Mitoferrin is essential for erythroid iron assimilation.

Iron has a fundamental role in many metabolic processes, including electron transport, deoxyribonucleotide synthesis, oxygen transport and many essential redox reactions involving haemoproteins and Fe-S cluster proteins. Defective iron homeostasis results in either iron deficiency or iron overload. Precise regulation of iron transport in mitochondria is essential for haem biosynthesis, haemoglobin production and Fe-S cluster protein assembly during red cell development. Here we describe a zebrafish mutant, frascati (frs), that shows profound hypochromic anaemia and erythroid maturation arrest owing to defects in mitochondrial iron uptake. Through positional cloning, we show that the gene mutated in the frs mutant is a member of the vertebrate mitochondrial solute carrier family (SLC25) that we call mitoferrin (mfrn). mfrn is highly expressed in fetal and adult haematopoietic tissues of zebrafish and mouse. Erythroblasts generated from murine embryonic stem cells null for Mfrn (also known as Slc25a37) show maturation arrest with severely impaired incorporation of 55Fe into haem. Disruption of the yeast mfrn orthologues, MRS3 and MRS4, causes defects in iron metabolism and mitochondrial Fe-S cluster biogenesis. Murine Mfrn rescues the defects in frs zebrafish, and zebrafish mfrn complements the yeast mutant, indicating that the function of the gene may be highly conserved. Our data show that mfrn functions as the principal mitochondrial iron importer essential for haem biosynthesis in vertebrate erythroblasts.

The role and regulation of friend of GATA-1 (FOG-1) during blood development in the zebrafish.

The nuclear protein FOG-1 binds transcription factor GATA-1 to facilitate erythroid and megakaryocytic maturation. However, little is known about the function of FOG-1 during myeloid and lymphoid development or how FOG-1 expression is regulated in any tissue. We used in situ hybridization, gain- and loss-of-function studies in zebrafish to address these problems. Zebrafish FOG-1 is expressed in early hematopoietic cells, as well as heart, viscera, and paraspinal neurons, suggesting that it has multifaceted functions in organogenesis. We found that FOG-1 is dispensable for endoderm specification but is required for endoderm patterning affecting the expression of late-stage T-cell markers, independent of GATA-1. The suppression of FOG-1, in the presence of normal GATA-1 levels, induces severe anemia and thrombocytopenia and expands myeloid-progenitor cells, indicating that FOG-1 is required during erythroid/myeloid commitment. To functionally interrogate whether GATA-1 regulates FOG-1 in vivo, we used bioinformatics combined with transgenic assays. Thus, we identified 2 cis-regulatory elements that control the tissue-specific gene expression of FOG-1. One of these enhancers contains functional GATA-binding sites, indicating the potential for a regulatory loop in which GATA factors control the expression of their partner protein FOG-1.

Ca2+ signaling in mouse cardiomyocytes with ablated S100A1 protein.

S100A1 is a Ca2+-binding protein expressed at high levels in the myocardium. It is thought to modulate the Ca2+ sensitivity of the sarcoplasmic reticulum (SR) Ca2+ release channels (ryanodine receptors or RyRs) and its expression has been shown to be down regulated in various heart diseases. In this study we used S100A1 knock-out (KO) mice to investigate the consequences of chronic S100A1 deficiency on Ca2+ cycling in ventricular cardiomyocytes. Confocal Ca2+ imaging showed that field-stimulated KO myocytes had near normal Ca2+ signaling under control conditions but a blunted response to beta-adrenergic stimulation with 1 micromol/l isoproterenol (ISO). Voltage-clamp experiments revealed that S100A1-deficient cardiomyocytes have elevated ICa under basal conditions. This larger Ca2+ influx was accompanied by augmented Ca2+ transients and elevated SR Ca2+ content, without changes in macroscopic excitation-contraction coupling gain, which suggests impaired fractional Ca2+ release. Exposure of KO and WT cells to ISO led to similar maximal ICa. Thus, the stimulation of the ICa was less pronounced in KO cardiomyocytes, suggesting that changes in basal ICa could underlie the reduced beta-adrenergic response. Taken together, our findings indicate that chronic absence of S100A1 results in enhanced L-type Ca2+ channel activity combined with a blunted SR Ca2+ release amplification. These findings may have implications in a variety of cardiac pathologies where abnormal RyR Ca2+ sensitivity or reduced S100A1 levels have been described.

S100A1-deficient male mice exhibit increased exploratory activity and reduced anxiety-related responses.

S100 proteins comprise a family of Ca(2+) binding proteins of at least 21 members. They are distinctly expressed in a variety of cell types and tissues and are thought to play unique roles, although they share a high degree of sequence homology and expression overlap. S100A1 is prominently expressed in the heart, where it takes part in Ca(2+)-cycling. Its role in the central nervous system (CNS) is largely unknown. We have generated S100A1-deficient mice by gene trap mutagenesis to study the involvement of S100A1 in the cytoarchitecture of the brain, in learning and memory, and in avoidance-approach behavior. S100A1 knock out (KO) mice develop well and their brains present with normal morphology. In wild type (Wt) mice, S100A1 protein was found in the hippocampus, cerebral cortex and amygdala, and partially co-localized with the astrocyte marker glial fibrillary acidic protein (GFAP) in the stratum radiatum of the hippocampus. Astrocytes and neurons of S100A1KO mice did not differ from those of Wt mice regarding shape, distribution and density. In the water maze, S100A1KO mice performed equally well as Wt, implying that S100A1 is not involved in spatial learning and memory. In avoidance-approach tests, predominantly male S100A1KO mice showed reduced anxiety-like responses and enhanced explorative activities. We conclude that S100A1 plays a role in modulating innate fear and exploration of novel stimuli.

Zebrafish: a genetic model for vertebrate organogenesis and human disorders.

Mutations may be tolerated without noticeable effect or may present with a specific phenotype that reveals information about the function of the mutated gene. This information is an inexhaustible source for understanding biology and let us ask particular questions about the molecular mechanisms of development, degeneration and disease. The zebrafish (Danio rerio) has been proven to be instrumental in the genetic analysis of spontaneous and induced mutations and has provided invaluable clues to the elucidation of complex molecular processes in vertebrate biology. Since completion of the two large-scale mutagenesis screens carried out at the Max-Planck Institute in Tuebingen and at the Massachusetts General Hospital in Boston, many of the recovered mutations have been cloned and the function of the mutated genes studied. Special interest laid in the analysis of mutations affecting structures and organ systems characteristic for vertebrates such as the notochord, neural crest, heart, vasculature, blood and kidney. This review updates our knowledge of heart, vessel, blood and kidney organogenesis in zebrafish and extrapolates our insights to human disorders by assessing common genetic pathways.

Effects of long-term nonylphenol exposure on gonadal development and biomarkers of estrogenicity in juvenile rainbow trout Oncorhynchus mykiss.

Environmental pollutants with estrogenic activity including nonylphenol (NP) have the potential to alter gonadal development and reproduction of wild fish. To investigate the estrogenic action of environmentally relevant concentrations of NP, rainbow trout (Oncorhynchus mykiss) were continuously exposed during the embryonic, larval and juvenile life stage to 1.05 and 10.17 microg/l NP for 1 year, and sexual differentiation, vitellogenin (VG), VG mRNA, and zona radiata protein (ZRP) expression were examined after that period. The applied NP concentrations did not affect mortality and hatching rates, and did not have an influence on the body weight of 1-year-old fish. No occurrence of testis-ova was observed and sex-ratios of NP exposed groups of fish were unchanged when compared with control groups. The induction of VG and ZRP expression was a more sensitive reaction to the presence of NP than the formation of testis-ova and the reversal of sex. Increased VG expression in trout liver occurred already at 1.05 microg/l NP, whereas VG mRNA levels, quantified by competitive RT-PCR, were not significantly elevated in NP exposed fish. ZRP contents were significantly higher at 10.17 microg/l NP. Since induction of VG did not occur in all fish exposed to 1 or 10 microg/l NP and ZRP induction did not occur in all fish exposed to 10 microg/l, some individuals may be more affected by exposure to NP than others. This study demonstrates that NP concentrations typically found in sewage treatment effluents and some rivers do not affect sexual differentiation in rainbow trout, but induce VG and ZRP expression in the liver of exposed fish.

Development of a fish reporter gene system for the assessment of estrogenic compounds and sewage treatment plant effluents.

This study reports on the development and application of a fish-specific estrogen-responsive reporter gene assay. The assay is based on the rainbow trout (Oncorhynchus mykiss) gonad cell line RTG-2 in which an acute estrogenic response is created by cotransfecting cultures with an expression vector containing rainbow trout estrogen receptor a complementary DNA (rtERalpha cDNA) in the presence of an estrogen-dependent reporter plasmid and an estrogen receptor (ER) agonist. In a further approach, RTG-2 cells were stably transfected with the rtERalpha cDNA expression vector, and clones responsive to 17beta-estradiol (E2) were selected. The estrogenic activity of E2, 17alpha-ethinylestradiol, 4-nonylphenol, nonylphenoxy acetic acid, 4-tert-octylphenol, bisphenol A, o,p'-DDT, p,p'-DDT, o,p'-2,2-bis(chlorophenyl)-1,1-dichloroethylene (o,p'-DDE), p,p'-DDE, o,p'-2,2-bis(chlorophenyl)-1,1-di-chloroethane (o,p'-DDD), p,p'-DDD, and p,p'-2,2-bis(chlorophenyl)acetic acid (p,p'-DDA) was assessed at increasing concentrations. All compounds except o,p'-DDT, p,p'-DDE, and p,p'-DDA showed logistic dose-response curves, which allowed the calculation of lowest-observed-effect concentrations and the concentrations at which half-maximal reporter gene activities were reached. To check whether estrogen-responsive RTG-2 cells may be used to detect the estrogenic activity of environmental samples, an extract from a sewage treatment plant (STP) effluent was assessed and found to have estrogenic activity corresponding to the transcriptional activity elicited by 0.05 nM of E2. Dose-response curves of nonylphenol, octylphenol, bisphenol A, and o,p'-DDD revealed that the RTG-2 reporter gene assay is more sensitive for these compounds when compared to transfection systems recombinant for mammalian ERs. These differences may have an effect on the calculation of E2 equivalents when estrogenic mixtures of known constitution, or environmental samples, such as STP effluents, are assessed.

Comparative analysis of estrogenic activity in sewage treatment plant effluents involving three in vitro assays and chemical analysis of steroids.

In this study, we assessed and compared the suitability of three in vitro screening tools for the measurement of estrogenic activity in sewage treatment plant effluents (STPEs). These assays were the yeast estrogen screen (YES), production of zona radiata proteins (ZRPs) in trout hepatocytes, and the induction of reporter gene expression in the transfected rainbow trout gonad cell line RTG-2. Data obtained with the YES were additionally compared with calculated estrogenicity, based on steroid analysis data of the effluents. For comparison purposes, the response of the in vitro systems toward the estrogenic chemicals beta-estradiol, ethinyl estradiol, bisphenol-A, nonylphenol, and octylphenol was assessed. All three assays showed sensitivities in the same order of magnitude in response to the steroid compounds tested, with ZRP production being the least sensitive. Regarding the estrogenic environmental chemicals tested, the RTG-2 assay was more than an order of magnitude more sensitive than the other two assays. Despite their different sensitivities toward selected test chemicals, the three in vitro systems indicated estrogenic activity in the same concentration range for the tested STPEs. Calculated estrogenicity (chemical analysis) and measured estrogenicity (YES) were of the same order of magnitude for the STPEs tested. The present study indicates that all three in vitro systems, with the yeast-based system being the easiest and most robust, are applicable for the screening of estrogenic activity in effluent samples.

Vascular endothelial growth factor receptor signaling is required for cardiac valve formation in zebrafish.

Vascular endothelial growth factor-receptors (VEGF-Rs) are pivotal regulators of vascular development, but a specific role for these receptors in the formation of heart valves has not been identified. We took advantage of small molecule inhibitors of VEGF-R signaling and showed that blocking VEGF-R signaling with receptor selective tyrosine kinase inhibitors, PTK 787 and AAC 787, from 17-21 hr post-fertilization (hpf) in zebrafish embryos resulted in a functional and structural defect in cardiac valve development. Regurgitation of blood between the two chambers of the heart, as well as a loss of cell-restricted expression of the valve differentiation markers notch 1b and bone morphogenetic protein-4 (bmp-4), was readily apparent in treated embryos. In addition, microangiography revealed a loss of a definitive atrioventricular constriction in treated embryos. Taken together, these data demonstrate a novel function for VEGF-Rs in the endocardial endothelium of the developing cardiac valve.

Zebrafish slc4a2/ae2 anion exchanger: cDNA cloning, mapping, functional characterization, and localization.

Although the zebrafish has been used increasingly for the study of pronephric kidney development, studies of renal ion transporters and channels of the zebrafish remain few. We report the cDNA cloning and characterization of the AE2 anion exchanger ortholog from zebrafish kidney, slc4a2/ae2. The ae2 gene in linkage group 2 encodes a polypeptide of 1,228 aa exhibiting 64% aa identity with mouse AE2a. The exon-intron boundaries of the zebrafish ae2 gene are nearly identical to those of the rodent and human genes. Whole-mount in situ hybridization detects ae2 mRNA in prospective midbrain as early as the five-somite stage, then later in the pronephric primordia and the forming pronephric duct, where it persists through 72 h postfertilization (hpf). Zebrafish Ae2 expressed in Xenopus laevis oocytes mediates Na(+)-independent, electroneutral (36)Cl(-)/Cl(-) exchange moderately sensitive to inhibition by DIDS, is inhibited by acidic intracellular pH and by acidic extracellular pH, but activated by (acidifying) ammonium and by hypertonicity. Zebrafish Ae2 also mediates Cl(-)/HCO(3)(-) exchange in X. laevis oocytes and accumulates in or near the plasma membrane in transfected HEK-293 cells. In 24-48 hpf zebrafish embryos, the predominant but not exclusive localization of Ae2 polypeptide is the apical membrane of pronephric duct epithelial cells. Thus Ae2 resembles its mammalian orthologs in function, mechanism, and acute regulation but differs in its preferentially apical expression in kidney. These results will inform tests of the role of Ae2 in zebrafish kidney development and function.