7,17-diHDPAn-3 enhances primitive and definitive erythrocyte production. (A,D) WISH for hbbe1 in embryos treated with 7,17-diHDPAn-3 from 11 to 24 hpf and from 11 to 36 hpf, compared to controls (n = 9 embryos per group). Images were taken at original magnification ×5 (24 hpf, lateral whole embryos) and 10× magnification (24 hpf, lateral tails; 36 hpf, ventral yolk sacs; and 36 hpf, lateral tails). (B,E) Quantification of WISH signal intensity using Fiji software revealed increased hbbe1-positive cells in the ICM at 24 hpf (B) and ventral yolk sac at 36 hpf (E) in treated embryos (n = 4 embryos per group; unpaired Student t test). (C,F) RT-qPCR analysis demonstrated a 38.5% increase in hbae3 expression at 24 hpf (C) and a 21% increase in hbbe1 expression at 36 hpf (F) in treated groups vs controls (n = 3 biological replicates per group; technical triplicates; 30 embryos per sample). (G) FACS analysis of erythroid progenitor and mature erythroid cells in 36-hpf Tg(gata1:DsRed) embryos shows increased erythroid cell number after 7,17-diHDPAn-3 exposure (n = 3 biological replicates per conditions, with 75 embryos per sample). (H) Quantification of erythroid cell percentages normalized to total live single cells showed a ∼52% increase in the treated group (n = 3 biological replicates per group; unpaired Student t test). (I) WISH for hbbe1 after 5 to 6 dpf of exposure revealed increased signal in the ventral head and CHT with 9 embryos per group. Ventral images (top) showing an increase in staining in the heart and branchial region (white arrows); lateral trunk (bottom panel) showing both an increase and anterior expansion of staining, marked by the red box and black arrows. (J) Quantification of hbbe1 signal intensity in the ventral head and CHT showed significantly higher signal in treated embryos (n = 3; unpaired Student t test). (K) RT-qPCR at 6 dpf confirmed increased hbbe1 expression (n = 3 biological replicates per group; technical triplicates; 30 embryos per sample). Bar plots represent mean ± SD. Statistical comparisons between control and treated groups were performed using unpaired Student t tests. ∗P < .05; ∗∗∗P < .001. ctrl, control; ICM, intermediate cell mass.

7,17-diHDPAn-3 enhances primitive and definitive erythrocyte production. (A,D) WISH for hbbe1 in embryos treated with 7,17-diHDPAn-3 from 11 to 24 hpf and from 11 to 36 hpf, compared to controls (n = 9 embryos per group). Images were taken at original magnification ×5 (24 hpf, lateral whole embryos) and 10× magnification (24 hpf, lateral tails; 36 hpf, ventral yolk sacs; and 36 hpf, lateral tails). (B,E) Quantification of WISH signal intensity using Fiji software revealed increased hbbe1-positive cells in the ICM at 24 hpf (B) and ventral yolk sac at 36 hpf (E) in treated embryos (n = 4 embryos per group; unpaired Student t test). (C,F) RT-qPCR analysis demonstrated a 38.5% increase in hbae3 expression at 24 hpf (C) and a 21% increase in hbbe1 expression at 36 hpf (F) in treated groups vs controls (n = 3 biological replicates per group; technical triplicates; 30 embryos per sample). (G) FACS analysis of erythroid progenitor and mature erythroid cells in 36-hpf Tg(gata1:DsRed) embryos shows increased erythroid cell number after 7,17-diHDPAn-3 exposure (n = 3 biological replicates per conditions, with 75 embryos per sample). (H) Quantification of erythroid cell percentages normalized to total live single cells showed a ∼52% increase in the treated group (n = 3 biological replicates per group; unpaired Student t test). (I) WISH for hbbe1 after 5 to 6 dpf of exposure revealed increased signal in the ventral head and CHT with 9 embryos per group. Ventral images (top) showing an increase in staining in the heart and branchial region (white arrows); lateral trunk (bottom panel) showing both an increase and anterior expansion of staining, marked by the red box and black arrows. (J) Quantification of hbbe1 signal intensity in the ventral head and CHT showed significantly higher signal in treated embryos (n = 3; unpaired Student t test). (K) RT-qPCR at 6 dpf confirmed increased hbbe1 expression (n = 3 biological replicates per group; technical triplicates; 30 embryos per sample). Bar plots represent mean ± SD. Statistical comparisons between control and treated groups were performed using unpaired Student t tests. ∗P < .05; ∗∗∗P < .001. ctrl, control; ICM, intermediate cell mass.

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