Mechanisms of copper deficiency in the zebrafish embryo.

摘要

Proper maternal nutrition is critical for early embryonic development. Despite overwhelming epidemiologic data indicating the benefits nutrient supplementation for the developing organism we do not fully understand the genetics of predisposition to abnormal developmental phenotypes when faced with suboptimal nutrient levels.;Copper is an essential nutrient required for critical biochemical processes. Severe defects in copper homeostasis lead to significant disease typified by the X-linked recessive disorder Menkes disease. Patients with Menkes disease have cutis laxa, bone deformities, hypopigmentation, arterial malformation, and neurodegeneration due to copper deficiency caused by loss-of-function mutations in ATP7A, a copper transport protein. Despite the critical requirement for adequate copper nutrition and the characterization of key copper transport proteins there remain significant gaps in our understanding of copper metabolism.;In order to better understand both the cell metabolic and developmental requirements for copper our laboratory has defined a zebrafish model of severe copper deficiency. The copper chelator neocuproine causes a Menkes-like phenotype in wild-type zebrafish embryos and the mutant calamity which phenocopies neocuproine treated embryos contains a null allele of atp7a.;In this thesis we build upon this previous model in two ways in order to address the problem of treatment of Menkes disease and to define novel pathways important for copper metabolism. The first body of work uses small-molecule modifiers of mRNA processing (morpholinos) to correct splicing defects which cause Menkes disease phenotypes in zebrafish embryos. Since about 20% of human disease causing mutations are due to splicing defects this approach may be directly applicable to a wide array of human diseases. The second body of work uses a forward chemical-genetic screen for zebrafish mutants sensitive to mild copper deficiency. Screening mutagenized embryos exposed to subthreshold doses of the chelator neocuproine for copper deficiency phenotypes resulted in two mutants. The first, containing a hypomorphic allele of atp7a demonstrates the effect of maternal nutrition and genetics on embryonic development. The second contains a loss-of-function mutation in the vacuolar proton pump leading to a severe embyronic lethal phenotype which is sensitive to copper chelation. This implicates pH gradients in the metabolism of copper in zebrafish.