Fig. 3

(A) Fusion construct encoding p2A-linked proteins mito-APEX2, APEX2-H2B, and mCherry that localize to the mitochondria, nucleus, and cytoplasm, respectively. The draculin promoter was used to transiently drive strong mosaic expression in HSPCs. Random insertion in the genome was by Tol2-mediated transgenesis. (B) Tol2 draculin:mito-APEX2_p2A_APEX2-H2B_p2A_mCherry (drl:APEX2-mCherry) fusion construct was injected together with tol2 mRNA in one cell wild type zebrafish embryos. (C) At 5 days post fertilization (dpf), embryos with circulating mCherry⁺ HSPCs were visually screened and retro-orbitally injected with alpha bungarotoxin to paralyze the embryo, and Oregon Green dye to label the vasculature. (D) Dye-injected mCherry⁺ double positive embryos were visually screened and used for light sheet microscopy (example shows a 439 × 439 × 115 µm³ volume of the anterior kidney marrow (KM); ISVs, intersegmental vessels; yellow dotted line, DA, dorsal aorta; gut AF, gut autofluorescence). (E) Brightfield example of a single embryo after fixation and DAB (3,3'-diaminobenzidine) staining to label APEX2⁺ HSPCs that are located within the dotted box (dotted line marks DA, dorsal aorta; Y, yolk; D, dorsal; V, ventral; A, anterior; P, posterior). (F) After embedding, the sample was oriented and trimmed based on images acquired using micro-computed tomography (microCT) (example shows orthogonal sections in three planes, N; notochord, G; glomerulus, S; swim bladder). (G) Single plane from ~3000 sections of SBEM data (example shows a 233 × 331 × 213 µm³ volume; s1-s5, somites 1–5; G, glomerulus; PD, pneumatic duct).