FUNCTIONAL CHARACTERIZATION OF HEME TRANSPORTERS IN ZEBRAFISH
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Hrg1 and Mrp5 are identified as eukaryotic heme importer and exporter, respectively. Two Hrg1 paralogs have been annotated in zebrafish genome, Hrg1a (Slc48a1b) and Hrg1b (Slc48a1a) with 84% homology in protein sequences. Hrg1a and hrg1b are widely expressed in embryonic and adult zebrafish. Yeast growth assays reveal that zebrafish Hrg1a and Hrg1b are both capable of heme import. However, hrg1a and hrg1b double knockout (hrg1 DKO) zebrafish generated by CRISPR/Cas9 has no overt defects in differentiation and maturation of erythroid cells. Knockdown of hrg1a in hrg1b mutants or vice versa does not impair erythroid lineage in zebrafish embryos. These genetic results suggest that Hrg1 is not required for maturation and hemoglobinization of primitive erythroid cells. Hrg1a and hrg1b mRNA are upregulated in adult kidneys and spleens upon PHZ-induced hemolysis, together with hmox1, a downstream heme degrading enzyme, suggesting that Hrg1 is involved in adult heme-iron recycling during erythrophagcytosis in kidney and spleen of adult zebrafish. DAB-enhanced Perl’s iron staining reveals that iron is accumulated in macrophages in the kidney and spleen in adult wild-type zebrafish. However, macrophages with positive Perl’s staining are rarely found in the kidney of hrg1 DKO and instead large amount of iron is deposited in renal tubules, suggesting defects in heme-iron recycling by kidney macrophages in hrg1 DKO under PHZ-induced hemolysis. Whole transcriptome sequencing of mRNA extracted from spleens and kidneys reveals massive differentially expressed genes in hrg1 DKO involved in immune response, lipid transport, oxidation-reduction process and proteolysis. These indicate that hrg1 DKO are deficient in recycling heme-iron derived from damaged RBCs in the absence of functional Hrg1. Phylogenetic analysis reveals that Mrp5 and Mrp9 are closed homologs in the zebrafish genome. Yeast growth assays reveal that both zebrafish Mrp5 and Mrp9 are capable of heme export. Morpholino knockdown of mrp5 and mrp9 in zebrafish showed severe anemia in developing embryos indicating their involvements in erythropoietic development. Subsequent generation and characterization of mrp5 and mrp9 mutants by CRISPR/Cas9 will further define the function of Mrp5 and Mrp9 during zebrafish development.