Kimberly Dooley

Zebrafish: a model system for the study of human disease.

The zebrafish (Danio rerio) is a powerful model organism for the study of vertebrate biology, being well suited to both developmental and genetic analysis. Large-scale genetic screens have identified hundreds of mutant phenotypes, many of which resemble human clinical disorders. The creation of critical genetic reagents, coupled with the rapid progress of the zebrafish genome initiative directed by the National Institutes of Health, are bringing this model system to its full potential for the study of vertebrate biology, physiology and human disease.

Deficiency of glutaredoxin 5 reveals Fe-S clusters are required for vertebrate haem synthesis.

Iron is required to produce haem and iron–sulphur (Fe–S) clusters, processes thought to occur independently. Here we show that the hypochromic anaemia in shiraz (sir) zebrafish mutants is caused by deficiency of glutaredoxin 5 (grx5), a gene required in yeast for Fe–S cluster assembly. We found that grx5 was expressed in erythroid cells of zebrafish and mice. Zebrafish grx5 rescued the assembly of Δgrx5 yeast Fe–S, showing that the biochemical function of grx5 is evolutionarily conserved. In contrast to yeast, vertebrates use iron regulatory protein 1 (IRP1) to sense intracellular iron and regulate mRNA stability or the translation of iron metabolism genes. We found that loss of Fe–S cluster assembly in sir animals activated IRP1 and blocked haem biosynthesis catalysed by aminolaevulinate synthase 2 (ALAS2). Overexpression of ALAS2 RNA without the 5′ iron response element that binds IRP1 rescued sir embryos, whereas overexpression of ALAS2 including the iron response element did not. Further, antisense knockdown of IRP1 restored sir embryo haemoglobin synthesis. These findings uncover a connection between haem biosynthesis and Fe–S clusters, indicating that haemoglobin production in the differentiating red cell is regulated through Fe–S cluster assembly.

The chianti zebrafish mutant provides a model for erythroid-specific disruption of transferrin receptor 1.

Iron is a crucial metal for normal development, being required for the production of heme, which is incorporated into cytochromes and hemoglobin. The zebrafish chianti (cia) mutant manifests a hypochromic, microcytic anemia after the onset of embryonic circulation, indicative of a perturbation in red blood cell hemoglobin production. We show that cia encodes tfr1a, which is specifically expressed in the developing blood and requisite only for iron uptake in erythroid precursors. In the process of isolating zebrafish tfr1, we discovered two tfr1-like genes (tfr1a and tfr1b) and a single tfr2 ortholog. Abrogation of tfr1b function using antisense morpholinos revealed that this paralog was dispensable for hemoglobin production in red cells. tfr1b morphants exhibited growth retardation and brain necrosis, similar to the central nervous system defects observed in the Tfr1 null mouse, indicating that tfr1b is probably used by non-erythroid tissues for iron acquisition. Overexpression of mouse Tfr1, mouse Tfr2, and zebrafish tfr1b partially rescued hypochromia in cia embryos, establishing that each of these transferrin receptors are capable of supporting iron uptake for hemoglobin production in vivo. Taken together, these data show that zebrafish tfr1a and tfr1b share biochemical function but have restricted domains of tissue expression, and establish a genetic model to study the specific function of Tfr1 in erythroid cells.

Transferrin-a modulates hepcidin expression in zebrafish embryos

The iron regulatory hormone hepcidin is transcriptionally up-regulated in response to iron loading, but the mechanisms by which iron levels are sensed are not well understood. Large-scale genetic screens in the zebrafish have resulted in the identification of hypochromic anemia mutants with a range of mutations affecting conserved pathways in iron metabolism and heme synthesis. We hypothesized that transferrin plays a critical role both in iron transport and in regulating hepcidin expression in zebrafish embryos. Here we report the identification and characterization of the zebrafish hypochromic anemia mutant, gavi, which exhibits transferrin deficiency due to mutations in transferrin-a. Morpholino knockdown of transferrin-a in wild-type embryos reproduced the anemia phenotype and decreased somite and terminal gut iron staining, while coinjection of transferrin-a cRNA partially restored these defects. Embryos with transferrin-a or transferrin receptor 2 (TfR2) deficiency exhibited low levels of hepcidin expression, however anemia, in the absence of a defect in the transferrin pathway, failed to impair hepcidin expression. These data indicate that transferrin-a transports iron and that hepcidin expression is regulated by a transferrin-a-dependent pathway in the zebrafish embryo.

montalcino, A zebrafish model for variegate porphyria

OBJECTIVE Inherited or acquired mutations in the heme biosynthetic pathway leads to a debilitating class of diseases collectively known as porphyrias, with symptoms that can include anemia, cutaneous photosensitivity, and neurovisceral dysfunction. In a genetic screen for hematopoietic mutants, we isolated a zebrafish mutant, montalcino (mno), which displays hypochromic anemia and porphyria. The objective of this study was to identify the defective gene and characterize the phenotype of the zebrafish mutant. MATERIALS AND METHODS Genetic linkage analysis was utilized to identify the region harboring the mno mutation. Candidate gene analysis together with reverse transcriptase polymerase chain reaction was utilized to identify the genetic mutation, which was confirmed via allele-specific oligo hybridizations. Whole mount in situ hybridizations and o-dianisidine staining were used to characterize the phenotype of the mno mutant. mRNA and morpholino microinjections were performed to phenocopy and/or rescue the mutant phenotype. RESULTS Homozygous mno mutant embryos have a defect in the protoporphyrinogen oxidase (ppox) gene, which encodes the enzyme that catalyzes the oxidation of protoporphyrinogen. Homozygous mutant embryos are deficient in hemoglobin, and by 36 hours post-fertilization are visibly anemic and porphyric. The hypochromic anemia of mno embryos was partially rescued by human ppox, providing evidence for the conservation of function between human and zebrafish ppox. CONCLUSION In humans, mutations in ppox result in variegate porphyria. At present, effective treatment for acute attacks requires the administration intravenous hemin and/or glucose. Thus, mno represents a powerful model for investigation, and a tool for future screens aimed at identifying chemical modifiers of variegate porphyria.