Figure 7
Figure 7. NPMc+ increases the number of definitive hematopoietic cells in zebrafish embryos. (A-B) Brightfield images of a 32-hpf embryo (lateral view, anterior to the left, dorsal upwards) where a black box shows the region of the AGM (A) or of the PBI (B), where subsequent images were taken. (C,F,I,L) Confocal images taken from the AGM region of live 32-hpf Tg(cd41:EGFP) transgenic zebrafish embryos (black box in panel A), uninjected (C), or injected with NPM1 10 pg (F) or NPMc+ 50 pg (I) mRNA. Green cells indicate expression of EGFP under the control of the cd41 promoter. The bracket highlights cd41low EGFP+ cells in the AGM region, while the arrow points at the renal EGFP+ population located in the region of the pronephric ducts. EGFP+ cells in the AGM field were counted and showed increased numbers upon NPMc+ expression (L). Images were captured on a Yokogawa spinning disk confocal microscope using a 10×/0.3 NA Plan-fluor phase objective. (Di-iv, Gi-iv, Ji-iv, M-O) Confocal images taken from the AGM region of live 32-hpf Tg(c-myb:EGFP);(gata1:DsRed) transgenic zebrafish embryos (black box in panel A), uninjected (Di-Div), or injected with NPM1 10 pg (Gi-Giv) or NPMc+ 50 pg (Ji-Jiv) mRNA. Green indicates cells expressing EGFP under the control of the c-myb promoter; red indicates cells expressing DsRed under the control of the gata1 promoter; yellow indicates cells coexpressing both genes (highlighted by arrowheads). The white boxes in panels Diii, Giii, and Jiii indicate an area, magnified in a single Z-stack slice (Div,Giv,Jiv), showing how single slices were used to quantify the number of yellow cells. NPMc+ expression causes an increase in EGFP+ cell numbers in the field of analysis (quantified in panel M) without affecting numbers of cells expressing DsRed under the control of the gata1 promoter (quantified in panel N) or double-positive cells (quantified in panel O by counting yellow cells in 3 different Z-stack slices in each of 4 embryos). Images were taken as for Tg(cd41:EGFP) embryos. (E,H,K,P) Confocal images taken from the PBI region of live 32-hpf Tg(gata1:EGFP);(lmo2:DsRed) transgenic zebrafish embryos (black box in panel B), uninjected (E), or injected with NPM1 10 pg (H) or NPMc+ 50 pg (K) mRNA. Green indicates cells expressing EGFP under the control of the gata1 promoter; red indicates cells expressing DsRed under the control of the lmo2 promoter; yellow indicates cells coexpressing both genes (highlighted by arrowheads). NPMc+ expression causes an increase in cells expressing both genes (K), quantified in panel P by counting yellow cells in 3 different Z-stack slices in each of 4 embryos. Images are single Z-stack slices, and the white boxes in panels Ei, Hi, and Ki indicates an area magnified in panels Eii, Hii, and Kii. Images were acquired on a Yokogawa spinning disk confocal microscope using a 20×/0.75 NA Plan-Apo DIC objective. All confocal images were acquired using the Andor IQ software. Analysis of images was performed using ImageJ software. Error bars represent SEM. *Statistically significant differences between the NPMc+-injected and both NPM1-injected and uninjected embryos (*P < .05, **P < .005; Student t test). In histograms blue indicates uninjected control embryos; green, NPM1-injected embryos; and red, NPMc+-injected embryos.

NPMc+ increases the number of definitive hematopoietic cells in zebrafish embryos. (A-B) Brightfield images of a 32-hpf embryo (lateral view, anterior to the left, dorsal upwards) where a black box shows the region of the AGM (A) or of the PBI (B), where subsequent images were taken. (C,F,I,L) Confocal images taken from the AGM region of live 32-hpf Tg(cd41:EGFP) transgenic zebrafish embryos (black box in panel A), uninjected (C), or injected with NPM1 10 pg (F) or NPMc+ 50 pg (I) mRNA. Green cells indicate expression of EGFP under the control of the cd41 promoter. The bracket highlights cd41low EGFP+ cells in the AGM region, while the arrow points at the renal EGFP+ population located in the region of the pronephric ducts. EGFP+ cells in the AGM field were counted and showed increased numbers upon NPMc+ expression (L). Images were captured on a Yokogawa spinning disk confocal microscope using a 10×/0.3 NA Plan-fluor phase objective. (Di-iv, Gi-iv, Ji-iv, M-O) Confocal images taken from the AGM region of live 32-hpf Tg(c-myb:EGFP);(gata1:DsRed) transgenic zebrafish embryos (black box in panel A), uninjected (Di-Div), or injected with NPM1 10 pg (Gi-Giv) or NPMc+ 50 pg (Ji-Jiv) mRNA. Green indicates cells expressing EGFP under the control of the c-myb promoter; red indicates cells expressing DsRed under the control of the gata1 promoter; yellow indicates cells coexpressing both genes (highlighted by arrowheads). The white boxes in panels Diii, Giii, and Jiii indicate an area, magnified in a single Z-stack slice (Div,Giv,Jiv), showing how single slices were used to quantify the number of yellow cells. NPMc+ expression causes an increase in EGFP+ cell numbers in the field of analysis (quantified in panel M) without affecting numbers of cells expressing DsRed under the control of the gata1 promoter (quantified in panel N) or double-positive cells (quantified in panel O by counting yellow cells in 3 different Z-stack slices in each of 4 embryos). Images were taken as for Tg(cd41:EGFP) embryos. (E,H,K,P) Confocal images taken from the PBI region of live 32-hpf Tg(gata1:EGFP);(lmo2:DsRed) transgenic zebrafish embryos (black box in panel B), uninjected (E), or injected with NPM1 10 pg (H) or NPMc+ 50 pg (K) mRNA. Green indicates cells expressing EGFP under the control of the gata1 promoter; red indicates cells expressing DsRed under the control of the lmo2 promoter; yellow indicates cells coexpressing both genes (highlighted by arrowheads). NPMc+ expression causes an increase in cells expressing both genes (K), quantified in panel P by counting yellow cells in 3 different Z-stack slices in each of 4 embryos. Images are single Z-stack slices, and the white boxes in panels Ei, Hi, and Ki indicates an area magnified in panels Eii, Hii, and Kii. Images were acquired on a Yokogawa spinning disk confocal microscope using a 20×/0.75 NA Plan-Apo DIC objective. All confocal images were acquired using the Andor IQ software. Analysis of images was performed using ImageJ software. Error bars represent SEM. *Statistically significant differences between the NPMc+-injected and both NPM1-injected and uninjected embryos (*P < .05, **P < .005; Student t test). In histograms blue indicates uninjected control embryos; green, NPM1-injected embryos; and red, NPMc+-injected embryos.

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