Figure 4. mrp5 is required for zebrafish erythropoiesis.

(A) Phylogenetic analysis of MRP5/ABCC5 clade in C. elegans (orange), zebrafish (red), mice (green), and humans (blue). Sequences were aligned using ClustalW and a phylogenetic tree was generated using the Neighbor-Joining method in MEGA5. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances are in the units of the number of amino acid substitutions per site. (B) Lateral view of zebrafish mrp5 expression by whole mount in situ hybridization using anti-sense probe, 4 days post-fertilization. Anterior is to the left. Sense probe image is shown to indicate background staining. B, brain, N, neural tube. (C) Knockdown of zebrafish mrp5 using morpholinos (MO^mrp5) results in severe anemia, as indicated by reduced staining of o-dianisidine-positive red cells, as indicated by black arrowheads. (D) Knockdown of zebrafish mrp-5 using MO^mrp5 results in reduced red cell formation. Transgenic embryos expressing GFP from the globin locus control region (LCR-GFP) were injected with control MO or MO^mrp5. LEFT: On day 2 post-fertilization, percent GFP-positive RBCs was analyzed by FACS. X and Y axes measure GFP and forward scatter, respectively; boxed area indicates gate for RBCs. RIGHT: Quantification of morphants shown at left. For MO^mrp5 injection, n=4. **P<0.01 for MO^mrp5 morphants compared to control morphants under identical conditions. (One-way ANOVA, Bonferroni post-test.) (E) Lateral view of zebrafish gata1 expression in wildtype, control MO, and MO^mrp5 morphants by whole mount in situ hybridization using anti-sense probe, 24 hpf. Anterior is to the left. (F) Quantification of anemia rescue in zebrafish coinjected with mrp5 cRNA. ***P<0.001 for MO^mrp5 morphants compared to control morphants under identical conditions, n=4. **P<0.01 for mrp5 morphants co-injected with rescue cRNA when compared to mrp5 morphants with no rescue cRNA under identical conditions, n=3 (One-way ANOVA, Bonferroni post-test.) See also Figure S4 and Table S1.