemokine signaling in neutrophils, preparation of embryos for live imaging, a wound assay for studying neutrophil signaling and the protocol for acquisition and analysis of images. We also provide a side-protocol to test chemokine receptor responses to candidate ligands, which is useful when trying to establish ligand recognition patterns in uncharacterized receptors. These techniques can be used in combination with further genetic manipulations, such as inhibition of endogenous chemokine expression or generation of mutant receptors with altered ligand-induced trafficking, to interrogate how specific signaling dynamics affect leukocyte behavior in vivo. The transgenic lines expressing fluorescently tagged chemokine receptors can also be used as reporters for endogenous chemokine gradients, which are otherwise difficult to detect by direct antibody staining. The described methodology provides scope for expanding the generation of reporters to other immuno-signaling receptors.

<table><tbody><tr><td>1-phenyl-2-thiourea</td><td>Sigma-Aldrich</td><td>P7629-25G</td><td></td></tr><tr><td>50mL CellStar Centrifuge Tube</td><td>Grenier Bio-One Limited</td><td>210270</td><td></td></tr><tr><td>Clark capillary glass</td><td>Harvard Apparatus</td><td>30-0020</td><td></td></tr><tr><td>Cleanline Thin Bleach</td><td>Scientific Laboratory Supplies</td><td>CLE0301</td><td></td></tr><tr><td>Dissecting scope</td><td>Nikon</td><td>SMZ645</td><td></td></tr><tr><td>Dri Block heater</td><td>Techne</td><td>FDB02AD</td><td></td></tr><tr><td>Fiji</td><td>National Institutes of Health, Bethesda, MD</td><td></td><td></td></tr><tr><td>Forceps, Jewelers Dumont No. 5</td><td>Sigma-Aldrich</td><td>F6521</td><td></td></tr><tr><td>Glass bottom microwell dishes</td><td>MatTek</td><td>P35G-1.5-14-C</td><td></td></tr><tr><td>Glass pasteur pipette</td><td>Fisherbrand</td><td>11795098</td><td></td></tr><tr><td>Invitrogen Ambion mMESSAGE mMACHINE SP6 Transcription Kit</td><td>Invitrogen</td><td>10391175</td><td></td></tr><tr><td>Leica SP8 confocal microscope</td><td>Leica</td><td>N/A</td><td></td></tr><tr><td>Low melting point agarose</td><td>Invitrogen</td><td>16520-100</td><td></td></tr><tr><td>Magnetic stand</td><td>Narishige</td><td>GJ-1</td><td></td></tr><tr><td>MATLAB</td><td>The MathWorks, Inc., Natick, MA</td><td></td><td></td></tr><tr><td>Methylene blue</td><td>Sigma-Aldrich</td><td>M9140-25G</td><td></td></tr><tr><td>MgSO4</td><td>Sigma-Aldrich</td><td>M-2643</td><td></td></tr><tr><td>Microinjection system</td><td>Parker</td><td>Picospritzer III</td><td></td></tr><tr><td>Microloader pipette tips</td><td>Eppendorf</td><td>5242956003</td><td></td></tr><tr><td>Micromanipulator</td><td>Narishige</td><td>M-152</td><td></td></tr><tr><td>Micropipette Puller</td><td>Sutter Instrument Company</td><td>Flaming/Brown P-97</td><td></td></tr><tr><td>Microscope slide</td><td>Thermo-Scientific</td><td>J1800AMNZ</td><td></td></tr><tr><td>PerkinElmer Spinning Disk UltraVIEW ERS, Olympus IX81 spinnng disk confocal microscope</td><td>Olympus</td><td>N/A</td><td></td></tr><tr><td>Petri dish (120mm)</td><td>Grenier Bio-One Limited</td><td>632180</td><td></td></tr><tr><td>Phenol red</td><td>Sigma-Aldrich</td><td>P0290-100ML</td><td></td></tr><tr><td>Poly(A) Tailing Kit</td><td>Invitrogen</td><td>AM1350</td><td></td></tr><tr><td>Prism</td><td>GraphPad Software</td><td></td><td></td></tr><tr><td>Small paintbrush</td><td>N/A</td><td></td><td></td></tr><tr><td>Spectral Applied Research LMM5 laser module</td><td></td><td>N/A</td><td></td></tr><tr><td>Surgical Scalpel Blade No. 23</td><td>Swann-Morton</td><td>233-5528</td><td></td></tr><tr><td>Tricaine MS222</td><td>Sigma-Aldrich</td><td>E10521-50G</td><td></td></tr><tr><td>Volocity software</td><td></td><td>N/A</td><td></td></tr><tr><td>Yokogawa CSU10 spinning disc confocal scanner</td><td>Yokogawa</td><td>N/A</td><td></td></tr></tbody></table>

live imaging of chemokine receptors in zebrafish neutrophils during wound responses

Live Imaging of Chemokine Receptors in Zebrafish Neutrophils During Wound Responses

Leukocyte guidance by chemical gradients is essential for immune responses. Neutrophils are the first cells to be recruited to sites of tissue damage where they execute crucial antimicrobial functions. Their trafficking to these loci is orchestrated by several inflammatory chemoattractants, including chemokines. At the molecular level, chemoattractant signaling is regulated by the intracellular trafficking of the corresponding receptors. However, it remains unclear how subcellular changes in chemokine receptors affect leukocyte migration dynamics at the cell and tissue level. Here we describe a methodology for live imaging and quantitative analysis of chemokine receptor dynamics in neutrophils during inflammatory responses to tissue damage. These tools have revealed that differential chemokine receptor trafficking in zebrafish neutrophils coordinates neutrophil clustering and dispersal at sites of tissue damage. This has implications for our understanding of how inflammatory responses are self-resolved. The described tools could be used to understand neutrophil migration patterns in a variety of physiological and pathological settings and the methodology could be expanded to other signaling receptors.

Ventral fin wounding is followed by rapid neutrophil mobilization from the CHT into the ventral fin and clustering at the wound margin, within 30-60 min (Figure 1). We visualized the distribution of two chemokine receptors, Cxcr1 and Cxcr2, which are expressed by zebrafish neutrophils24 and recognize Cxcl8a and Cxcl8b14, using spinning-disk confocal microscopy. We generated two corresponding transgenic lines, Tg(lyz:Cxcr1-FT) and Tg(lyz:Cxcr2-FT), in which neutrophils express a fluorescent timer (FT) construct of the receptor, i.e. a fusion with a tandem of sfGFP and tagRFP (Figure 2 and reference14). The use of the two fluorophores was intended to allow monitoring of a broad range of receptor fates and provide estimates of protein turnover time at the plasma membrane, as newly synthesized receptors would fluoresce in green and progressively become red as they age8,14. However, these receptors were found to have fast constitutive turnover at the neutrophil plasma membrane and that the residence time was shorter than the maturation time of tagRFP, with sfGFP showing membrane localisation and tagRFP showing vesicular localization at steady state (Supplementary Video 1 and ref14). Therefore, we focused on the distribution of sfGFP to monitor ligand-induced internalization at sites of tissue damage. The pattern of receptor distribution was quantified using the contrast metric, which reports differences in intensity between neighboring pixels. The rationale is that when the receptor distribution is membranous and smooth, the contrast value is low. When the receptor distribution is vesicular and more punctate, then the contrast value is high (Figure 3).
An alternative method is to quantify the ratio of receptor levels (sfGFP intensity) over the levels of a control membrane marker e.g. membrane CFP (mCFP) (Figure 3). Both methods could detect receptor internalization, as indicated by more vesicular receptor distribution pattern globally in the cell (higher contrast value) or lower receptor levels at the membrane (lower sfGFP/mCFP ratio). However, the contrast metric could also detect receptor internalization in neutrophil clusters at the wound, in which membrane segmentation was less accurate and not applicable (Figure 3). Using this metric, we were able to quantify visible differences between Cxcr1 and Cxcr2 trafficking in neutrophils at wounds (Figure 4 and Supplementary Video 2). Cxcr1-FT internalized in cells located at the wound whereas Cxcr2-FT remained membranous in neutrophils at the wound (Figure 4A-C, Supplementary Video 2 and Supplementary Video 3). Suppression of Cxcl8a and Cxcl8b, through morpholino treatment, differentially affected Cxcr1-FT internalization at wounds (Figure 4C,D). To further validate that Cxcr1-FT responds to Cxcl8a, we performed chemokine response assays in early embryos. We found that Cxcr1-FT markedly internalized in embryos in which Cxcl8a was co-expressed (Figure 5). Altogether these results indicate that the described methods can be deployed to measure chemokine-induced receptor internalization in neutrophils and establish the identity of the ligand mediating these effects.
<img alt="Neutrophil migration diagram and microscope image showing wound response in zebrafish vasculature." class="xfigimg" src="/files/ftp_upload/61679/61679fig01.jpg" data-altupdatedat="1788250015000" data-alt="Figure 1"> 
Figure 1: Neutrophil migration to ventral fin wounds. (A) (Left) Cartoon of 3 dpf larva showing the location of the caudal hematopoietic tissue (CHT), the venus circulation (VC, blue), the ventral fin (VF) and the wound site. (Right) Cartoon depicting the area of the wound (W) with neutrophils getting mobilized from the CHT and clustering at the wound. The caudal vein plexus (CVP) of the CHT tissue is drawn in blue. (B) Bright field image (left) and confocal projection (right) showing the ventral fin wound and the distribution of neutrophils in Tg(mpx:GFP) larvae at 2 h post-wounding. Dashed lines show VF and CHT outlines. Scale bar = 25 µm. Cartoon and fluorescent image modified from ref.14 (http://creativecommons.org/licenses/by/4.0/). <a href="https://www.jove.com/files/ftp_upload/61679/61679fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Transgenic zebrafish chemotaxis and mobilization, lyz promoter diagram, fluorescence microscopy." class="xfigimg" src="/files/ftp_upload/61679/61679fig02.jpg" data-altupdatedat="1788250015000" data-alt="Figure 2"> 
Figure 2: Live imaging of chemokine receptor trafficking in neutrophils. (A) Constructs used for neutrophil-specific transgenic expression of Cxcr1-FT (Fluorescent Timer) and Cxcr2-FT. Confocal projections of neutrophils in the head of a 3 dpf transgenic larva (Tg(lyz:Cxcr1-FT), top; Tg(lyz:Cxcr2-FT), bottom) showing tRFP (magenta) and sfGFP (green) channels. Scale bar = 20 µm. (B) Anatomical scheme of 3 dpf larva as in Figure 1A. Below the larva are schemes depicting the area of the wound (W) with neutrophils getting mobilized from the CHT (top) or performing chemotaxis upon entering the ventral fin (bottom). Dashed square indicates area imaged in snapshots on the right. (C) Neutrophils in Tg(lyz:Cxcr1-FT) larvae (sfGFP is shown) upon mobilization from the CHT (top panels) or chemotaxis towards the wound (bottom panels). Arrows show the same cells over time. Time points on the right image are minutes elapsed after image on the left. Scale bar = 10 µm. (D) Schematic representation of experimental approach for live imaging of chemokine receptor trafficking. Panels A-C modified from ref.14 (http://creativecommons.org/licenses/by/4.0/). <a href="https://www.jove.com/files/ftp_upload/61679/61679fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Cell contour segmentation process; GFP contrast and CFP/GFP ratio analysis; microscopy images." class="xfigimg" src="/files/ftp_upload/61679/61679fig03.jpg" data-altupdatedat="1788250015000" data-alt="Figure 3"> 
Figure 3: Quantification examples of receptor dynamics. Single (blue) or clustered neutrophils (green) at wounds or non-mobilized neutrophils in the CHT (red, orange) were segmented and analyzed by different methods to compare results. The same example cells shown were analyzed with two methods to relate what is seen in the image with the range of values extracted. (A) The surface of the selected, example cells were segmented based on contour definition in the sfGFP channel. (B) Contrast was computed from the example cells shown in A. (C) The membrane of the selected, example cells were segmented based on contour definition in the CFP channel. Ratiometric analysis of sfGFP/CFP followed. (D) The ratio of sfGFP/CFP was computed on the example cells shown in C. Error bars represent S.E.M. from individual cells, in cases of n&gt;1, values here were not used for statistical analysis but merely to exemplify measurements obtained with the different quantification methods. Scale bar = 10 µm. Figure modified from ref.14 (http://creativecommons.org/licenses/by/4.0/). <a href="https://www.jove.com/files/ftp_upload/61679/61679fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Microscopy images and graph of zebrafish wound response; sfGFP expression; Cxcr1/Cxcr2 analysis." class="xfigimg" src="/files/ftp_upload/61679/61679fig04v2.jpg" data-altupdatedat="1788250015000" data-alt="Figure 4"> 
Figure 4: Differential dynamics of Cxcr1 and Cxcr2 in response to wounding. (A) Confocal projection of neutrophils in Tg(lyz:Cxcr1-FT) or Tg(lyz:Cxcr2-FT) larvae at the wound at 80 min post-wounding (sfGFP channel shown). Scale bar = 10 µm.  (B) Magnified Cxcr1-FT neutrophil (left) and Cxcr2-FT (right) at the wound. Green receptor is shown in gray. Scale bar = 5 µm. (C) Normalized contrast (contrast per individual neutrophil normalized to the mean contrast of non-mobilized cells in the CHT). cxcl8a refers to injection of a splice-blocking together with a translation-blocking morpholino for cxcl8a. cxcl8b refers to injection with a splice-blocking morpholino for cxcl8b. For Tg(lyz:Cxcr1-FT): n=24 cells (CHT), n=47 cells (wound) from 8 larvae. For Tg(lyz:Cxcr1-FT) with morpholinos: n=28 cells (Cxcl8a-MO) from 5 larvae, n=16 cells (Cxcl8b-MO) from 5 larvae. For Tg(lyz:Cxcr2-FT): n=10 cells (CHT) and n=20 cells (wound) from 3 larvae. Data were pooled from independent larvae acquired in 1-5 imaging sessions. Kruskal-Wallis test with Dunn’s multiple comparisons test for Tg(lyz:Cxcr1-FT), two-tailed unpaired Mann-Whitney test for Tg(lyz:Cxcr2-FT). (D) Confocal projection of neutrophils in Tg(lyz:Cxcr1-FT) transgenic larvae treated with cxcl8a morpholino (MO) (left) and cxcl8b MO (right) responding to fin wounds (sfGFP channel shown in green). Snapshot taken at timepoints of equivalent neutrophil accumulation (85 min post-wounding in left image and 45 min post-wounding in right image). Scale bar = 10 µm. Figure modified from ref.14 (http://creativecommons.org/licenses/by/4.0/). <a href="https://www.jove.com/files/ftp_upload/61679/61679fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Fluorescence microscopy images: tagged proteins, receptor-ligand binding, cellular localization." class="xfigimg" src="/files/ftp_upload/61679/61679fig05.jpg" data-altupdatedat="1788250015000" data-alt="Figure 5"> 
Figure 5: Chemokine response assay in early embryos. Laser-scanning confocal slices of gastrulating embryos showing expression and distribution of Cxcr1-FT. 100 pg of Cxcr1-FT mRNA was injected into one cell-stage eggs with or without 150 pg Cxcl8a mRNA. Green and red receptors are shown in separate channels. Control membrane CFP marker (mCFP) is shown in the cyan channel. Scale bar = 20 μm. Figure modified from ref.14 (http://creativecommons.org/licenses/by/4.0/). <a href="https://www.jove.com/files/ftp_upload/61679/61679fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplementary Movie 1: Transgenic neutrophils in the head of a Tg(lyz:Cxcr1-FT) (left) and Tg(lyz:Cxcr2-FT) (right) larva at 3 dpf. sfGFP(green), tagRFP (magenta). Frame interval is 30 sec and frame rate is 5 fps. Scale bar = 20 μm. Video originates from ref.14 (http://creativecommons.org/licenses/by/4.0/). <a href="https://cloudflare.jove.com/files/ftp_upload/61679/Supplementary_Movie1.avi" target="_blank">Please click here to download this video.</a>
Supplementary Movie 2: Neutrophils in Tg(lyz:Cxcr1-FT) (left) and Tg(lyz:Cxcr2-FT) (right) transgenic larvae responding to fin wounds. Movie starts within 10 min post-wounding and lasts 60 min. sfGFP (green), tagRFP (magenta). Frame interval is 30 sec and frame rate is 10 fps. CHT = caudal hematopoietic tissue. VF = ventral fin. Scale bar = 25 μm. Video originates from ref.14 (http://creativecommons.org/licenses/by/4.0/). <a href="https://cloudflare.jove.com/files/ftp_upload/61679/Supplementary_Movie2.avi" target="_blank">Please click here to download this video.</a>
Supplementary Movie 3. Additional examples of neutrophils from a wounded Tg(lyz:Cxcr1- FT) transgenic larva (different larva to that shown in Video 2), acquired at higher resolution, showing receptor internalization (sfGFP channel shown in green) upon mobilization in the CHT or upon entry and chemotaxis in the ventral fin. Frame interval is 30 sec and frame rate is 2 fps. Scale bar = 10 μm. Video originates from ref.14 (http://creativecommons.org/licenses/by/4.0/). <a href="https://cloudflare.jove.com/files/ftp_upload/61679/Supplementary_Movie3.avi" target="_blank">Please click here to download this video.</a>
Supplementary File 1: <a href="https://cloudflare.jove.com/files/ftp_upload/61679/Supplementary%20file%201.m" target="_blank">Please click here to download this file.</a>
Supplementary File 2: <a href="https://cloudflare.jove.com/files/ftp_upload/61679/Supplementary%20file%202.m" target="_blank">Please click here to download this file.</a>
Supplementary File 3: <a href="https://cloudflare.jove.com/files/ftp_upload/61679/Supplementary%20file%203.m" target="_blank">Please click here to download this file.</a>
Supplementary File 4: <a href="https://cloudflare.jove.com/files/ftp_upload/61679/Supplementary%20file%204.m" target="_blank">Please click here to download this file.</a>

Here we describe protocols to perform live imaging and quantitative analysis of chemoattractant receptor dynamics in zebrafish neutrophils

Leukocyte guidance by chemical gradients is essential for immune responses. Neutrophils are the first cells to be recruited to sites of tissue damage ...

live-imaging-chemokine-receptors-zebrafish-neutrophils-during-wound

Research

JoVE Journal

Immunology and Infection

The rapid development, small size and transparency of zebrafish are tremendous advantages for the study of innate immune control of infection1-4. Here we demonstrate techniques for infecting zebrafish larvae using the fungal pathogen Candida albicans by microinjection, methodology recently used to implicate phagocyte NADPH oxidase activity in control of fungal dimorphism5.

Non-invasive Imaging of Disseminated Candidiasis in Zebrafish Larvae

Here we describe a novel high-content chemically induced inflammation assay aiming at the identification of immune-modulatory bioactives. We have successfully combined automated microscopy with custom developed software scripts enabling automated quantification of the inflammatory response as well as further data processing, analysis, mining, and storage.

Facilitating Drug Discovery: An Automated High-content Inflammation Assay in Zebrafish

This video demonstrates the tracking of leukocyte recruitment using transgenic zebrafish larvae expressing photoconvertible green fluorescent protein. Pre-injecting red fluorescent dye-labeled bacteria into the otic vesicle and inducing photoconversion enables observation of leukocytes' migration, phagocytosis, and dispersal throughout the body, providing valuable insights into immune response dynamics.

Photoconversion-Based Leukocyte Tracking in Bacterial-Infected Transgenic Zebrafish

The video demonstrates a zebrafish wounding and entrapment device technique for observing fluorescently labeled fungal pathogen infection in zebrafish larvae. Initial infection attracts macrophages, and as infection persists, neutrophil recruitment progresses at the injection site.

A zWEDGI Technique to Visualize Fungal Pathogen-Infected Zebrafish Larvae

Studying the earliest events of preneoplastic cell progression and innate immune cell interaction is pivotal to understand and treat cancer. Here we describe a method to conditionally induce epithelial cell transformations and the subsequent live imaging of innate immune cell interaction with HRASG12V expressing skin cells in zebrafish larvae.

Live Imaging of Innate Immune and Preneoplastic Cell Interactions Using an Inducible Gal4/UAS Expression System in Larval Zebrafish Skin

Here, we present a protocol for the generation and imaging of a localized bacterial infection in the zebrafish otic vesicle.

Non-invasive Imaging of the Innate Immune Response in a Zebrafish Larval Model of Streptococcus iniae Infection

Systemic and localized zebrafish infection models for human influenza A virus are demonstrated. Using a systemic infection model, zebrafish can be used to screen antiviral drugs. Using a localized infection model, zebrafish can be used to characterize host immune cell responses.

Using Zebrafish Models of Human Influenza A Virus Infections to Screen Antiviral Drugs and Characterize Host Immune Cell Responses

Microinjection of zebrafish embryos and larvae is a crucial but challenging technique used in many zebrafish models. Here, we present a range of microscale tools to aid in the stabilization and orientation of zebrafish for both microinjection and imaging.

Microstructured Devices for Optimized Microinjection and Imaging of Zebrafish Larvae

Here we present a protocol for live-imaging wound repair and the associated inflammatory response at high spatio-temporal resolution in vivo. This method utilizes the pupal stage of Drosophila development to enable long-term imaging and tracking of specific cell populations over time and is compatible with efficient RNAi-mediated gene inactivation.

Long-term In Vivo Tracking of Inflammatory Cell Dynamics Within Drosophila Pupae

To test the effect of a chemokine on macrophage recruitment in vivo, the whole mount in situ hybridization was used to detect the ectopic expression of the chemokine, and immunostaining was used to label macrophages. Live imaging was used for real-time observation of macrophage migration.

An Ectopic Chemokine Expression Model for Testing Macrophage Recruitment In Vivo

Zebrafish targeting reactive electrophiles and oxidants (Z-REX) is a chemical biology-based method for the investigation of reactive small-molecule signaling. This technique can be applied to live fish of different developmental stages. Here, we couple standard assays in zebrafish with Z-REX for signaling pathway analysis.

Monitoring On-Target Signaling Responses in Larval Zebrafish - Z-REX Unmasks Precise Mechanisms of Electrophilic Drugs and Metabolites

Learn live imaging of chemokine receptor dynamics in zebrafish neutrophils during wound responses. This protocol enables visualization of CXCR1 and CXCR2 trafficking to study immune cell behavior in vivo.

Live Imaging of Chemokine Receptors in Zebrafish Neutrophils During Wound Responses

Explore More Videos