Figure 3
Figure 3. Gain-of-function mutations in the zebrafish HIF2A ortholog hif2aa cause a dominant active-like response in neutrophil behavior and result in increased collagen deposition following zebrafish tailfin transection. hif2aa G487R or G487W RNA (177 pg) or dominant active (da) hif1ab control was injected into 1-cell stage zebrafish mpx:GFP embryos. (A) Whole body total neutrophil numbers at 2 dpf were not altered by injection of hif2aa G487 variants; n = 44 performed as 3 independent experiments. (B-E) Tailfin transection was performed at 2 dpf, and neutrophils were counted at 6 and 24 hours postinjury (hpi). Data shown are mean ± SEM. (B) Injection of dominant active hif2aa variants did not alter the recruitment of neutrophils to the tailfin injury site after 6 hpi. n = 24 performed as 3 independent experiments. (C-D) hif2aa G487 mutations caused a significant increase in neutrophil number at 24 hpi compared with phenol red injected negative controls. (C) Representative overlaid fluorescence and brightfield micrographs (original magnification ×4) imaged on a TE2000U inverted microscope (Nikon, Kingston upon Thames, United Kingdom) at constant exposure. (D) Neutrophil numbers at 24 hpi. n = 24 performed as 3 independent experiments. (E) Injection of hif2aa G487 mutations significantly decreased the percentage of neutrophils at the injury site colabeled with terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling apoptosis staining at 12 hpi. n = 3 performed as independent experiments containing 17 to 36 embryos per injection group per repeat. (F) Evidence of hif2aa target gene activation in zebrafish embryos with overexpressed G487 mutant constructs. Photomicrographs of 24 hpf embryos after injection of dominant active forms of hif2aa RNA (177pg) or dominant negative (dn) hif2aa at the 1-cell stage. Embryos were stained for phd3 expression by in situ hybridization as a representative hif2aa target gene. (G-H) hif2aa G487R or G487W RNA (177pg) or dominant active (da) hif1ab control was injected into 1-cell stage zebrafish mpx:GFP embryos. Tailfins were transected 2 dpf. (G) Representative bright field and fluorescence micrographs (original magnification ×4) of tailfins taken 72 hpi and stained with anti-collagen1 antibody and an anti-mouse Alexa 488 secondary antibody. (H) Mean fluorescence intensity was analyzed using Volocity 5 software. Data represent n = 45 embryos per group performed as 3 independent experiments.

Gain-of-function mutations in the zebrafish HIF2A ortholog hif2aa cause a dominant active-like response in neutrophil behavior and result in increased collagen deposition following zebrafish tailfin transection.hif2aa G487R or G487W RNA (177 pg) or dominant active (da) hif1ab control was injected into 1-cell stage zebrafish mpx:GFP embryos. (A) Whole body total neutrophil numbers at 2 dpf were not altered by injection of hif2aa G487 variants; n = 44 performed as 3 independent experiments. (B-E) Tailfin transection was performed at 2 dpf, and neutrophils were counted at 6 and 24 hours postinjury (hpi). Data shown are mean ± SEM. (B) Injection of dominant active hif2aa variants did not alter the recruitment of neutrophils to the tailfin injury site after 6 hpi. n = 24 performed as 3 independent experiments. (C-D) hif2aa G487 mutations caused a significant increase in neutrophil number at 24 hpi compared with phenol red injected negative controls. (C) Representative overlaid fluorescence and brightfield micrographs (original magnification ×4) imaged on a TE2000U inverted microscope (Nikon, Kingston upon Thames, United Kingdom) at constant exposure. (D) Neutrophil numbers at 24 hpi. n = 24 performed as 3 independent experiments. (E) Injection of hif2aa G487 mutations significantly decreased the percentage of neutrophils at the injury site colabeled with terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling apoptosis staining at 12 hpi. n = 3 performed as independent experiments containing 17 to 36 embryos per injection group per repeat. (F) Evidence of hif2aa target gene activation in zebrafish embryos with overexpressed G487 mutant constructs. Photomicrographs of 24 hpf embryos after injection of dominant active forms of hif2aa RNA (177pg) or dominant negative (dn) hif2aa at the 1-cell stage. Embryos were stained for phd3 expression by in situ hybridization as a representative hif2aa target gene. (G-H) hif2aa G487R or G487W RNA (177pg) or dominant active (da) hif1ab control was injected into 1-cell stage zebrafish mpx:GFP embryos. Tailfins were transected 2 dpf. (G) Representative bright field and fluorescence micrographs (original magnification ×4) of tailfins taken 72 hpi and stained with anti-collagen1 antibody and an anti-mouse Alexa 488 secondary antibody. (H) Mean fluorescence intensity was analyzed using Volocity 5 software. Data represent n = 45 embryos per group performed as 3 independent experiments.

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