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    • br Experimental Procedures br Author

    2018-10-20


    Experimental Procedures
    Author Contributions O.G. performed the analysis, visualization, qRT-PCR, and writing of the manuscript; K.M. carried out Hlf functional experiments; N.B. helped with the computational analysis; Y.G. performed the cell culture and functional assays; M.F. did some of the qRT-PCR; H.N.G. and T.S. undertook part of the initial analysis of the ImmGen arrays and most of the RNA-seq analysis; R.G. supervised the study and wrote the manuscript.
    Acknowledgments We thank Ayla Ergun for initial discussions; Dan Peer for expert advice; Marianna Romzova for qRT-PCR advice; Vered Chalifa-Caspi and Inbar Plaschkes for their essential bioinformatics analysis of arrays and help with RNA-seq visualization on the UCSC browser. This study was supported by ISF grants 1690/13 and 2348/15, and CIG grant 618647.
    Introduction The zebrafish is a useful organism to model hematopoietic cell transplantation (HCT). As in mammals, transplanted zebrafish hematopoietic stem/progenitor MG 262 (HSPC) collected from whole kidney marrow (WKM) are able to repopulate all hematopoietic lineages and provide long-term reconstitution in irradiated recipient fish (Traver et al., 2004). Furthermore, many of the biological aspects important for successful HCT in mammals are conserved in zebrafish. Two recent examples include the role of major histocompatibility complex matching and our prior work demonstrating the role of stromal derived factor-1 (SDF-1) in HSPC homing activity (de Jong et al., 2011; Glass et al., 2011, 2013). Cellular engraftment in adult zebrafish is determined by analyzing the WKM of the recipients, typically by measuring the fluorophore-labeled donor cells using flow cytometry. With the development of transparent Casper fish (White et al., 2008), the fluorescent hematopoietic cells from the donor can also be monitored in vivo via live imaging, which could provide a more complete picture of the hematopoietic recovery process after transplant. However, despite its rapid acquisition time and high resolution, the sensitivity of fluorescent imaging can be severely reduced by high background noise and limited tissue penetration, preventing the detection of low signals in deep tissue, such as those during hematopoietic cell homing and early engraftment in the kidney within the first few days after HCT. Bioluminescence imaging (BLI), on the other hand, has an excellent signal-to-noise ratio, as there is virtually no background in the tissues (Lin et al., 2008). In murine HCT, donor cell tracking by non-invasive BLI can reveal the dynamics of different hematopoietic cell repopulation in the recipients (Cao et al., 2004; Wang et al., 2003). Although in mice, robust BLI is generated 7–8 days post-HCT, the optical clarity of the zebrafish is ideal for the development of BLI to track hematopoietic cell homing function within the first few days after HCT. To explore the suitability of BLI for tracking the transplanted donor hematopoietic cells, we generated ubi:luc zebrafish that ubiquitously expressed firefly luciferase under control of the luciferase promoter and used this transgenic line as a WKM donor in HCT. We showed that, using BLI, luciferase-expressing donor hematopoietic cells could be continuously monitored in the same individual to demonstrate the kinetics of the hematopoietic reconstitution following transplantation in adult zebrafish. Furthermore, we demonstrate that this BLI-based system has use as a “functional” chemical screen of small molecules that enhance homing and engraftment.
    Results
    Discussion The ability to perform HCT in zebrafish has expanded our ability to study vertebrate HSPC and transplant biology. In vivo optical imaging, in contrast to post-mortem analysis of donor cell engraftment, can non-invasively analyze temporal-spatial information of the transplanted hematopoietic cells over time. Unlike fluorescence-based screens, biological tissues do not have inherent bioluminescence giving BLI the advantage of a high signal-to-noise ratio. Furthermore, the wavelength of the light emitted from firefly luciferase-expressing cells is sufficient to produce good tissue penetration in small animals. BLI can be performed with short exposure times allowing a greater number of animals to undergo image acquisition simultaneously and increase throughput. The sensitivity of BLI can be further increased when the transparent adult zebrafish, Casper (derived from mating roy and nacre pigment mutants), are used as their light absorption is minimal (White et al., 2008). BLI, being a rapid and sensitive quantitative technique, can facilitate a variety of medium-throughput screens and will be particularly advantageous to use with zebrafish, which is a logistically and financially attractive model for drug discovery.