Isolation of HSPCs from the zebrafish kidney. FCM analysis of KMCs was performed in a gata2a:GFP; runx1:mCherry double-transgenic zebrafish. (a) A scatter profile of KMCs. The SSC^low non-granulocytic cell fraction is gated. (b) SSC^low cells are subdivided into three distinct hematopoietic populations, gata2a⁺runx1⁺ (orange gate), gata2a⁻runx1⁺ (red gate), and gata2a⁺runx1⁻ (green gate). (c) Contour plot of FSC vs. SSC in KMCs, gata2a⁺runx1⁺, gata2a⁻runx1⁺, and gata2a⁺runx1⁻ cells. Dotted lines separate the “low”, “mid”, and “high” intensity of FSC.

Transcriptome analysis of three distinct hematopoietic populations. (a) Hierarchical clustering of selected HSC- (upper), erythroid-myeloid- (middle), and lymphoid-related genes (lower). (b) Gene ontology enrichment analysis of highly expressed genes in gata2a⁺runx1⁺ (upper), gata2a⁻runx1⁺ (middle), or gata2a⁺runx1⁻ cells (lower).

Expression of hematopoietic marker genes in HSPC populations. Relative expression levels of hematopoietic marker genes are shown. Orange, red, and green bars denote gata2a⁺runx1⁺, gata2a⁻runx1⁺, and gata2a⁺runx1⁻ cells, respectively. The expression level in the kidney tissue is shown as 1.0 in each panel. Error bars, s.d.

Transplantation assays of HSPC populations. (a) Experimental procedure of a competitive repopulation assay. One hundred BFP-labeled donor cells were co-transplanted with 50,000 of DsRed-labeled KMCs (competitors) into a sublethally irradiated recipient animal. At 16 wpt, donor chimerism was determined by the percentage of BFP⁺ cells within the total BFP⁺ and DsRed⁺ cells in each recipient. (b) Representative result of FCM analysis in a recipient transplanted with gata2a⁺runx1⁺ cells. (c) Percentage of donor chimerism in each recipient group (mean ± s.e.m).

Multilineage differentiation of gata2a⁺runx1⁺ cells. (a) Representative result of FCM analysis of KMCs in a recipient co-transplanted with BFP-labeled gata2a⁺runx1⁺ cells and DsRed-labeled competitors. G, granulocyte; P, precursor; L, lymphoid. (b) Percent distribution of gata2a⁺runx1⁺-derived BFP⁺ cells in “granulocyte”, “precursor”, and “lymphoid”. Data show the mean ± s.e.m. in the recipients (n = 6). (c) Relative expression level of lineage marker genes in isolated gata2a⁺runx1⁺-derived BFP⁺ cells in a recipient. Red dotted line indicates the expression level of each gene in competitor-derived DsRed⁺ cells.

Colony-forming assays of HSPC populations. (a) Representative images of a colony formed in the presence of Epoa (CFU-E) or Gcsfb (CFU-G). (b,c) Percentage (b) and absolute number (c) of CFU-E (red bars) and CFU-G (blue bars) in gata2a⁺runx1⁺, gata2a⁻runx1⁺, or gata2a⁺runx1⁻ cells in the kidney. Data are shown as mean ± s.e.m. *p < 0.01; **p < 0.001 by one-way ANOVA with Dunnett’s test. (d) Representative image of a large colony observed in the well plated with gata2a⁺runx1⁺ cells in the presence of Gcsfb. The image shows cells expressing BFP. (e,f) Representative images of a colony formed by gata2a⁺runx1⁺ cells in the presence of both Epoa and Gcsfb. Orange, red, green, and black arrowheads in (f) indicate gata2a⁺runx1⁺, gata2a⁻runx1⁺, gata2a⁺runx1⁻, and gata2a⁻runx1⁻ cells, respectively. (g) Percent distribution of individual GFP⁺ mCherry⁺ (orange bar), GFP⁻ mCherry⁺ (red bar), GFP⁺ mCherry⁻ (green bar), and GFP⁻ mCherry⁻ (black bar) cells at day 0, 3, and 7 of culture. Bars, 20 μm (a), 100 μm (d), 5 μm (e,f).

Hematopoietic differentiation in the zebrafish kidney. Schematic diagram of hematopoietic differentiation in the zebrafish kidney is shown. The orange, red, and green area denote the phenotypic transgene expression of gata2a:GFP and runx1:mCherry. Cross-hatched area shows the putative expression of gata2a:GFP and runx1:mCherry in hematopoietic progenitors. Long-term repopulating HSCs show gata2a⁺runx1⁺. In contrast, gata2a⁻runx1⁺ cells are mostly erythroid- and thrombocyte-primed progenitors, but also some myeloid-primed progenitors. Additionally, some myeloid-primed progenitors are gata2a⁺runx1⁻. Lymphoid-primed progenitors/precursors may express gata2a:GFP, but not runx1:mCherry.