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. 2011 Aug 4;118(5):1274-82.
doi: 10.1182/blood-2011-01-331199. Epub 2011 Mar 17.

Clonal Analysis of Hematopoietic Progenitor Cells in the Zebrafish

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Free PMC article

Clonal Analysis of Hematopoietic Progenitor Cells in the Zebrafish

David L Stachura et al. Blood. .
Free PMC article

Abstract

Identification of hematopoietic progenitor cells in the zebrafish (Danio rerio) has been hindered by a lack of functional assays to gauge proliferative potential and differentiation capacity. To investigate the nature of myeloerythroid progenitor cells, we developed clonal methylcellulose assays by using recombinant zebrafish erythropoietin and granulocyte colony-stimulating factor. From adult whole kidney marrow, erythropoietin was required to support erythroid colony formation, and granulocyte colony-stimulating factor was required to support the formation of colonies containing neutrophils, monocytes, and macrophages. Myeloid and erythroid colonies showed distinct morphologies and were easily visualized and scored by their expression of lineage-specific fluorescent transgenes. Analysis of the gene-expression profiles after isolation of colonies marked by gata1:DsRed or mpx:eGFP transgenes confirmed our morphological erythroid and myeloid lineage designations, respectively. The majority of progenitor activity was contained within the precursor light scatter fraction, and more immature precursors were present within the lymphoid fraction. Finally, we performed kinetic analyses of progenitor activity after sublethal irradiation and demonstrated that recovery to preirradiation levels occurred by 14 days after irradiation. Together, these experiments provide the first report of clonal hematopoietic progenitor assays in the zebrafish and establish the number, characteristics, and kinetics of myeloerythroid progenitors during both steady-state and stress hematopoiesis.

Figures

Figure 1
Figure 1
Zebrafish G-csf and Epo increase colony formation from unfractionated mpx:eGFP WKM. (A) CFUs/100 000 unfractionated WKM cells plated in methylcellulose with combinations of carp serum, G-csf, or Epo added. Bars represent average of at least 3 independent experiments, with error bars representing SD. Black bars represent Mpx:eGFP colonies and green bars represent Mpx:eGFP+ colonies. (B) Brightfield (top rows) and GFP fluorescent images (bottom rows) of representative colonies enumerated in panel A. All images were taken at 50×. Scale bar in top left panel is 50 μm.
Figure 2
Figure 2
Precursor and lymphoid fractions of WKM contain myeloid and erythroid progenitors. (A) Experimental schematic for isolation and culture of mpx:eGFP; gata1:DsRed cells from precursor (blue) and lymphoid (purple) fraction of adult WKM. (B) CFUs/100 000 mpx:eGFP, gata1:DsRed precursor cells plated in methylcellulose with combinations of carp serum, G-csf, or Epo added. Red bars represent gata1:DsRed+ colonies, green bars represent mpx:eGFP+ colonies, and black bars represent negative colonies generated from the precursor fraction. (C) CFUs/100 000 mpx:eGFP, gata1:DsRed lymphoid cells plated in methylcellulose with combinations of carp serum, G-csf, or Epo added. Red bars represent gata1:DsRed+ colonies, green bars represent mpx:eGFP+ colonies, and black bars represent negative colonies generated from the lymphoid fraction. Bars represent average of at least 3 independent experiments, with error bars representing SD.
Figure 3
Figure 3
G-csf encourages myeloid differentiation, whereas Epo encourages erythroid differentiation from zebrafish hematopoietic progenitors. (A) Brightfield images (top row), mpx:eGFP fluorescence (middle row), and gata1:DsRed fluorescence (bottom row) of colonies grown in various growth factor conditions from the precursor fraction of WKM (conditions listed along top row of images). (B) Brightfield images (top row), mpx:eGFP fluorescence (middle row), and gata1:DsRed fluorescence (bottom row) of colonies grown in various growth factor conditions from the lymphoid fraction of WKM (conditions listed along top row of images). All images photographed at magnification 50×. Scale bars in top left panels are 50 μm. Arrowheads in top right panels denote mixed (CFU-granulocyte, erythroid, macrophage colonies) colonies.
Figure 4
Figure 4
G-csf encourages myeloid differentiation as assayed by morphology and gene expression of isolated colonies. (A) Cytocentrifuged colonies isolated from precursor (top) and lymphoid (bottom) fraction methylcellulose cultures were stained with May-Grünwald-Giemsa. Tight colonies were isolated from cultures with only carp serum and Epo (left column), whereas ruffled and spread colonies were isolated from cultures containing carp serum and G-csf (right column). All images were photographed at magnification 1000×; scale bar in bottom right is 20 μm. After photographing, cells were cut and pasted from multiple fields to create a composite image. (B) Reverse transcription-PCR analysis of colonies isolated from precursor (top) and lymphoid (bottom) fraction of methylcellulose cultures. Colony morphology is listed on left, and genes assayed are listed along top of gel images.
Figure 5
Figure 5
Kinetic analysis of myeloerythroid progenitor recovery after sublethal ionizing radiation. (A) CFUs generated from mpx:eGFP; gata1:DsRed DN cells after recovery from 25 Gy irradiation. (B) Ratio of CFUs generated to number of mpx:eGFP; gata1:DsRed DN cells isolated and cultured from the lymphoid (●) and precursor (■) light scatter fractions (as diagrammed in Figure 2A). The x-axis is days after 25 Gy irradiation; day 0 fish were not irradiated. Data points represent the average of 3 individual biologic replicates performed in triplicate, and error bars represent SD.

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