Creation And Analysis Of Zebrafish Mutants As Models For Human Porphyrias And Anemia

Heme is an important molecule in almost all organisms, as a component of numerous proteins such as hemoglobin, cytochrome P450 and vitamin B1. In addition to being the prosthetic group of proteins, heme also modulates a number of molecular and cellular processes as a signaling molecule. Heme biosynthesis is catalyzed by a cascade of eight enzymes that are highly conserved from bacteria to humans. Porphyria is a kind of complex inherited metabolic disorder, resulting from the deficiency of a specific enzyme in the heme biosynthetic pathway. Porphyrias are classified into 3 groups: the acute hepatic porphyrias, the hepatic cutaneous porphyria and the erythropoietic cutaneous porphyrias. The pathogenesis of porphyrias has not been understood very well. The zebrafish(Danio rerio) has become a bona fide vertebrate model for studying numerous human diseases. Heme biosynthesis is conserved throughout vertebrate evolution, all 8 of human genes involved in heme biosynthesis have zebrafish orthologues. Four zebrafish mutants sauternes, yquem, montalcino and dracula were reported for human porphyrias, which were identified in large-scale forward genetic mutagenesis screens. However, no hereditable zebrafish models with genetic mutations for other porphyrias have been generated to date.In this research we created two zebrafish mutants alad-/- and cpox-/- as models for human ADP(ALA-dehydratase-deficient porphyria) and HCP(hereditary coproporphyria) using TALEN or CRISPR-cas9 mediated gene targeting. Characterization of these two zebrafish mutants shows homozygous alad-/- and cpox-/-fishes display phenotypes of hypochromia, heme deficiency, abnormal erythrocytic maturation and accumulation of heme precursor intermediates, mimicking human ALA dehydratase deficient porphyria(ADP) and hereditary coproporphyrian(HCP). The rescue experiment also indicated that the hypochromic anemia phenotype of alad-/- and cpox-/- can be rescued by human ALAD and CPOX, suggesting that the functions of these two enzymes are conserved between zebrafish and human.There are two genes, hmbsa and hmbsb, encoding the third enzymes in heme biosynthesis in zebrafish. Phylogenetic analysis shows that zebrafish hmbsa and hmbsb are co-orthologs of human HMBS. In this study we also generated two zebrafish mutants hmbsa-/- and hmbsb-/- as models for human AIP(acute intermittent porphyria). These two mutant all displays hypochromia and abnormal behavior.I conducted deep sequencing transcriptome analysis of previously reported heme-deficient mutants alas2-/- and urod-/-. The results show that a large number of differentially expressed genes in alas2-/- and urod-/-, indicating the complexity of porphyric syndrome. Expression of the gene encoding ALAS1, the rate-limiting enzyme in heme biosynthesis, is controlled by the circadian clock system. Heme provides a mechanism for coordinating the circadian clock system and energy metabolism. This study also generated zebrafish mutants with deficiency of different per genes, and shows the altered expression pattern of alas1 in per1a-/-.Generation of these heme-deficient mutants provide animal models for investigate novel aspects of porphyria pathogenesis and drug screening for human porphyrias.