Gcsfa and Gcsfb ligands both expand primitive neutrophils and mφs in the zebrafish embryo. (A) Fluorescence images of 24-hpf lyz:GFP embryos injected at the 1-cell stage of development with PBS (mock) and either gcsfa or gcsfb mRNA. Fluorescent images taken on a Leica DMI-6000 inverted fluorescent scope with a Hamamatsu Photonics Orca 3CCD color digital camera (Hamamatsu, Japan) at ×50 and processed by Volocity (Perkin Elmer, MA) and Photoshop (Adobe Systems, San Jose, CA) software. (B) Numbers of lyz:GFP⁺ cells at 22 to 24 hpf (y-axis) after injection of scrambled MO (mock, circles) and in vitro transcribed gcsfa (squares) or gcsfb (triangles) mRNA. Mean (dashed red line) with 95% confidence interval (red error bars) and level of statistical significance. *P < .02; **P < .0001. (C) Percentage of mpeg1:GFP⁺ cells at 22 to 24 hpf (y-axis) after injection of PBS (mock, circles) or in vitro transcribed gcsfa (squares) or gcsfb (triangles) mRNA. 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. *P < .02; **P < .001. N.S., no significance. (D) Primitive mφs and neutrophils express the gcsf receptor. qRT-PCR analysis of gcsfr in FACS-isolated primitive mφs (mpeg1:GFP⁺ cells at 20-24 hpf), primitive neutrophils (mpx:GFP⁺ and lyz:GFP⁺ cells at 20-24 hpf), and adult zebrafish kidney (Whole Kidney). Levels are relative to the housekeeping gene ef1α. All samples are at least biological duplicate preparations. Bars represent the mean, and error bars represent standard error of the mean (SEM). *P < .05.