Characterization of the hypochromic blood mutants zinfandel, chianti and sauternes: a genetic analysis of hemoglobin synthesis in the zebrafish, Danio rerio

Disorders of hemoglobin production in mammals may be caused by congenital defects in iron metabolism, heme biosynthesis or globin gene expression. A common histologic feature of these diseases is a microcytic, hypochromic anemia in which red blood cells have both a reduced volume and a reduced hemoglobin content. I have studied three zebrafish blood mutants, zinfandel (zin), chianti ( cia) and sauternes (sau ), that display a similar phenotype. This thesis describes the characterization of these three mutants and focuses on the positional cloning of the sauternes gene. I cloned a number of embryonic globin genes from the zebrafish and examined their expression in both wild-type and mutant embryos. These experiments showed that sau mutants have a specific defect in the expression of one of these genes. In contrast, cia and zin only show defects in embryonic globin gene expression that are secondary to a decrease in red blood cell number. The expression of the adult globin genes does not appear to be perturbed by these mutations, although the hypochromic anemia observed in cia and sau embryos persists throughout ontogeny. zin mutants are also viable, but do not display a hematologic phenotype as adults. I have also studied the structure of the zebrafish globin loci. The globin genes form two clusters in the genome. One cluster maps to linkage group (LG) 12 and appears to consist solely of embryonic globin genes. The second locus is found on LG 3 and contains both embryonic and adult globin genes. Linkage analysis demonstrated that the zin mutation maps to the LG 3 globin locus. zin likely represents a mutation within an embryonic globin gene or a defect in the regulation of these genes. The cia gene has been mapped to LG 2. This localization eliminated a number of known genes as candidates for cia. However, poor marker density in this region of the genome has hampered efforts to isolate cia via a positional cloning approach. Strategies aimed towards the eventual identification of this gene will be discussed. sau encodes the erythroid specific isoform of the heme biosynthetic enzyme d -amino levulinic acid synthase. In humans, mutations in this gene cause congenital sideroblastic anemia. sau represents the first animal model of this disease. This work further establishes the feasibility of positional cloning in the zebrafish, and highlights the application of zebrafish genetics to the study of human disease.