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Transplantation and in vivo imaging of multilineage engraftment in zebrafish bloodless mutants

Nature Immunology volume 4, pages 1238–1246 (2003)Cite this article

Abstract

The zebrafish is firmly established as a genetic model for the study of vertebrate blood development. Here we have characterized the blood-forming system of adult zebrafish. Each major blood lineage can be isolated by flow cytometry, and with these lineal profiles, defects in zebrafish blood mutants can be quantified. We developed hematopoietic cell transplantation to study cell autonomy of mutant gene function and to establish a hematopoietic stem cell assay. Hematopoietic cell transplantation can rescue multilineage hematopoiesis in embryonic lethal gata1−/− mutants for over 6 months. Direct visualization of fluorescent donor cells in embryonic recipients allows engraftment and homing events to be imaged in real time. These results provide a cellular context in which to study the genetics of hematopoiesis.

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Figure 1: Separation of major blood cell lineages from WKM by light-scatter characteristics.
Figure 2: Analysis of zebrafish transgenic lines by flow cytometry.
Figure 3: Flow cytometry profiling of zebrafish blood mutants.
Figure 4: Hematopoietic cell transplantation rescues lethality in gata1−/− mutants.
Figure 5: Transplantation of WKM from double-transgenic donors allows independent visualization of leukocytes and erythrocytes in translucent recipient embryos.

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Acknowledgements

We thank A. Kiger, N. Trede, and P. Ernst for critical evaluation of the manuscript; H. Stern, J. Amatruda, and M. Fleming for providing sections and review of histology; A. Flint and M. Handley for assistance with flow cytometry; and D. Langenau and T. Look, and H.F. Lin and R. Handin, for providing rag2eGFP and itga2beGFP transgenic zebrafish, respectively, prior to publication. We thank R. Wingert and J. Cope for providing mutant lines; H. Zhu for assistance with the gata1dsRED line; and A. Winzeler for technical assistance. Supported by the Irvington Institute for Immunological Research (D.T.); the Howard Hughes Medical Institute (B.P. and L.I.Z.); the National Institutes of Health (B.P. and L.I.Z.) and the William Randolph Hearst Foundation (B.P.); and the Grousbeck family and Legal Sea Foods (L.I.Z.).

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Authors and Affiliations

  1. Children's Hospital Boston and the Howard Hughes Medical Institute, 320 Longwood Avenue, Enders 720, Boston, 02115, Massachusetts, USA

    David Traver, Barry H Paw & Leonard I Zon

  2. Department of Cell Biology, Box 3709, Duke University Medical Center, Durham, 27710, North Carolina, USA

    Kenneth D Poss

  3. University of California at Los Angeles, 621 Charles Young Drive South, LS4325, Los Angeles, 90095, California, USA

    W Todd Penberthy & Shuo Lin

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Correspondence to Leonard I Zon.

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Supplementary information

Supplementary Video 1.

Hematopoietic cell transplantation rescues erythropoiesis and survival of gata-1-/- mutants. Individual gata-1-/- recipient at 8 weeks post-transplantation, showing numbers of donor-derived GFP+ cells similar to those in age-matched GATA-1eGFP donor animals. (MOV 1013 kb)

Supplementary Video 2.

Transplantation of whole kidney marrow from double transgenic donors allows independent visualization of leukocytes (GFP+) and erythrocytes (dsRED+) in translucent recipient embryos. On day 1 post-transplantation, videomicroscopy of tail vessels in gata-1-/- recipient showed slow-moving, round leukocytes, larger leukocytes displaying an end-over-end tumbling migration, and rapidly circulating erythrocytes. (MOV 1855 kb)

Supplementary Video 3.

Transplantation of DP WKM leads to rapid engraftment of hematopoietic sites in bloodless recipients. Ventral view of a bloodless recipient at 5 days post-transplantation. Robust engraftment of one thymic lobe by GFP+ leukocytes is seen between the eye and the pectoral fin on the right side (top) of the larva. (MOV 3048 kb)

Supplementary Video 4.

bloodless recipients display sustained, multilineage hematopoiesis from donor-derived cells. Robust reconstitution of dsRED+ erythrocytes and GFP+ leukocytes as observed in the tail capillaries of a bloodless recipient at 8 weeks post-transplantation. (MOV 2593 kb)

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Traver, D., Paw, B., Poss, K. et al. Transplantation and in vivo imaging of multilineage engraftment in zebrafish bloodless mutants. Nat Immunol 4, 1238–1246 (2003). https://doi.org/10.1038/ni1007

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