A genetic analysis of vertebrate iron metabolism: Characterization of the zebrafish blood mutants weissherbst and chardonnay

My thesis project focused on the study of hemoglobin production using zebrafish as a model system. A large scale mutagenesis screen performed in Tubingen, Germany identified five zebrafish mutants, zinfandel, sauternes, chianti, weissherbst and chardonnay, with a hypochromic anemia due to decreased hemoglobin production. I undertook the study two of these mutants, weissherbst (weh) and chardonnay (cdy). Positional and candidate cloning approaches identified the chardonnay gene as the intestinal and red blood cell iron transporter Dmt1, demonstrating that the hypochromic anemia of cdy mutants results from both inefficient intestinal iron absorption and defective red blood cell iron utilization. I took a positional cloning approach to cloning the weh mutant gene. Both mutant alleles of weh are embryonic lethal, thus it was likely that the weh gene played a role not only in the production of hemoglobin, but in other vital systems. This thesis describes the identification of the weh mutant gene as a gene encoding a novel multiple-transmembrane domain protein called Ferroportin1. I performed rescue experiments to characterize the defect in weh embryos and showed that ferroportin1 is required for the transport of iron from the maternally derived stores in the yolk to the embryonic circulation. I subsequently demonstrated that the Ferroportin1. protein can function as an iron exporter in a Xenopus oocyte expression system. Analysis of the expression of the mouse and human homologs; of ferroportin1 demonstrated expression in tissues that play a key role in mammalian iron metabolism, placenta, intestine, liver and macrophages. These experiments also demonstrated localized expression of Ferroportin1 at the basal and basolateral surface of the placental syncytiotrophoblast and the intestinal enterocyte, respectively. This localized expression pattern suggested that mammalian Ferroportin1 plays a role in iron export from these cell types into the circulation. Analysis of weh mutants rescued from embryonic lethality by injection of iron-dextran demonstrated the requirement for the weh gene in intestinal iron absorption. The studies in this thesis will serve as a basis for future studies of vertebrate iron metabolism using zebrafish as a model system. In addition, the identification of the novel iron exporter Ferroportin1 has provided researchers that study iron metabolism in all organisms with a new piece in the puzzle.