FIG 1 

FIG 1 

Development of a Shigella-zebrafish infection model to study emergency granulopoiesis. (A to C) At 2 dpf, lyz::dsRed zebrafish larvae with red neutrophils were injected in the HBV with PBS or a low dose (0.5 × 10³ to 2.0 × 10³ CFU) of GFP⁺S. flexneri M90T and imaged by fluorescence stereomicroscopy. (A) Representative images of the infection site in a single larva over time (Movie S1). Scale bars, 100 µm. (B) Schematic of the zebrafish larva, highlighting the AGM region of neutrophil and HSC quantifications. Shown are representative images of the AGM of PBS-injected or Shigella-infected larvae at 48 hpi. Scale bars, 100 µm. (C) Quantifications of neutrophils from PBS-injected (open circles) or Shigella-infected (closed circles) larvae as in panel B. Circles represent counts from individual larvae. Data were pooled from 4 independent experiments using n ≥ 10 larvae per condition per experiment. Means ± SEM are shown (horizontal bars). P values between conditions at cognate time points were determined by unpaired two-tailed Student’s t test. Significance was defined as P < 0.05. ***, P < 0.001. (D) Tg(runx::mCherry)/Tg(mpx::GFP) larvae with red HSPCs and green neutrophils were injected at 2 dpf in the HBV with a low dose of S. flexneri M90T, and images of the AGM were captured by confocal microscopy (100× objective). Dashed arrows represent vasculature. DA, dorsal aorta; CV, cardinal vein. Cells expressing high levels of mCherry (*) were considered HSCs; cells expressing lower levels of mCherry were considered HPCs (i.e., HSC progeny [arrowheads]). Maximum-intensity z-projection images are shown. Scale bars, 20 µm. (E) Tg(runx::GFP) larvae with green HSCs were injected with a low dose of mCherry⁺S. flexneri M90T as in panel D. HSCs in the AGM of PBS-injected (open circles) or Shigella-infected (closed circles) larvae were quantified at 48 hpi. Circles represent counts from individual larvae. Data were pooled from 3 independent experiments using n ≥ 6 larvae per condition per experiment. Means ± SEM are shown (horizontal bars). P values between conditions were determined by unpaired two-tailed Student’s t test. Significance was defined as P < 0.05. **, P < 0.01. (F) lyz::dsRed larvae were treated with control (Ctrl) or Gcsfr morpholino oligonucleotide (Mo). Morphants were injected in the HBV at 2 dpf with PBS (open circles) or a low dose of GFP⁺S. flexneri M90T (closed circles). Larvae were imaged by fluorescent stereomicroscopy, and neutrophils in the AGM were quantified at 48 h following treatment. Each circle represents a count from an individual larva. Means ± SEM (horizontal bars) are shown. Data were pooled from three independent experiments using n ≥ 6 larvae per condition per experiment. P values between conditions were determined by unpaired two-tailed Student’s t test. Significance was defined as P < 0.05. ***, P < 0.001. (G) lyz::dsRed larvae were treated with control or Irf8 morpholino oligonucleotide. Morphants were injected in the HBV at 2 dpf with a low dose of GFP⁺S. flexneri M90T. Neutrophil quantifications from the AGM of infected larvae are shown. Circles represent counts from individual larvae. Data were pooled from 3 independent experiments using n ≥ 4 larvae per condition per experiment. Means ± SEM are shown (horizontal bars). P values between conditions were determined by unpaired two-tailed Student’s t test. Significance was defined as P < 0.05.