Figure 3.
Expression of zebrafish Jagn1bΔT12-G14 results in neutropenia, without affecting other hematopoietic cells. (A) Top panel shows a schematic representation of identified CN-associated mutations in human JAGN1 gene. The bottom panel shows the tested mutations with the corresponding approach in this study. Note, to mimic the deletion (ΔT12-G14) we overexpressed jagn1bΔT12-G14 mRNA. Further, we targeted the transmembrane domain using CRISPR/Cas9 gene editing to introduce a truncating mutation. (B-C) Quantitative analysis of the cells expressing the neutrophil marker lysozyme C (lyz), stained with WISH in (B) jagn1b TM crispants and (C) after jagn1bΔT12-G14 overexpression alone or in combination with jagn1b morpholino. Each dot represents the number of cells in the hematopoietic tissue of a 2.25 dpf individual embryo. Data are presented as mean ± standard deviation. The data represent combined results from 2 independent experiments. N represents the total number of analyzed embryos. (D-E) Representative images of lyz+ neutrophils in (D) TM crispants and (E) after overexpression of jagn1bΔT12-G14 alone or in combination with jagn1b morpholino. Images were taken with 10× magnification. Scale bars represent 100 μm. (F-I) Effect of overexpression of jagn1bΔT12-G14 on other hematopoietic cells. The left panels show quantitative analyses of stained cells with WISH against hematopoietic cell-specific marker. The right panels show representative images of the WISH-stained embryos. The cells were stained against (F) cmyb, (G) spi1b, (H) mpeg1.1 all 3 in 2.25 dpf embryos and (I) hbae1.1 in 1 dpf embryos. (F,I) Comparison of the percentage (%) of normal gene expression pattern between wild-type embryos compared to that carrying jagn1bΔT12-G14 mutation. (G-H) Numbers of stained cells in the hematopoietic region. Each dot represents the number of cells in the hematopoietic tissue of an individual 2.25 dpf embryo. Data are presented as mean ± standard deviation. The data represent combined results from 2 independent experiments. N represents the total number of analyzed embryos. Pictures were taken with 8× to 10× magnification. Scale bars represent 100 μm. (J) Representative images of intracellular Jagn1 protein localization (GFP signal) after overexpression of gfp-jagn1bWT and gfp-jagn1bΔT12-G14 mRNA in draculin:mCherry-positive HSPCs at 20 hours postfertilization (hpf). ns, not significant; ∗∗P < .01; ∗∗∗∗P < .0001.

Expression of zebrafish Jagn1bΔT12-G14 results in neutropenia, without affecting other hematopoietic cells. (A) Top panel shows a schematic representation of identified CN-associated mutations in human JAGN1 gene. The bottom panel shows the tested mutations with the corresponding approach in this study. Note, to mimic the deletion (ΔT12-G14) we overexpressed jagn1bΔT12-G14 mRNA. Further, we targeted the transmembrane domain using CRISPR/Cas9 gene editing to introduce a truncating mutation. (B-C) Quantitative analysis of the cells expressing the neutrophil marker lysozyme C (lyz), stained with WISH in (B) jagn1b TM crispants and (C) after jagn1bΔT12-G14 overexpression alone or in combination with jagn1b morpholino. Each dot represents the number of cells in the hematopoietic tissue of a 2.25 dpf individual embryo. Data are presented as mean ± standard deviation. The data represent combined results from 2 independent experiments. N represents the total number of analyzed embryos. (D-E) Representative images of lyz+ neutrophils in (D) TM crispants and (E) after overexpression of jagn1bΔT12-G14 alone or in combination with jagn1b morpholino. Images were taken with 10× magnification. Scale bars represent 100 μm. (F-I) Effect of overexpression of jagn1bΔT12-G14 on other hematopoietic cells. The left panels show quantitative analyses of stained cells with WISH against hematopoietic cell-specific marker. The right panels show representative images of the WISH-stained embryos. The cells were stained against (F) cmyb, (G) spi1b, (H) mpeg1.1 all 3 in 2.25 dpf embryos and (I) hbae1.1 in 1 dpf embryos. (F,I) Comparison of the percentage (%) of normal gene expression pattern between wild-type embryos compared to that carrying jagn1bΔT12-G14 mutation. (G-H) Numbers of stained cells in the hematopoietic region. Each dot represents the number of cells in the hematopoietic tissue of an individual 2.25 dpf embryo. Data are presented as mean ± standard deviation. The data represent combined results from 2 independent experiments. N represents the total number of analyzed embryos. Pictures were taken with 8× to 10× magnification. Scale bars represent 100 μm. (J) Representative images of intracellular Jagn1 protein localization (GFP signal) after overexpression of gfp-jagn1bWT and gfp-jagn1bΔT12-G14 mRNA in draculin:mCherry-positive HSPCs at 20 hours postfertilization (hpf). ns, not significant; ∗∗P < .01; ∗∗∗∗P < .0001.

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