Hematopoiesis with multilineage potential in the kidney and blood of adult runx1^W84X/W84X zebrafish. (A) Histologic sections of kidneys from WT and runx1^W84X/W84X zebrafish. Red arrows indicate hematopoietic cells; and yellow arrows, kidney tubules. The pictures are representative of kidneys from 10 each of the WT and runx1^W84X/W84X fish (original magnification ×400). (B) Flow cytometric analysis of kidney cells. FSC-A indicates forward scatter; and SSC-A, side scatter. Cell populations labeled with red circles are erythroid, blue for lymphoid, green for progenitors, and brown for myeloid. The numbers next to the circles indicate percentages for each cell population. The panels are representative of kidneys from 3 each of WT and runx1^W84X/W84X fish. (C) Peripheral blood cells in the WT and runx1^W84X/W84X zebrafish. Red arrows indicate erythrocytes; brown arrows, monocytes; blue arrows, lymphocytes; purple arrow, thrombocytes; green arrow, progenitor cell. Original magnifications ×100.

cd41-GFP^low cells in the AGM and kidney regions of runx1^W84X/W84X and E4I4-MO–injected embryos. (A) cd41-GFP⁺ cells in the WT and runx1^W84X/W84X larvae at 6 days after fertilization. The red and yellow arrows indicate stationary cd41-GFP⁺ cells in the pronephric duct and the AGM, respectively; and white arrow, cd41-GFP⁺ cells in circulation in the WT larva. (B-E) E4I4-MO data showing that the phenotype of the morphants is similar to that of the runx1^W84X/W84X larvae at 6 days after fertilization. (B) Schematic of the genomic organization of the runx1 gene: rectangles represent exons 1 to 9; lines connecting the exons (not to scale), introns; red line, E4I4-MO; and black arrows, RT-PCR primers. (C) RT-PCR with runx1 primers (top panel) and β-actin primers (bottom panel) from 2 embryos of each of the following groups: uninjected (UI), E4I4-2.5 ng injected, and E4I4-5 ng injected. (D) Table showing the number of injected embryos with different phenotypes at 2.5-ng and 5-ng doses of E4I4-MO. (E) cd41-GFP⁺ cells in the AGM (yellow arrow) and pronephric duct (red arrow) in the 2.5-ng injected morphant embryo at 6 days after fertilization (A,E: original magnifications ×50). (F-G) Lineage tracing showing colocalization of uncaged fluorescence and cd41-GFP in the same cells in the pronephros. (F) Schematic view of embryos 42 hours after fertilization, indicating the uncaged position of cd41-GFP⁺ cells in the ventral to dorsal aorta region (VDA), marked by a green cross. (G) Schematic view of embryos 5 days after fertilization, showing the pronephros region (marked red), which were imaged in panels H to M. (H-J) Double staining of flu (H), cd41:GFP (I), and merged view (J) of pronephros in WT embryos. (K-M) Double staining of flu (K), cd41:GFP (L), and merged view (M) of pronephros in runx1^W84X/W84X embryos. Arrows indicate flu, cd41:GFP costained cells. Flu indicates antiflu antibody detected using TSA-cy3 as substrate; and GFP, goat anti-GFP antibody as primary and anti–goat Alexa 488 antibody a secondary. The numbers of uncaged embryos exhibiting kidney signals were 3 of 12 and 8 of 12 for runx1^W84X/W84X and WT embryos, respectively; on average, there were 2 signals in each runx1^W84X/W84X embryo and 5 signals in each WT embryo. The GFP expression intensity in the runx1^W84X/W84X embryos seemed to be lower than that in the WT embryos, which may reflect a distinct cd41-GFP⁺ population that were formed or maintained in the presence of a truncated runx1.