Key Points Two Gcsf ligands function redundantly through the Gcsf receptor to promote myelopoiesis in zebrafish. Gcsf signaling is required for HSPC emergence and expansion in zebrafish.

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 lineagespecific 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. (Blood. 2011;118(5): 1274-1282) IntroductionThe majority of cells within the hematopoietic system are postmitotic and relatively short lived, requiring continuous replenishment throughout life. The production of all blood cells is dependent on the actions of hematopoietic stem cells (HSCs), exceedingly rare cells that both self-renew and generate lineage-restricted progenitors. It is through the geometric amplification of these committed progenitors that the vast numbers of mature cells required to sustain life are produced daily. Commitment of HSCs to each of the hematopoietic lineages occurs through a hierarchy of progenitors and precursors, with lineage potential lost with each stepwise differentiation event. The development of mature effector cells from upstream HSCs, multipotent, oligopotent, and unipotent precursors has served as a paradigm for tissue-replenishing stem cell systems.Whereas long-term reconstitution of lethally irradiated mice remains the standard for HSC function, in vitro culture methods have been instrumental in determining the branchpoints of the hematopoietic tree. The development of clonal in vitro cultures by Metcalf and colleagues in the 1960s enabled the growth of murine bone marrow progenitors 1 and the study and quantitation of progenitor number during hematologic disease 2 and exposure to irradiation. 3 These assays were used to investigate the ontogeny of the developing murine hematopoietic system 4 and refined to stud...

Forward genetic screens in zebrafish have been utilized to identify genes essential for the generation of primitive blood and the emergence of hematopoietic stem cells (HSCs), but have not elucidated genes essential for hematopoietic stem and progenitor cell (HSPC) proliferation and differentiation due to a lack of methodologies to functionally assess these processes. We previously described techniques to test the developmental potential of HSPCs by culturing them on zebrafish kidney stromal (ZKS) cells, derived from the main site of hematopoiesis in the adult teleost. Here we describe an additional primary stromal cell line we refer to as zebrafish embryonic stromal trunk (ZEST) cells, derived from tissue surrounding the embryonic dorsal aorta, the site of HSC emergence in developing fish. ZEST cells encouraged HSPC differentiation towards the myeloid, lymphoid, and erythroid pathways when assessed by morphological and qRT-PCR analyses. Additionally, ZEST cells significantly expanded the number of cultured HSPCs in vitro, indicating that these stromal cells are supportive of both HSPC proliferation and multilineage differentiation. Examination of ZEST cells indicates that they express numerous cytokines and Notch ligands, and possess endothelial characteristics. Further characterization of ZEST cells should prove to be invaluable in understanding the complex signaling cascades instigated by the embryonic hematopoietic niche required to expand and differentiate HSPCs. Elucidating these processes and identifying possibilities for the modulation of these molecular pathways should allow the in vitro expansion of HSPCs for a multitude of therapeutic uses.