We previously showed that side population (SP) cells, characterized by specific Hoechst dye efflux pattern in flow cytometric analysis, were present in teleost kidney hematopoietic tissue, and that kidney SP cells were enriched in hematopoietic stem cells (HSCs). ABCG2/Abcg2 is an ATP-binding cassette (ABC) transporter that is known to be associated with Hoechst dye efflux activity of mammalian HSCs. In the present study, we examined the expression and function of Abcg2 in kidney SP cells from zebrafish (Danio rerio). Although the zebrafish genome has 4 paralogous copies of ABCG2 (zAbcg2a, b, c, and d), zAbcg2a and zAbcg2c mRNA was expressed in kidney SP cells. Transfection of COS-7 cells with zAbcg2a and zAbcg2c showed that zAbcg2a was directly associated with the SP phenotype. These results indicate that zAbcg2a mRNA is a useful marker for zebrafish HSCs. In situ hybridization in kidney tissue showed that zAbcg2a-posi-
IntroductionSide population (SP) cells are identified from bone marrow based on their capacity to efflux of the fluorescent dye Hoechst 33342 (Hoechst), and are enriched in hematopoietic stem cells (HSCs). 1 Since the Hoechst dye efflux activity of HSCs is widely conserved in mammals, SP cell purification has been extensively used to enrich mammalian HSCs. [2][3][4] Recently, we showed that the hematopoietic activity of teleost SP cells isolated from kidney, the main hematopoietic organ in teleosts, 5,6 using the transplantation model system of clonal ginbuna (Carassius auratus langsdorfii, S3n strain) and ginbuna goldfish (Carassius auratus) hybrids (S4n strain). The SP cell population was clearly identified from ginbuna kidney. When the kidney SP cells from S3n were injected into S4n, all types of donor-derived blood cells (erythrocytes, neutrophils, basophils, monocytes, thrombocytes, and T and B lymphocytes) were detected in the recipient blood over a period of 9 months. 7 These findings indicate that SP cell purification can be used to enrich HSCs from not only mammals but also teleosts.Zebrafish (Danio rerio) has recently emerged as a powerful genetic model in which forward genetic screens can uncover novel genes involved in development, hematopoiesis, and human disease. 8 Although many homologues of human genes, including hematopoietic genes, have been identified, 9,10 stem-cell markers have not been shown in zebrafish. Therefore, little is known about the localization of HSCs and about stem-cell niche, a microenvironment supporting the maintenance and self-renewal of stem cells, in teleost kidney hematopoietic tissue. In murine bone marrow, ABCG2/Abcg2, an ATP-binding cassette (ABC) transporter associated with Hoechst dye efflux activity of HSCs, 11-15 is highly expressed in the most primitive HSCs and is sharply downregulated with differentiation. 11 In addition, the expression of Abcg2 gene in stem cells is highly conserved from various sources such as mouse skin, skeletal muscle, mammary grand, and testis, as well as human bone marrow. 11,[16][17][18] Abcg2 is thus used as a gener...