Gcsfa and Gcsfb expand HSCs in the zebrafish embryo. (A) Maximum projection of multiple z-stack images of the dorsal aorta region of 48 hpf cmyb:GFP⁺; flk1:mCherry⁺ transgenic animals after injection of PBS (mock, top), gcsfa-MO, gcsfb-MO, and gcsfr-MO, in vitro–transcribed gcsfa, and in vitro transcribed gcsfb at the single-cell stage of development. White arrowheads (yellow cells) denote double-positive HSCs located between the dorsal aorta and cardinal vein. Fluorescence images taken on a Leica TCS SP5 inverted confocal system at ×250 and processed by Volocity (Perkin Elmer) and Photoshop (Adobe Systems) software. (B) Numbers of HSCs (cmyb:GFP⁺; flk1:mCherry⁺) were enumerated from individual confocal z-stacks from the dorsal aorta region of 48 hpf cmyb:GFP⁺; flk1:mCherry⁺ transgenic animals as shown in panel A. Mean (dashed red line) with 95% confidence interval (red error bars) and level of statistical significance. *P < .05; **P < .01; ***P < .0002. (C) Maximum projection of multiple z-stack images of the CHT region of embryos shown in panel A. White arrowheads (yellow cells) denote double-positive HSCs located in contact with the vascular plexus of the CHT. Fluorescence images taken on a Leica TCS SP5 inverted confocal system at ×250 and processed by Volocity (Perkin Elmer) and Photoshop (Adobe Systems) software. (D) Numbers of HSCs (cmyb:GFP⁺; flk1:mCherry⁺) were enumerated from individual confocal z-stacks from the CHT region of 48 hpf cmyb:GFP⁺; flk1:mCherry⁺ transgenic animals as shown in panel C. Mean (dashed red line) with 95% confidence interval (red error bars) and level of statistical significance. *P < .003; **P < .006; ***P < .0004; ****P < .0001.