Accumulation of Cd41-eGFP⁺ HSPCs in cmyb^hkz3 mutants results from crippled egress of HSPCs from the VDA. (A-B) Still image sequence extracted from confocal microscopic recording of red boxed VDA area in cmyb^hkz3/Tg(cd41:eGFP) mutant embryo (B) and sibling (A) between 50 and 60 hpf in each 3-minute interval. Corresponding movies for panels A and B are shown in supplemental Videos 1 and 2, respectively. T0 is the still confocal image taken at the beginning of imaging with black number indicating cells eventually undergoing egress within the recording period. Numbered HSPCs (yellow arrows) were released into circulation at time points indicated above each panel. A, aorta; V, vein. (C) A corresponding summary of the sequence of cells exiting from the VDA in sibling (A) and cmyb^hkz3 mutant (B). (D) Quantification of the numbers of Cd41-eGFP⁺ HSPCs exiting from the VDA in a 5-hour recording period in 2- to 2.5-hpf embryos (n = 5, mean ± SD). An asterisk indicates significant difference with wild type (t test, P < .05).