Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 5 de 5
Filter
Add more filters










Database
Language
Publication year range
1.
Exp Hematol ; 36(9): 1132-42, 2008 Sep.
Article in English | MEDLINE | ID: mdl-18550261

ABSTRACT

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.


Subject(s)
Anemia, Hypochromic/genetics , Disease Models, Animal , Porphyria, Variegate/genetics , Protoporphyrinogen Oxidase/genetics , Zebrafish Proteins/genetics , Zebrafish/genetics , Amino Acid Sequence , Animals , Codon, Nonsense , Conserved Sequence , DNA, Complementary/genetics , Embryo, Nonmammalian/pathology , Hemoglobins/biosynthesis , Hemoglobins/deficiency , Homozygote , Humans , Mice , Molecular Sequence Data , Phenotype , Porphyria, Variegate/blood , Porphyria, Variegate/embryology , Recombinant Fusion Proteins/physiology , Sequence Alignment , Sequence Homology, Amino Acid , Species Specificity , Zebrafish/embryology , Zebrafish Proteins/deficiency
2.
Nature ; 436(7053): 1035-39, 2005 Aug 18.
Article in English | MEDLINE | ID: mdl-16110529

ABSTRACT

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 grx5 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.


Subject(s)
Glutaredoxins/deficiency , Glutaredoxins/metabolism , Heme/biosynthesis , Iron-Sulfur Proteins/metabolism , Oxidoreductases/deficiency , Oxidoreductases/metabolism , Zebrafish/metabolism , 5-Aminolevulinate Synthetase/genetics , Amino Acid Sequence , Animals , Cloning, Molecular , Erythrocytes/cytology , Erythrocytes/metabolism , Gene Expression Regulation , Glutaredoxins/chemistry , Glutaredoxins/genetics , Homeostasis , Iron/metabolism , Iron Regulatory Protein 1/metabolism , Iron-Sulfur Proteins/biosynthesis , Iron-Sulfur Proteins/genetics , Mice , Molecular Sequence Data , Oxidoreductases/chemistry , Oxidoreductases/genetics , Response Elements/genetics , Saccharomyces cerevisiae/enzymology , Saccharomyces cerevisiae/genetics , Zebrafish/genetics
3.
Development ; 130(20): 5043-52, 2003 Oct.
Article in English | MEDLINE | ID: mdl-12952905

ABSTRACT

The van gogh (vgo) mutant in zebrafish is characterized by defects in the ear, pharyngeal arches and associated structures such as the thymus. We show that vgo is caused by a mutation in tbx1, a member of the large family of T-box genes. tbx1 has been recently suggested to be a major contributor to the cardiovascular defects in DiGeorge deletion syndrome (DGS) in humans, a syndrome in which several neural crest derivatives are affected in the pharyngeal arches. Using cell transplantation studies, we demonstrate that vgo/tbx1 acts cell autonomously in the pharyngeal mesendoderm and influences the development of neural crest-derived cartilages secondarily. Furthermore, we provide evidence for regulatory interactions between vgo/tbx1 and edn1 and hand2, genes that are implicated in the control of pharyngeal arch development and in the etiology of DGS.


Subject(s)
DiGeorge Syndrome/genetics , T-Box Domain Proteins/genetics , Zebrafish/metabolism , Amino Acid Sequence , Animals , Branchial Region/metabolism , Ear/embryology , Endoderm/metabolism , Humans , Mesoderm/metabolism , Molecular Sequence Data , Mutation , Sequence Deletion , T-Box Domain Proteins/metabolism
4.
Physiol Genomics ; 11(2): 91-8, 2002 Oct 29.
Article in English | MEDLINE | ID: mdl-12388799

ABSTRACT

Vertebrate hematopoiesis is regulated by distinct cell-specific transcription factors such as GATA-1 and SCL. Mammalian p45-NFE2 was characterized for its ability to bind the hypersensitive sites of the globin locus control region. NFE2 is a member of a cap'n'collar (CNC) and basic zipper (BZIP) superfamily that regulates gene transcription. It has been implicated in diverse processes such as globin gene expression, oxidative stress, and platelet lineage differentiation. Here, we have isolated the zebrafish ortholog of NFE2. The gene is highly homologous, particularly in the DNA-binding domain. Mapping the zebrafish NFE2 to linkage group 23 establishes a region of chromosomal synteny with human chromosome 12, further suggesting evolutionary conservation. During embryogenesis, the zebrafish gene is expressed specifically in erythroid cells and also in the developing ear. NFE2 expression is lacking in zebrafish mutants that have no hematopoietic cells. An analysis of the sauternes mutant, which carries a mutation in the ALAS-2 gene and thus has defective heme synthesis, demonstrates higher levels of NFE2 expression than normal. This further establishes the block to erythroid differentiation in the sauternes mutant. Our studies demonstrate conservation of the vertebrate genetic program for the erythroid lineage.


Subject(s)
DNA-Binding Proteins/chemistry , DNA-Binding Proteins/genetics , Transcription Factors/chemistry , Transcription Factors/genetics , Zebrafish/genetics , Animals , Base Sequence , Chromosomes, Human, Pair 21/genetics , Erythroid-Specific DNA-Binding Factors , GATA1 Transcription Factor , Gene Expression Profiling/methods , Gene Expression Regulation, Developmental/genetics , Humans , Kidney/chemistry , Molecular Sequence Data , Mutation/genetics , NF-E2 Transcription Factor, p45 Subunit , Synteny/genetics , Zebrafish Proteins/chemistry , Zebrafish Proteins/genetics
5.
Development ; 129(18): 4359-70, 2002 Sep.
Article in English | MEDLINE | ID: mdl-12183387

ABSTRACT

The red blood cell membrane skeleton is an elaborate and organized network of structural proteins that interacts with the lipid bilayer and transmembrane proteins to maintain red blood cell morphology, membrane deformability and mechanical stability. A crucial component of red blood cell membrane skeleton is the erythroid specific protein 4.1R, which anchors the spectrin-actin based cytoskeleton to the plasma membrane. Qualitative and quantitative defects in protein 4.1R result in congenital red cell membrane disorders characterized by reduced cellular deformability and abnormal cell morphology. The zebrafish mutants merlot (mot) and chablis (cha) exhibit severe hemolytic anemia characterized by abnormal cell morphology and increased osmotic fragility. The phenotypic analysis of merlot indicates severe hemolysis of mutant red blood cells, consistent with the observed cardiomegaly, splenomegaly, elevated bilirubin levels and erythroid hyperplasia in the kidneys. The result of electron microscopic analysis demonstrates that mot red blood cells have membrane abnormalities and exhibit a severe loss of cortical membrane organization. Using positional cloning techniques and a candidate gene approach, we demonstrate that merlot and chablis are allelic and encode the zebrafish erythroid specific protein 4.1R. We show that mutant cDNAs from both alleles harbor nonsense point mutations, resulting in premature stop codons. This work presents merlot/chablis as the first characterized non-mammalian vertebrate models of hereditary anemia due to a defect in protein 4.1R integrity.


Subject(s)
Anemia, Hemolytic/genetics , Membrane Proteins/deficiency , Membrane Proteins/genetics , Mutation , Neuropeptides , Zebrafish/genetics , Amino Acid Sequence , Animals , Base Sequence , Chromosome Mapping , Cloning, Molecular , Codon, Nonsense , Cytoskeletal Proteins/deficiency , Cytoskeletal Proteins/genetics , Cytoskeletal Proteins/metabolism , DNA Primers , DNA, Complementary/genetics , DNA, Complementary/isolation & purification , Disease Models, Animal , Erythrocyte Membrane/physiology , Erythrocyte Membrane/ultrastructure , Genetic Linkage , Membrane Proteins/metabolism , Polymerase Chain Reaction , Recombinant Proteins/metabolism , Sequence Alignment , Sequence Deletion , Sequence Homology, Amino Acid , Zebrafish/embryology
SELECTION OF CITATIONS
SEARCH DETAIL
...