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PubMed2006

Mycobacterium marinum infection of adult zebrafish causes caseating granulomatous tuberculosis and is moderated by adaptive immunity.

The zebrafish, a genetically tractable model vertebrate, is naturally susceptible to tuberculosis caused by Mycobacterium marinum, a close genetic relative of the causative agent of human tuberculosis, Mycobacterium tuberculosis. We previously developed a zebrafish embryo-M. marinum infection model to study host-pathogen interactions in the context of innate immunity. Here, we have constructed a flowthrough fish facility for the large-scale longitudinal study of M. marinum-induced tuberculosis in adult zebrafish where both innate and adaptive immunity are operant. We find that zebrafish are exquisitely susceptible to M. marinum strain M. Intraperitoneal injection of five organisms produces persistent granulomatous tuberculosis, while the injection of approximately 9,000 organisms leads to acute, fulminant disease. Bacterial burden, extent of disease, pathology, and host mortality progress in a time- and dose-dependent fashion. Zebrafish tuberculous granulomas undergo caseous necrosis, similar to human tuberculous granulomas. In contrast to mammalian tuberculous granulomas, zebrafish lesions contain few lymphocytes, calling into question the role of adaptive immunity in fish tuberculosis. However, like rag1 mutant mice infected with M. tuberculosis, we find that rag1 mutant zebrafish are hypersusceptible to M. marinum infection, demonstrating that the control of fish tuberculosis is dependent on adaptive immunity. We confirm the previous finding that M. marinum DeltaRD1 mutants are attenuated in adult zebrafish and extend this finding to show that DeltaRD1 predominantly produces nonnecrotizing, loose macrophage aggregates. This observation suggests that the macrophage aggregation defect associated with DeltaRD1 attenuation in zebrafish embryos is ongoing during adult infection.

Animals↗

PubMed2001

Isolation and characterization of a zebrafish homologue of the cone rod homeobox gene.

PURPOSE: To isolate and characterize a zebrafish CRX: homologue. Mammalian CRX: genes are expressed specifically in photoreceptors and pinealocytes, regulate photoreceptor gene expression, are necessary for normal photoreceptor differentiation, and when mutated cause a variety of photoreceptor degenerations. METHODS: A zebrafish retinal cDNA library was screened with a human CRX cDNA probe. Radiation hybrid mapping, Northern blot analysis, in situ hybridization, and transient transfection studies were performed using standard methods. RESULTS: Based on amino acid sequence comparisons, zebrafish crx shows 50% identity with human CRX, and 85% identity in the homeodomain. A phylogenetic analysis indicates that zebrafish crx is most closely related to the mammalian Crx proteins, and more distantly related to the Otx proteins. Zebrafish crx maps between 49.6 and 54.5 cm from the top of linkage group LG05C, a map position consistent with the location of the mouse and human CRX genes. Northern blot analysis and in situ hybridization indicate that zebrafish crx is expressed in the retina and pineal gland. In adult zebrafish, crx is expressed by both rods and cones in the outer nuclear layer, and in cells in the outer zone of the inner nuclear layer, in the region occupied by bipolar cells. Similar to mammalian Crx, zebrafish crx interacts with neural retinal leucine zipper (Nrl) to activate, although weakly, rhodopsin promoter activity. CONCLUSIONS: Based on molecular phylogeny, chromosomal location, expression pattern, and ability to activate rhodopsin promoter activity in transient transfection assays, zebrafish crx appears to be an orthologue and functional homologue of mammalian CRX:

Amino Acid Sequence↗

PubMed2003

Hematologic and serum biochemical values for zebrafish (Danio rerio).

The zebrafish (Danio rerio) has proven an excellent model for study of vertebrate development and genetics. Mutagenesis studies have produced many blood mutants with defects ranging from hematopoiesis to coagulation. The overwhelming majority of zebrafish studies have focused on development and mutational effects in embryos, whereas effects in mature zebrafish have gone largely unexplored. We believe that zebrafish will prove a valuable model for study of aging and age-related diseases, and we have sought to characterize some of the basic features of mature zebrafish. Accordingly, blood was collected from adult zebrafish and was analyzed to determine reference hematologic and biochemical parameters. White blood cell differential counts indicated predominantly lymphocytes, with mean proportion of 82.95%. Total red blood cell counts averaged 3.02 x 10(6) cells/microl. Except for increases in alanine transaminase (ALT), amylase, and phosphorus values, serum biochemical analytes were within the range of reported values for mammals and other species of fish. Accurate analysis of the many zebrafish mutants generated requires determination of normal characteristics of zebrafish. We believe results such as these will help define normal adult zebrafish, which have a tremendous potential for use in the study of human disease and aging.

Animals↗

PubMed2002

In silico and in situ characterization of the zebrafish (Danio rerio) gnrh3 (sGnRH) gene.

BACKGROUND: Gonadotropin releasing hormone (GnRH) is responsible for stimulation of gonadotropic hormone (GtH) in the hypothalamus-pituitary-gonadal axis (HPG). The regulatory mechanisms responsible for brain specificity make the promoter attractive for in silico analysis and reporter gene studies in zebrafish (Danio rerio). RESULTS: We have characterized a zebrafish [Trp7, Leu8] or salmon (s) GnRH variant, gnrh3. The gene includes a 1.6 Kb upstream regulatory region and displays the conserved structure of 4 exons and 3 introns, as seen in other species. An in silico defined enhancer at -976 in the zebrafish promoter, containing adjacent binding sites for Oct-1, CREB and Sp1, was predicted in 2 mammalian and 5 teleost GnRH promoters. Reporter gene studies confirmed the importance of this enhancer for cell specific expression in zebrafish. Interestingly the promoter of human GnRH-I, known as mammalian GnRH (mGnRH), was shown capable of driving cell specific reporter gene expression in transgenic zebrafish. CONCLUSIONS: The characterized zebrafish Gnrh3 decapeptide exhibits complete homology to the Atlantic salmon (Salmo salar) GnRH-III variant. In silico analysis of mammalian and teleost GnRH promoters revealed a conserved enhancer possessing binding sites for Oct-1, CREB and Sp1. Transgenic and transient reporter gene expression in zebrafish larvae, confirmed the importance of the in silico defined zebrafish enhancer at -976. The capability of the human GnRH-I promoter of directing cell specific reporter gene expression in zebrafish supports orthology between GnRH-I and GnRH-III.

Journal Article↗

PubMed2005

Faithful expression of living color reporter genes in transgenic medaka under two tissue-specific zebrafish promoters.

To test tissue specificity of zebrafish gene promoters in a heterologous fish species, two transgenic medaka lines under two zebrafish promoters were generated. Under the zebrafish skeletal muscle-specific mylz2 promoter, transgenic medaka expressed green fluorescent protein (GFP) exclusively in skeletal muscles, mimicking the endogenous medaka mylz2 mRNA expression and also identical to GFP expression in mylz2:gfp transgenic zebrafish. A madaka mylz2 promoter was also capable of directing skeletal muscle-specific GFP expression in transient transgenic zebrafish embryos. In the krt8:rfp transgenic medaka line with the zebrafish epithelial krt8 promoter, red fluorescent protein was specifically expressed in the skin epithelia as well as the epithelial lining cells of the anterior digestive tract, which was also identical to GFP expression in krt8:gfp transgenic zebrafish. Therefore, the two zebrafish promoters faithfully function in a heterologous fish species, and it is likely that the mechanisms of tissue-specific expression are largely conserved among fish species.

Amino Acid Sequence↗

PubMed1999

Conservation of intracellular Wnt signaling components in dorsal-ventral axis formation in zebrafish.

The mechanism of early dorso-ventral axis specification in zebrafish embryos is not well understood. While beta-catenin has been clearly implicated as a determinant of the axis, the factors upstream and downstream of beta-catenin in this system are not defined. Unlike in Xenopus, where a sperm-induced cortical rotation is used to localize beta-catenin on the future dorsal side of the embryo, zebrafish do not have an obviously similar morphogenetic movement. Recently, a GSK-3 (Glycogen Synthase Kinase-3) binding protein (GBP) was identified as a novel member of the Wnt pathway required for maternal dorsal axis formation in Xenopus. GBP stabilizes beta-catenin levels by inhibiting GSK-3 and potentially provides a link between cortical rotation and beta-catenin regulation. Since zebrafish may use a different mechanism for regulating beta-catenin, we asked whether zebrafish also express a maternal GBP. We report the isolation of the zebrafish GBP gene and show that it is maternally expressed and is present as mRNA ubiquitously throughout early embryonic development. Over-expression of zebrafish GBP in frogs and fish leads to hyper-dorsalized phenotypes, similar to the effects resulting from over-expression of beta-catenin, indicating that components upstream of beta-catenin are conserved between amphibians and teleosts. We also examined whether Tcf (T cell factor) functions in zebrafish embryos. As in frogs, ectopic expression of a dominant negative form of XTcf-3 ventralizes zebrafish embryos. In addition, ectopic beta-catenin expression activates the promoter of the Tcf-dependent gene siamois, indicating that the step immediately downstream of beta-catenin is also conserved between fish and frogs.

Amino Acid Sequence↗

PubMed2025

Loss of responses to odorants and pheromones in mPRγ (paqr5b)-knockout zebrafish.

In our previous study, we generated a membrane-type progesterone receptor γ (paqr5b) knockout zebrafish line. Knocking out paqr5b by genome editing resulted in the loss of neurons in the olfactory rosette (OR). These findings indicated that Paqr5b plays an essential role in the formation of olfactory neurons. In this study, we investigated the extent to which paqr5b-/- fish lacking olfactory neurons retain their sense of smell. We used a reported tank for zebrafish olfactory analysis with a dividing plate in the middle. The tank was divided into three zones: the right zone, where test substances were added; the neutral zone; and the left zone. The fish were released into the neutral zone at the beginning of each test, the chemical was added to the corner of the right zone, and a 3-min video was taken to track the movements of the fish. The video was then played back, and the time spent in the three zones was counted manually. Both male and female paqr5b+/+ and paqr5b-/- zebrafish were separately analyzed for time spent in the three zones after exposure to ATP, cadaverine, and the pheromone 17α,20β-dihydroxy-4-pregnen-3-one (DHP). Both male and female paqr5b+/+ zebrafish were strongly attracted to ATP and stayed in the right zone for approximately 2 min (67%). In addition, paqr5b+/+ zebrafish avoided cadaverine and stayed longer in the left zone than in any other zone. In contrast, paqr5b-/- zebrafish stayed in all three zones for approximately the same amount of time, even after exposure to ATP and cadaverine. The paqr5b+/+ fish were attracted to DHP and stayed in the right zone longer. Paqr5b-/- zebrafish of either sex were not reactive to DHP exposure. These results showed that paqr5b-/- zebrafish lacking olfactory neurons lost responsiveness to odorants and pheromones.

Animals↗

PubMed2004

Molecular cloning of zebrafish and medaka vitellogenin genes and comparison of their expression in response to 17beta-estradiol.

In the present study, both zebrafish and medaka vitellogenin genes have been isolated and used as a biomarker to compare the two small aquarium fish in response to estrogen treatment and thus to evaluate the two fish models in development of a biomonitoring system for environmental estrogens. The isolated zebrafish vitellogenin gene, zvtg1, is the most abundantly expressed vitellogenin gene in zebrafish and its complete protein sequence of 1360 amino acids was deduced from a genomic and a cDNA clone. The isolated medaka vitellogenin (mvtg1) genomic clone covers 1053 amino acids in the N-terminal. Both zebrafish zvtg1 and medaka mvtg1 are specifically expressed in female liver and their expression can be induced by 17beta-estradiol (E2) in male fish both by intramuscular injection and immersion treatment. A real-time reverse transcriptase polymerase chain reaction (RT-PCR) assay was developed for quantification of vitellogenin mRNA level in both control fish and E2-treated fish. The lowest-observed-effect concentrations of E2 for the induction of vitellogenin mRNAs were observed at 1 microg/l for zebrafish and 0.1 microg/l for medaka in a 2-day exposure experiment. Further kinetics studies of the two fish models indicated that medaka was able to respond much faster to E2 treatment than zebrafish, while the zebrafish can attain a much higher level of vitellogenin mRNAs than medaka after a long-term E2 treatment. The implication of these observations may be that the medaka system is better in monitoring acute treatment while the zebrafish system is better in monitoring chronic exposure.

Amino Acid Sequence↗

PubMed2006

Flounder and fugu have a single lefty gene that covers the functions of lefty1 and lefty2 of zebrafish during L-R patterning.

The lefty gene encodes a member of the TGF-beta superfamily that regulates L-R axis formation during embryogenesis via antagonistic activity against Nodal, another TGF-beta superfamily member. Both mouse and zebrafish have two lefty genes, lefty1 and lefty2. Interestingly, the expression domains of mouse and zebrafish lefty are different from one another. At present, the orthology and functional diversity of the mouse and zebrafish lefty genes are not clear. Here, we report that flounder and two fugu species, Takifugu and Tetraodon, have a single lefty gene in their genomes. In addition, we provide evidence that the mouse lefty genes were duplicated on a single chromosome but the zebrafish lefty genes arose from a whole-genome duplication that occurred early in the divergence of ray-finned fishes. These independent origins likely explain the difference in the expression domains of the mouse and zebrafish lefty gene pairs. Furthermore, we found that the duplication corresponding to the zebrafish lefty2 gene was lost from the fugu genome, suggesting that loss of lefty2 in the fugu/flounder lineage occurred after its divergence from the zebrafish lineage. During L-R patterning, the single lefty gene of flounder covers two expression domains, the left side of the dorsal diencephalon and the left LPM, which are regulated separately by lefty1 and lefty2 in zebrafish. We infer that the lefty genes of the ray-finned fishes and mammals underwent independent gene duplication events that resulted in independent regulation of lefty expression.

Amino Acid Sequence↗

PubMed2003

Unique and conserved aspects of gut development in zebrafish.

Although the development of the digestive system of humans and vertebrate model organisms has been well characterized, relatively little is known about how the zebrafish digestive system forms. We define developmental milestones during organogenesis of the zebrafish digestive tract, liver, and pancreas and identify important differences in the way the digestive endoderm of zebrafish and amniotes is organized. Such differences account for the finding that the zebrafish digestive system is assembled from individual organ anlagen, whereas the digestive anlagen of amniotes arise from a primitive gut tube. Despite differences of organ morphogenesis, conserved molecular programs regulate pharynx, esophagus, liver, and pancreas development in teleosts and mammals. Specifically, we show that zebrafish faust/gata-5 is a functional ortholog of gata-4, a gene that is essential for the formation of the mammalian and avian foregut. Further, extraembryonic gata activity is required for this function in zebrafish as has been shown in other vertebrates. We also show that a loss-of-function mutation that perturbs sonic hedgehog causes defects in the development of the esophagus that parallel those associated with targeted disruption of this gene in mammals. Perturbation of sonic hedgehog also affects zebrafish liver and pancreas development, and these effects occur in a reciprocal fashion, as has been described during mammalian liver and ventral pancreas development. Together, these data define aspects of digestive system development necessary for the characterization of zebrafish mutants. Given the similarities of teleost and mammalian digestive physiology and anatomy, these findings have implications for developmental and evolutionary studies as well as research of human diseases, such as diabetes, liver cirrhosis, and cancer.

Alleles↗

PubMed2006

Gene duplication and functional divergence of the zebrafish insulin-like growth factor 1 receptors.

Insulin-like growth factor (IGF) 1 receptor (IGF1R)-mediated signaling plays key roles in growth, development, and physiology. Recent studies have shown that there are two distinct ig f1r genes in zebrafish, termed ig f1ra and ig f1rb. In this study, we tested the hypothesis that zebrafish ig f1ra and ig f1rb resulted from a gene duplication event at the ig f1r locus and that this has led to their functional divergence. The genomic structures of zebrafish ig f1ra and ig f1rb were determined and their loci mapped. While zebrafish ig f1ra has 21 exons and is located on linkage group (LG) 18, zebrafish ig f1rb has 22 exons and mapped to LG 7. There is a strong syntenic relationship between the two zebrafish genes and the human IG F1R gene. Using a MO-based loss-of-function approach, we show that both Igf1ra and Igf1rb are required for zebrafish embryo viability and proper growth and development. Although Igf1ra and Igf1rb demonstrated a large degree of functional overlap with regard to cell differentiation in the developing eye, inner ear, heart, and muscle, they also exhibited functional distinction involving a greater requirement for Igf1rb in spontaneous muscle contractility. These findings suggest that the duplicated zebrafish ig f1r genes play largely overlapping but not identical functional roles in early development and provide novel insight into the functional evolution of the IGF1R/insulin receptor gene family.

Animals↗

PubMed2003

The zebrafish annexin gene family.

The Annexins (ANXs) are a family of calcium- and phospholipid-binding proteins that have been implicated in many cellular processes, including channel formation, membrane fusion, vesicle transport, and regulation of phospholipase A2 activity. As a first step toward understanding in vivo function, we have cloned 11 zebrafish anx genes. Four genes (anx1a, anx2a, anx5,and anx11a) were identified by screening a zebrafish cDNA library with a Xenopus anx2 fragment. For these genes, full-length cDNA sequences were used to cluster 212 EST sequences generated by the Zebrafish Genome Resources Project. The EST analysis revealed seven additional anx genes that were subsequently cloned. The genetic map positions of all 11 genes were determined by using a zebrafish radiation hybrid panel. Sequence and syntenic relationships between zebrafish and human genes indicate that the 11 genes represent orthologs of human anx1,2,4,5,6,11,13,and suggest that several zebrafish anx genes resulted from duplications that arose after divergence of the zebrafish and mammalian genomes. Zebrafish anx genes are expressed in a wide range of tissues during embryonic and larval stages. Analysis of the expression patterns of duplicated genes revealed both redundancy and divergence, with the most similar genes having almost identical tissue-specific patterns of expression and with less similar duplicates showing no overlap. The differences in gene expression of recently duplicated anx genes could explain why highly related paralogs were maintained in the genome and did not rapidly become pseudogenes.

Amino Acid Sequence↗

PubMed2005

Efficient RNA interference in zebrafish embryos using siRNA synthesized with SP6 RNA polymerase.

Double-stranded RNA (dsRNA) has been shown to be a useful tool for silencing genes in zebrafish (Danio rerio), while the blocking specificity of dsRNA is still of major concern for application. It was reported that siRNA (small interfering RNA) prepared by endoribonuclease digestion (esiRNA) could efficiently silence endogenous gene expression in mammalian embryos. To test whether esiRNA could work in zebrafish, we utilized Escherichia coli RNaseIII to digest dsRNA of zebrafish no tail (ntl), a mesoderm determinant in zebrafish and found that esi-ntl could lead to developmental defects, however, the effective dose was so close to the toxic dose that esi-ntl often led to non-specific developmental defects. Consequently, we utilized SP6 RNA polymerase to produce si-ntl, siRNA designed against ntl, by in vitro transcription. By injecting in vitro synthesized si-ntl into zebrafish zygotes, we obtained specific phenocopies of reported mutants of ntl. We achieved up to a 59%no tail phenotype when the injection concentration was as high as 4 microg/microL. Quantitative reverse transcription-polymerase chain reaction (RT-PCR) and whole-mount in situ hybridization analysis showed that si-ntl could largely and specifically reduce mRNA levels of the ntl gene. As a result, our data indicate that esiRNA is unable to cause specific developmental defects in zebrafish, while siRNA should be an alternative for downregulation of specific gene expression in zebrafish in cases where RNAi techniques are applied to zebrafish reverse genetics.

Animals↗

PubMed2007

D4 Dopamine receptor genes of zebrafish and effects of the antipsychotic clozapine on larval swimming behaviour.

Zebrafish, a model developmental genetic organism, is being increasingly used in behavioural studies. We have initiated studies designed to evaluate the response of zebrafish to antipsychotic drugs. This study focuses on characterization of zebrafish D4 dopamine receptors (D4Rs) and the response of larval zebrafish to the atypical antipsychotic clozapine. The D4R is of interest because of its high affinity for clozapine, while interest in clozapine stems from its effectiveness in reducing symptoms in acutely psychotic, treatment-resistant schizophrenic patients. By mining the zebrafish genomic database, we identified three distinct D4R genes, drd4a, drd4b and drd4c, and generated full-length open reading frames encoding each of the three D4Rs by reverse transcription-polymerase chain reaction. Gene mapping studies showed that each D4R gene mapped to a distinct chromosomal location in the zebrafish genome, and each gene exhibited a unique expression profile during embryogenesis. When administered to larval zebrafish, clozapine produced a rapid and profound effect on locomotor activity. The effect of clozapine was dose-dependent, resulted in hypoactivity and was prevented by the D4-selective agonist ABT-724. Our data suggest that the inhibitory effect of clozapine on the locomotor activity of larval zebrafish may be mediated through D4Rs.

Amino Acid Sequence↗

PubMed2006

Hierarchical subfunctionalization of fabp1a, fabp1b and fabp10 tissue-specific expression may account for retention of these duplicated genes in the zebrafish (Danio rerio) genome.

Fatty acid-binding protein type 1 (FABP1), commonly termed liver-type fatty acid-binding protein (L-FABP), is encoded by a single gene in mammals. We cloned and sequenced cDNAs for two distinct FABP1s in zebrafish coded by genes designated fabp1a and fabp1b. The zebrafish proteins, FABP1a and FABP1b, show highest sequence identity and similarity to the human protein FABP1. Zebrafish fabp1a and fabp1b genes were assigned to linkage groups 5 and 8, respectively. Both linkage groups show conserved syntenies to a segment of mouse chromosome 6, rat chromosome 4 and human chromosome 2 harboring the FABP1 locus. Phylogenetic analysis further suggests that zebrafish fabp1a and fabp1b genes are orthologs of mammalian FABP1 and most likely arose by a whole-genome duplication event in the ray-finned fish lineage, estimated to have occurred 200-450 million years ago. The paralogous fabp10 gene encoding basic L-FABP, found to date in only nonmammalian vertebrates, was assigned to zebrafish linkage group 16. RT-PCR amplification of mRNA in adults, and in situ hybridization to whole-mount embryos to fabp1a, fabp1b and fapb10 mRNAs, revealed a distinct and differential pattern of expression for the fabp1a, fabp1b and fabp10 genes in zebrafish, suggesting a division of function for these orthogolous and paralogous gene products following their duplication in the vertebrate genome. The differential and complementary expression patterns of the zebrafish fabp1a, fapb1b and fabp10 genes imply a hierarchical subfunctionalization that may account for the retention of both the duplicated fabp1a and fabp1b genes, and the fabp10 gene in the zebrafish genome.

Amino Acid Sequence↗

PubMed2002

Zebrafish mitotic kinesin-like protein 1 (Mklp1) functions in embryonic cytokinesis.

To understand the functions of microtubule motors in vertebrate development, we are investigating the kinesin-like proteins (KLPs) of the zebrafish, Danio rerio. Here we describe the structure, intracellular distribution, and function of zebrafish mitotic KLP1 (Mklp1). The zebrafish mklp1 gene that encodes this 867-amino acid protein maps to a region of zebrafish linkage group 18 that is syntenic with part of human chromosome 15. In zebrafish AB9 fibroblasts and in COS-7 cells, the zebrafish Mklp1 protein decorates spindle microtubules at metaphase, redistributes to the spindle midzone during anaphase, and becomes concentrated in the midbody during telophase and cytokinesis. The motor is detected consistently in interphase nuclei of COS cells and occasionally in those of AB9 cells. Nuclear targeting of Mklp1 is conferred by two basic motifs located in the COOH terminus of the motor. In cleaving zebrafish embryos, green fluorescent protein (GFP)-tagged Mklp1 is found in the nucleus in interphase and associates with microtubules of the spindle midbody in cytokinesis. One- or two-cell embryos injected with synthetic mRNAs encoding dominant-negative variants of GFP-Mklp1 frequently fail to complete cytokinesis during cleavage, resulting in formation of multinucleated blastomeres. Our results indicate that the zebrafish Mklp1 motor performs a critical function that is required for completion of embryonic cytokinesis.

Amino Acid Sequence↗

PubMed2005

Ferroportin1 is required for normal iron cycling in zebrafish.

Missense mutations in ferroportin1 (fpn1), an intestinal and macrophage iron exporter, have been identified between transmembrane helices 3 and 4 in the zebrafish anemia mutant weissherbst (weh(Tp85c-/-)) and in patients with type 4 hemochromatosis. To explore the effects of fpn1 mutation on blood development and iron homeostasis in the adult zebrafish, weh(Tp85c-/-) zebrafish were rescued by injection with iron dextran and studied in comparison with injected and uninjected WT zebrafish and heterozygotes. Although iron deposition was observed in all iron-injected fish, only weh(Tp85c-/-) zebrafish exhibited iron accumulation in the intestinal epithelium compatible with a block in iron export. Iron injections initially reversed the anemia. However, 8 months after iron injections were discontinued, weh(Tp85c-/-) zebrafish developed hypochromic anemia and impaired erythroid maturation despite the persistence of iron-loaded macrophages and elevated hepatic nonheme iron stores. Quantitative real-time RT-PCR revealed a significant decrease in mean hepatic transcript levels of the secreted iron-regulator hepcidin and increased intestinal expression of fpn1 in anemic weh(Tp85c-/-) adults. Injection of iron dextran into WT or mutant zebrafish embryos, however, resulted in significant increases in hepcidin expression 18 hours after injection, demonstrating that hepcidin expression in zebrafish is iron responsive and independent of fpn1's function as an iron exporter.

Anemia, Hypochromic↗

PubMed2002

Runx1 is required for zebrafish blood and vessel development and expression of a human RUNX1-CBF2T1 transgene advances a model for studies of leukemogenesis.

RUNX1/AML1/CBFA2 is essential for definitive hematopoiesis, and chromosomal translocations affecting RUNX1 are frequently involved in human leukemias. Consequently, the normal function of RUNX1 and its involvement in leukemogenesis remain subject to intensive research. To further elucidate the role of RUNX1 in hematopoiesis, we cloned the zebrafish ortholog (runx1) and analyzed its function using this model system. Zebrafish runx1 is expressed in hematopoietic and neuronal cells during early embryogenesis. runx1 expression in the lateral plate mesoderm co-localizes with the hematopoietic transcription factor scl, and expression of runx1 is markedly reduced in the zebrafish mutants spadetail and cloche. Transient expression of runx1 in cloche embryos resulted in partial rescue of the hematopoietic defect. Depletion of Runx1 with antisense morpholino oligonucleotides abrogated the development of both blood and vessels, as demonstrated by loss of circulation, incomplete development of vasculature and the accumulation of immature hematopoietic precursors. The block in definitive hematopoiesis is similar to that observed in Runx1 knockout mice, implying that zebrafish Runx1 has a function equivalent to that in mammals. Our data suggest that zebrafish Runx1 functions in both blood and vessel development at the hemangioblast level, and contributes to both primitive and definitive hematopoiesis. Depletion of Runx1 also caused aberrant axonogenesis and abnormal distribution of Rohon-Beard cells, providing the first functional evidence of a role for vertebrate Runx1 in neuropoiesis. To provide a base for examining the role of Runx1 in leukemogenesis, we investigated the effects of transient expression of a human RUNX1-CBF2T1 transgene [product of the t(8;21) translocation in acute myeloid leukemia] in zebrafish embryos. Expression of RUNX1-CBF2T1 caused disruption of normal hematopoiesis, aberrant circulation, internal hemorrhages and cellular dysplasia. These defects reproduce those observed in Runx1-depleted zebrafish embryos and RUNX1-CBF2T1 knock-in mice. The phenotype obtained with transient expression of RUNX1-CBF2T1 validates the zebrafish as a model system to study t(8;21)-mediated leukemogenesis.

Acute Disease↗