tfec overexpression augments the number and proliferation (G2/M phase) of HSPCs in the CHT. (A-B) ISH against cmyb and ikaros (markers of hematopoietic stem and progenitor cells [HSPCs]), prime panels depict the region indicated by the dashed outline box which highlights thymus staining. (C) Schematic indicating approximate imaging area in the tail, as indicated by the black box. (D) Confocal imaging in the CHT of double positive flk1:mCherry/cmyb :GPF embryos in NI or following hl-ztfec (+tfec) injection at 3 and 4 dpf. (F) Confocal imaging in the CHT of triple-positive flk1:eGPF/flk1:Cre/βactinSwitch:DsRed embryos in NI or after hl-ztfec (+tfec) injection at 3 and 4 dpf. (E,G) Analysis of embedded HSPCs at 3 dpf and HSPCs closely associated to endothelial cells at 4 dpf. (H) Imaging schematic at 3 or 4 dpf. (I-N) anti-GFP and pH3 immunostaining at 3 or 4 dpf of either noninjected (NI) or hl-ztfec-injected (+tfec) cmyb:eGPF embryos. (O) Quantification of the number of the pH3⁺ HSPCs in NI or hl-ztfec-injected embryos at 3 or 4 dpf. Scale bars represent 25 μm; error bars represent SEM. Statistical analysis was completed using an unpaired Student t test, comparing NI with hl-ztfec (+tfec)-injected embryos. NS, no significance. *P < .05; **P < .01. White arrows indicate embedded HSPCs at 3 dpf or closely associated HSPCs at 4 dpf in D and F and represent proliferating HSPCs in I-N.