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Blood, Vol. 112, Issue 7, 2969-2972, October 1, 2008
Carboxypeptidase A5 identifies a novel mast cell lineage in the zebrafish providing new insight into mast cell fate determination
Blood Dobson et al.
112: 2969
Supplemental materials for: Dobson et al
Files in this Data Supplement:
- Figure S1. Time course of cpa5 expression in zebrafish embryos (JPG, 77.3 KB)
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Whole mount in situ hybridization using a digoxigenin-labeled RNA anti-sense probe to zebrafish cpa5 in zebrafish embryos at (A) 24 hpf, (B) 28 hpf, (C) 72 hpf, and (D) 5 dpf. By 72 hpf, cpa5 expression is seen in both mast cells and in the pancreas (black arrowhead, hpf = hours post fertilization, dpf = days post fertilization). Lateral views, anterior left and dorsal at the top (5× objective, Leica, Wetzlar, Germany).
- Figure S2. Cytospin of FACS sorted FITC-labeled Fast Red-stained cpa5 positive cells isolated from zebrafish embryos demonstrate mast cell morphology (JPG, 48 KB)
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Cell suspensions from embryos at both 28 hpf and 7 dpf were sorted by FITC expression at 515–545 nm (FACSAria machine, Becton Dickinson, Franklin Lakes, NJ). The population with the highest fluorescent signal was collected and resorted by FITC and Fast Red at 600–620 nm and subjected to cytospin preparation. (A) Immature mast cells isolated at 28hpf. (B) Mature mast cells isolated at 7dpf. cpa5 positivity was confirmed by fluorescent imaging (100× objective/1.4NA oil immersion, Zeiss Axioplan 2, Zeiss, Jena, Germany, Zeiss Axiocam HRc digital camera, AxioVision 4.6 with Multi Channel Fluorescence Module, Diagnostic Instruments Inc., Sterling Heights, MI). Comparative imaging of cytospins of human mast cell progenitors and mature mast cells were originally published in Blood. Kirshenbaum AS, Goff JP, Semere T, Foster B, Scott LM, Metcalfe DD. Demonstration that human mast cells arise from a progenitor cell population that is CD34(+), c-kit(+), and expresses aminopeptidase N (CD13). Blood 1999;94(7): 2333–42 (c) the American Society of Hematology (hpf = hours post fertilization, dpf = days post fertilization, TB = toluidine blue, WG = Wright Giemsa).
- Figure S3. cpa5 positive cells colocalize with a proportion of cells expressing several myeloid markers (JPG, 188 KB)
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Double whole mount in situ hybridization using a digoxigenin-labeled RNA anti-sense probe to zebrafish cpa5 (blue) and FITC-labeled RNA anti-sense probe (red) to pu.1, mpo, l-plastin, and lysozyme C demonstrate coexpression of cpa5 in a proportion of cells expressing these myeloid markers (panel i yolk sac, panel ii tail, 5× objective, Leica MZ16F, Leica Application Suite V2.4.0R, Leica Microsystems (Switzerland) Ltd.). Evidence of coexpression is shown by colocalization observed in higher magnification images of selected cells (panel iii brightfield, panel iv fluorescence (10× objective, Leica MZ16F, Leica Application Suite V2.4.0R.). No colocalization is observed for fms and cebp-α (panel i brightfield, panel ii fluorescence (8× objective, Leica MZ16F, Leica Application Suite V2.4.0R. Panels assembled using Adobe Photoshop CS3 Extended V10.0, San Jose, CA).
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