Gcsfa and Gcsfb expand HSCs but not EMPs in the zebrafish embryo. (A) WISH of 24 hpf embryos for runx1 after injection of PBS (mock) or in vitro–transcribed gcsfa or gcsfb mRNA at the single-cell stage of development. Bright field images taken on a Leica M165C upright dissecting scope with a Leica DFC295 color digital camera at ×6 and processed by Photoshop (Adobe Systems) software. (B) Numbers of runx1⁺ cells along the dorsal aorta and PBI region quantitated from 3 independent experiments as in panel A. Mean (dashed red line) with 95% confidence interval (red error bars) and level of statistical significance. *P < .0001. (C) WISH of 36 hpf embryos for cmyb after injection of PBS (mock) or in vitro–transcribed gcsfa or gcsfb mRNA at the single-cell stage of development. Bright field images taken on a Leica M165C upright dissecting scope with a Leica DFC295 color digital camera at ×8 and processed by Photoshop (Adobe Systems) software. (D) Numbers of cmyb⁺ cells along the dorsal aorta and CHT region quantitated from 3 independent experiments as in panel C. Mean (dashed red line) with 95% confidence interval (red error bars) and level of statistical significance. *P < .02; **P < .0001. (E) Percentage of lmo2:GFP⁺; gata1:DsRed⁺ EMPs at 30 to 32 hpf (y-axis) after injection of PBS (mock, circles) or in vitro–transcribed gcsfa (squares) or gcsfb (triangles) mRNA at the single-cell stage of development. Each data point corresponds to 5 embryos pooled together before digestion and flow cytometry. Mean (dashed red line) with 95% confidence interval (red error bars) and level of statistical significance. (F) qRT-PCR analysis of gcsfr in FACS-isolated HSCs (cmyb:GFP⁺; flk1:DsRed⁺ cells at 48 hpf), EMPs (lmo2:GFP⁺; gata1:DsRed⁺ cells at 30 hpf), and adult zebrafish kidney (Whole Kidney). Levels are relative to the housekeeping gene ef1α. All samples are at least biological duplicate preparations. *P < .05; **P < .0003.