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.