Stachura, D.L., Svoboda, O., Lau, R.P., Balla, K.M., Zon, L.I., Bartunek, P., and Traver, D. (2011) Clonal analysis of hematopoietic progenitor cells in the zebrafish. Blood. 118(5):1274-82.
Identification of hematopoietic progenitor cells in the zebrafish 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 utilizing recombinant zebrafish erythropoietin (Epo) and granulocyte colony stimulating factor (Gcsf). From adult whole kidney marrow (WKM), Epo was required to support erythroid colony formation, and Gcsf 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 following 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 following sublethal irradiation and demonstrated that recovery to pre-irradiation levels occurred by 14 days post-irradiation. Together, these studies 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.