![Figure 4. DPY30 ortholog is essential for hematopoiesis in zebrafish. (A) Alignment of the human DPY30 and zebrafish dpy30 proteins. Identical amino acids are denoted in bold type and asterisks indicate stop codons. (B) Design of zebrafish dpy30 MOs in relationship to the zebrafish dpy30 gene. The red and gray bars represent the dpy30 MOs. (C and D) Effect of dpy30 MOs on zebrafish blood circulation. (C) Representative images of circulating blood cells (picture frames from the time-lapse video microscopy) in uninjected control zebrafish and zebrafish injected with 0.125× dose of dpy30 MO-1. Green arrows point at the blood cells. (D) Quantification of blood circulation status at indicated time (after fertilization) in uninjected control zebrafish (Control), zebrafish injected with 0.25× dose of dpy30 MO-1 (MO-1), with 0.25× dose of dpy30 MO-1 plus 50 to 100 pg of dpy30 mRNA (MO-1 + mRNA), 1× dose of control MO (cMO), or 0.375× dose of dpy30 MO-2 (MO-2). (E and F) Effect of dpy30 MOs on zebrafish hematopoiesis examined by o-dianisidine staining at 48 hpf. Zebrafish were uninjected (Control), injected with 0.25× dose of dpy30 MO-1 (MO-1), with 0.25× dose of dpy30 MO-1 plus 50 to 100pg of dpy30 mRNA (MO-1 + mRNA), or with 0.375× dose of dpy30 MO-2 (MO-2). (E) Typical results for o-dianisidine staining showing zebrafish with normal red blood cell production and with severe or moderate anemia. Red arrows point at the red blood cells. Note that although some residual red blood cells could sometimes be visualized at the heart region, none was seen in the periphery of the severely anemic fish. These images, from top to bottom, were selected from the Control, MO-1, and MO-1 + mRNA groups, respectively. (F) O-dianisidine staining results were quantified for each category of red blood cell levels (according to the images shown in [E]). Plotted are averages ± SD from 3 independent biological repeats for the left graph (also shown individually in supplemental Figure 10C), and from 2 biological repeats for the right graph (also shown individually in supplemental Figure 11B). **P < .01; ***P < .001 (Student t test). (G) Western blot analysis was used for H3K4 methylation using total extracts from 48 hpf uninjected control zebrafish and zebrafish injected with 0.25× dose of dpy30 MO-1. (H) In situ hybridization for rag1 at day 5 after fertilization in uninjected control zebrafish and zebrafish injected with 0.375× dose of dpy30 MO-2. Dorsal (top) and lateral (bottom) views are shown. The arrow points at the rag1 signal. (I) In situ hybridization for tal1, gata1a, and fli1a at 30 hpf in uninjected control zebrafish and zebrafish injected with 0.25× dose of dpy30 MO-1.](https://ash.silverchair-cdn.com/ash/content_public/journal/blood/124/13/10.1182_blood-2014-01-549220/4/2025f4.jpeg?Expires=1722390572&Signature=bdhWwSBeg4llClnXHvAHG7BBXP8MfqVkji9DmmFZWpPVdlX9GzyQl1FRI7Q8AlFTnUX-FeEq6juCBjCMHG-OiJSQv19lP1lVuTOF2I1hihIF5TUgNTBFalDszK2eaTZ3g2Hawry249G-ApyvSkjPDrJvCqn~A0YJTgAnHAF5mYeIdX0LzwSQRnSDFjn-u~60XFW4~oR7W6sS7nwRTlkf9GbdkKTOvArQEP2PYqgDRuFIM~5XsSIFxApKXPAavTU8b68xP9HzmoCPZ5W1zZHtM9hpbZIrwz2vH24J~ManBpXnBrwH9e5Clj9MUn7nuYA8bjT-V2MvK98Izdx9bCli4Q__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
DPY30 ortholog is essential for hematopoiesis in zebrafish. (A) Alignment of the human DPY30 and zebrafish dpy30 proteins. Identical amino acids are denoted in bold type and asterisks indicate stop codons. (B) Design of zebrafish dpy30 MOs in relationship to the zebrafish dpy30 gene. The red and gray bars represent the dpy30 MOs. (C and D) Effect of dpy30 MOs on zebrafish blood circulation. (C) Representative images of circulating blood cells (picture frames from the time-lapse video microscopy) in uninjected control zebrafish and zebrafish injected with 0.125× dose of dpy30 MO-1. Green arrows point at the blood cells. (D) Quantification of blood circulation status at indicated time (after fertilization) in uninjected control zebrafish (Control), zebrafish injected with 0.25× dose of dpy30 MO-1 (MO-1), with 0.25× dose of dpy30 MO-1 plus 50 to 100 pg of dpy30 mRNA (MO-1 + mRNA), 1× dose of control MO (cMO), or 0.375× dose of dpy30 MO-2 (MO-2). (E and F) Effect of dpy30 MOs on zebrafish hematopoiesis examined by o-dianisidine staining at 48 hpf. Zebrafish were uninjected (Control), injected with 0.25× dose of dpy30 MO-1 (MO-1), with 0.25× dose of dpy30 MO-1 plus 50 to 100pg of dpy30 mRNA (MO-1 + mRNA), or with 0.375× dose of dpy30 MO-2 (MO-2). (E) Typical results for o-dianisidine staining showing zebrafish with normal red blood cell production and with severe or moderate anemia. Red arrows point at the red blood cells. Note that although some residual red blood cells could sometimes be visualized at the heart region, none was seen in the periphery of the severely anemic fish. These images, from top to bottom, were selected from the Control, MO-1, and MO-1 + mRNA groups, respectively. (F) O-dianisidine staining results were quantified for each category of red blood cell levels (according to the images shown in [E]). Plotted are averages ± SD from 3 independent biological repeats for the left graph (also shown individually in supplemental Figure 10C), and from 2 biological repeats for the right graph (also shown individually in supplemental Figure 11B). **P < .01; ***P < .001 (Student t test). (G) Western blot analysis was used for H3K4 methylation using total extracts from 48 hpf uninjected control zebrafish and zebrafish injected with 0.25× dose of dpy30 MO-1. (H) In situ hybridization for rag1 at day 5 after fertilization in uninjected control zebrafish and zebrafish injected with 0.375× dose of dpy30 MO-2. Dorsal (top) and lateral (bottom) views are shown. The arrow points at the rag1 signal. (I) In situ hybridization for tal1, gata1a, and fli1a at 30 hpf in uninjected control zebrafish and zebrafish injected with 0.25× dose of dpy30 MO-1.
