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PubMed2004

Zebrafish as a model of human hematologic disorders.

PURPOSE OF REVIEW: This review summarizes the status of zebrafish as a genetic model to study human hematological disorders. Much of our current understanding of the function of genes modulating the process of hematopoietic stem cell generation, specification, and differentiation has come from mutant analysis. Because of the transparency of zebrafish embryos that allows for direct visualization of circulating erythroid cells, mutations affecting zebrafish erythropoiesis were among the first characterized mutants through positional cloning and candidate gene strategies. RECENT FINDINGS: New technologies have evolved that allow for generation, detection, and characterization of lineage specific alterations in the hematopoietic system. We will also briefly discuss the applications of several of these technologies such as targeted gene knockdown using antisense morpholinos, small molecule screen, transgenesis, and cell transplantation as related to blood disorders and hematopoietic development. SUMMARY: The combination of phenotype-driven forward genetic analyses and innovative technical advances has conferred zebrafish as a powerful genetic model to further dissect the function of hematopoietic genes. Through the use of available resources, the identification of novel genes or novel function for known hematopoietic genes will have important implications for our understanding of human disease pathogenesis, treatment, and prevention.

Animals↗

PubMed2000

The genetic basis of cardiac function: dissection by zebrafish (Danio rerio) screens.

The vertebrate heart differs from chordate ancestors both structurally and functionally. Genetic units of form, termed 'modules', are identifiable by mutation, both in zebrafish and mouse, and correspond to features recently acquired in evolution, such as the ventricular chamber or endothelial lining of the vessels and heart. Zebrafish (Danio rerio) genetic screens have provided a reasonably inclusive set of such genes. Normal cardiac function may also be disrupted by single-gene mutations in zebrafish. Individual mutations may perturb contractility or rhythm generation. The zebrafish mutations which principally disturb cardiac contractility fall into two broad phenotypic categories, 'dilated' and 'hypertrophic'. Interestingly, these correspond to the two primary types of heart failure in humans. These disorders of early cardiac function provide candidate genes to be examined in complex human heart diseases, including arrhythmias and heart failure.

Animals↗

PubMed1999

A large-scale insertional mutagenesis screen in zebrafish.

It is estimated that approximately 2500 genes are essential for the normal development of a zebrafish embryo. A mutation in any one of these genes can result in a visible developmental defect, usually followed by the death of the embryo or larva by days 5-7 of age. We are performing a large-scale insertional mutagenesis screen in the zebrafish with the goal of isolating approximately 1000 embryonic mutations. We plan to clone a significant fraction of the mutated genes, as these are the genes important for normal embryogenesis of a vertebrate. To achieve this goal, we prepared approximately 36, 000 founder fish by injecting blastula-stage embryos with one of two pseudotyped retroviruses. We estimate that together these fish harbor between 500,000-1,000,000 proviral insertions in their germ lines. The protocol we have devised and the size of our facility allow us to breed approximately 80,000-150,000 of these insertions to homozygosity within 2 years. Because a pilot screen conducted earlier in our laboratory revealed that the frequency of mutations obtained with this type of insertional mutagen is 1 embryonic lethal mutation per 70-100 proviral insertions, screening 100,000 insertions should yield at least 1000 mutants. Here we describe the protocol for the screen and initial results with the first of the two retroviral vectors used, a virus designated F(5). We screened an estimated 760 insertions among F(3) progeny from 92 F(2) families and obtained 9 recessive embryonic lethal mutations. Thus, the efficiency of mutagenesis with this viral vector is approximately one-ninth that observed with the chemical mutagen ENU in zebrafish. We have also obtained two dominant mutations, one of which is described here. As expected, mutated genes can be readily identified. So far, genes mutated in four of the nine recessive mutants and one of the two dominant mutants have been cloned. Further improvements to this technology could make large-scale insertional mutagenesis screening and rapid gene cloning accessible to relatively small zebrafish laboratories.

Alleles↗

PubMed2005

The developmental miRNA profiles of zebrafish as determined by small RNA cloning.

MicroRNAs (miRNAs) represent a family of small, regulatory, noncoding RNAs that are found in plants and animals. Here, we describe the miRNA profile of the zebrafish Danio rerio resolved in a developmental and cell-type-specific manner. The profiles were obtained from larger-scale sequencing of small RNA libraries prepared from developmentally staged zebrafish, and two adult fibroblast cell lines derived from the caudal fin (ZFL) and the liver epithelium (SJD). We identified a total of 154 distinct miRNAs expressed from 343 miRNA genes. Other experimental/computational sources support an additional 10 miRNAs encoded by 19 genes. The miRNAs can be classified into 87 distinct families. Cross-species comparison indicates that 81 families are conserved in mammals, 17 of which also have at least one member conserved in an invertebrate. Our analysis reveals that the zygotes are essentially devoid of miRNAs and that their expression begins during the blastula period with a zebrafish-specific family of miRNAs encoded by closely spaced multicopy genes. Computational predictions of zebrafish miRNA targets are provided that take into account the depth of evolutionary conservation. Besides miRNAs, we identified a prominent class of repeat-associated small interfering RNAs (rasiRNAs).

Animals↗

PubMed2000

Identification, characterization, and mapping of expressed sequence tags from an embryonic zebrafish heart cDNA library.

The generation of expressed sequence tags (ESTs) has proven to be a rapid and economical approach by which to identify and characterize expressed genes. We generated 5102 ESTs from a 3-d-old embryonic zebrafish heart cDNA library. Of these, 57.6% matched to known genes, 14.2% matched only to other ESTs, and 27.8% showed no match to any ESTs or known genes. Clustering of all ESTs identified 359 unique clusters comprising 1771 ESTs, whereas the remaining 3331 ESTs did not cluster. This estimates the number of unique genes identified in the data set to be approximately 3690. A total of 1242 unique known genes were used to analyze the gene expression patterns in the zebrafish embryonic heart. These were categorized into seven categories on the basis of gene function. The largest class of genes represented those involved in gene/protein expression (25.9% of known transcripts). This class was followed by genes involved in metabolism (18.7%), cell structure/motility (16.4%), cell signaling and communication (9.6%), cell/organism defense (7.1%), and cell division (4.4%). Unclassified genes constituted the remaining 17.91%. Radiation hybrid mapping was performed for 102 ESTs and comparison of map positions between zebrafish and human identified new synteny groups. Continued comparative analysis will be useful in defining the boundaries of conserved chromosome segments between zebrafish and humans, which will facilitate the transfer of genetic information between the two organisms and improve our understanding of vertebrate evolution.

Animals↗

PubMed2000

Genetic linkage mapping of zebrafish genes and ESTs.

Genetic screens in zebrafish (Danio rerio) have isolated mutations in hundreds of genes essential for vertebrate development, physiology, and behavior. We have constructed a genetic linkage map that will facilitate the identification of candidate genes for these mutations and allow comparisons among the genomes of zebrafish and other vertebrates. On this map, we have localized 771 zebrafish genes and expressed sequence tags (ESTs) by scoring single-stranded conformational polymorphisms (SSCPs) in a meiotic mapping panel. Of these sequences, 642 represent previously unmapped genes and ESTs. The mapping panel was comprised of 42 homozygous diploid individuals produced by heat shock treatment of haploid embryos at the one-cell stage (HS diploids). This "doubled haploid" strategy combines the advantages of mapping in haploid and standard diploid systems, because heat shock diploid individuals have only one allele at each locus and can survive to adulthood, enabling a relatively large quantity of genomic DNA to be prepared from each individual in the mapping panel. To integrate this map with others, we also scored 593 previously mapped simple-sequence length polymorphisms (SSLPs) in the mapping panel. This map will accelerate the molecular analysis of zebrafish mutations and facilitate comparative analysis of vertebrate genomes.

Animals↗

PubMed2002

A subtracted cDNA library from the zebrafish (Danio rerio) embryonic inner ear.

A database was built that consists of 4694 sequence contigs of approximately 18,000 reads of cDNAs isolated from the microdissected otocysts of zebrafish embryos at 20-30 hour postfertilization, following subtraction with a pool of liver cDNAs from adult fish. These sequences were compared with those of public databanks. Significant similarity were recorded and organized in a relational database at http://www.genoscope.cns.fr/zie. A first group of 2067 sequences correspond to 1428 known zebrafish genes or ESTs present in the Danio rerio section of UniGene. A second group of 302 sequences encode putative proteins that showed significant similarity (50%-100%) with 302 nonzebrafish proteins in the nr databank, a public databank containing an exhaustive nonredundant collection of protein sequences from different species (ftp://ftp.ncbi.nlm.nih.gov/blast/db/nr). The remaining 2325 (49.5%) sequence contigs or singletons showed no significant similarity with sequences available in public databanks. Several genes known to be expressed in the developing inner ear were represented in the present database, in particular genes involved in hair cell differentiation or innervation The occurrence of these genes validates the outcome of this study as the first collection of ESTs preferentially expressed in the zebrafish inner ear during the period of hair cell differentiation and neuroblast delamination from the otic vesicle epithelium. Novel zebrafish genes also involved in these processes are thus likely to be represented among the sequences obtained herein, for which no homology was found in the D. rerio section of UniGene. [The sequence data from this study have been submitted to EMBL under accession nos. AL714032-AL731531].

Animals↗

PubMed2002

Rapid mapping of zebrafish mutations with SNPs and oligonucleotide microarrays.

Large-scale genetic screens in zebrafish have identified thousands of mutations in hundreds of essential genes. The genetic mapping of these mutations is necessary to link DNA sequences to the gene functions defined by mutant phenotypes. Here, we report two advances that will accelerate the mapping of zebrafish mutations: (1) The construction of a first generation single nucleotide polymorphism (SNP) map of the zebrafish genome comprising 2035 SNPs and 178 small insertions/deletions, and (2) the development of a method for mapping mutations in which hundreds of SNPs can be scored in parallel with an oligonucleotide microarray. We have demonstrated the utility of the microarray technique in crosses with haploid and diploid embryos by mapping two known mutations to their previously identified locations. We have also used this approach to localize four previously unmapped mutations. We expect that mapping with SNPs and oligonucleotide microarrays will accelerate the molecular analysis of zebrafish mutations.

Animals↗

PubMed2005

Zebrafish embryos are susceptible to the dopaminergic neurotoxin MPTP.

The neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) induces selective loss of dopaminergic neurons in the mammalian midbrain, eliciting symptoms characteristic of Parkinson's disease. By exploiting the advantages of zebrafish embryos, we report here that dopaminergic neurons in this species are specifically perturbed when exposed to MPTP. In contrast to mammals, the zebrafish does not possess a midbrain dopaminergic system. Instead, the main population of neurons expressing the dopamine transporter is located in the posterior tuberculum of the diencephalon. Exposure of embryos to MPTP led to a pronounced reduction in the number of dopaminergic cells in the diencephalon. This effect can be reversed by deprenyl, a specific inhibitor of monoamine oxidase B that catalyses the conversion of MPTP to its active metabolite, MPP+. Similarly, direct treatment of embryos with MPP+ abolished the diencephalic dopaminergic neurons. These larvae also demonstrated behavioural defects in swimming responses. Thus, dopaminergic neurons in the posterior tuberculum of the zebrafish may be homologous to the midbrain dopaminergic system of mammals. In addition, the mechanism behind the loss of dopaminergic neurons following pharmacological perturbation may be conserved among vertebrates and suggest that the zebrafish can be used as a convenient and economical system to study the pathogenesis of Parkinson's disease and for testing potential therapeutic strategies.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine↗

PubMed2000

Combining physiology and genetics in the zebrafish retina.

The zebrafish has recently joined the ranks of Drosophila and C. elegans as a tractable model for genetic screens (Fishman, 1999). Zebrafish grow fast, can be kept in large numbers in a small space, and are efficiently mutagenized and screened. Genomic resources are made available at an increasing pace. These days, a mutation can be mapped and cloned in a matter of months. Because a mutant hunt is intrinsically unbiased in terms of the classes of genes that will be tagged, it holds the unique potential to discover novel genes or, in our era of genome sequencing, to identify novel functions for known genes. Zebrafish display dozens of innate behaviours in response to light, of which the optomotor and the optokinetic responses are the most widely studied (Brockerhoff et al. 1995; Easter & Nicola, 1996). Their retinae are crisply layered following the typical vertebrate pattern, and the retinal layers are tiled in an almost crystalline fashion by mosaics of different cell types. Electroretinograms are recorded routinely and therefore, not surprisingly, zebrafish are now also being used for a genetic approach to the visual system.

Animals↗

PubMed2006

pyewacket, a new zebrafish fin pigment pattern mutant.

Many mutants that disrupt zebrafish embryonic pigment pattern have been isolated, and subsequent cloning of the mutated genes causing these phenotypes has contributed to our understanding of pigment cell development. However, few mutants have been identified that specifically affect development of the adult pigment pattern. Through a mutant screen for adult pigment pattern phenotypes, we identified pyewacket (pye), a novel zebrafish mutant in which development of the adult caudal fin pigment pattern is aberrant. Specifically, pye mutants have fin melanocyte pigment pattern defects and fewer xanthophores than wild-type fins. We mapped pye to an interval where a single gene, the zebrafish ortholog of the human gene DHRSX, is present. pye will be an informative mutant for understanding how xanthophores and melanocytes interact to form the pigment pattern of the adult zebrafish fin.

Animals↗

PubMed2006

The Fugu tyrp1 promoter directs specific GFP expression in zebrafish: tools to study the RPE and the neural crest-derived melanophores.

In vertebrates, pigment cells account for a small percentage of the total cell population and they intermingle with other cell types. This makes it difficult to isolate them for analyzes of their functions in the context of development. To alleviate such difficulty, we generated two stable transgenic zebrafish lines (pt101 and pt102) that express green fluorescent protein (GFP) in melanophores under the control of the 1 kb Fugu tyrp1 promoter. In pt101, GFP is expressed in both retinal pigment epithelium (RPE) cells and the neural crest-derived melanophores (NCDM), whereas in pt102, GFP is predominately expressed in the NCDM. Our results indicate that the Fugu tyrp1 promoter can direct transgene expression in a cell-type-specific manner in zebrafish. In addition, our findings provide evidence supporting differential regulations of melanin-synthesizing genes in RPE cells and the NCDM in zebrafish. Utilizing the varying GFP expression levels in these fish, we have isolated melanophores via flow cytometry and revealed the capability of sorting the NCDM from RPE cells as well. Thus, these transgenic lines are useful tools to study melanophores in zebrafish.

Animals↗

PubMed2006

Aryl hydrocarbon receptor activation produces heart-specific transcriptional and toxic responses in developing zebrafish.

Proper regulation of the aryl hydrocarbon receptor (AHR), a ligand-activated transcription factor, is required for normal vertebrate cardiovascular development. AHR hyperactivation by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) during zebrafish (Danio rerio) development results in altered heart morphology and function, culminating in death. To identify genes that may cause cardiac toxicity, we analyzed the transcriptional response to TCDD in zebrafish hearts. Zebrafish larvae were exposed to TCDD for 1 h at 72 h after fertilization (hpf), and the hearts were extracted for microarray analysis at 1, 2, 4, and 12 h after exposure (73, 74, 76, and 84 h postfertilization). The remaining body tissue was also collected at each time for comparison. TCDD rapidly induced expression in 42 genes within 1 to2hof exposure. These genes function in xenobiotic metabolism, proliferation, heart contractility, and pathways that regulate heart development. Furthermore, these expression changes preceded signs of cardiovascular toxicity, characterized by decreased stroke volume, peripheral blood flow, and a halt in heart growth. This identifies strong candidates for important AHR target genes. It is noteworthy that the TCDD-induced transcriptional response in the hearts of zebrafish larvae was substantially different from that induced in the rest of the body tissues. One of the biggest differences included a cluster of genes that were down-regulated 12 h after exposure in heart tissue, but not in the body samples. More than 70% of the transcripts in this heart-specific cluster promote cellular growth and proliferation. Thus, the developing heart stands out as being responsive to TCDD at both the level of toxicity and gene expression.

Animals↗

PubMed1994

Integration and germ-line transmission of a pseudotyped retroviral vector in zebrafish.

The zebrafish is rapidly becoming a popular model system for the study of vertebrate development because it is ideal for both embryological studies and genetic analysis. To determine if a retroviral vector pseudotyped with the envelope glycoprotein of the vesicular stomatitis virus could infect zebrafish embryos, and in particular, the cells destined to become the germ line, a pseudotyped virus was injected into blastula-stage zebrafish embryos. Fifty-one embryos were allowed to develop and eight transmitted proviral DNA to their progeny. Founders were mosaic, but as expected, transgenic F1's transmitted proviral DNA in a Mendelian fashion to the F2 progeny. Transgenic F1 fish inherited a single integrated provirus, and a single founder could transmit more than one viral integration to its progeny. These results demonstrate that this pantropic pseudotyped vector, originally developed for human gene therapy, will make the use of retroviral vectors in zebrafish possible.

Animals↗

PubMed2002

Expression and genomic organization of the zebrafish chaperonin gene complex.

Chaperonin 10 and chaperonin 60 monomers exist within the multimeric mitochondrial chaperonin folding complex with a stoichiometry of 2:1. This complex is located in the mitochondrial matrix, where it aids in the folding and acquisition of the tertiary structure of proteins. We have previously isolated the cpn10 cDNA in zebrafish (Danio rerio), and demonstrated that it is ubiquitously expressed during embryonic development and transcriptionally upregulated after exposure to heat shock. In the present study, we have isolated a cDNA encoding chaperonin 60 (cpn60) from zebrafish, and have shown that it is similarly expressed uniformly and ubiquitously throughout early embryonic development of zebrafish. Upregulation of cpn60 expression was also observed after exposure of zebrafish embryos to a heat shock of 1 h at 37 degrees C compared with control embryos raised at 27 degrees C. The induction of the cpn60 heat shock response was greatest after 1 h of heat shock, whereas significant decreases of cpn60 mRNA were observed within 2 h following a return to 27 degrees C. We subsequently isolated genomic DNA sequences for both of these genes, and show that they are also arranged in a head-to-head organization and share a common bidirectional promoter that contains a single heat shock element (HSE). Our database analysis shows that this head-to-head organization is also found in human (Homo sapiens), rat (Rattus norvegicus), pufferfish (Fugu rubripes), and Caenorhabditis elegans, but not in Drosophila or yeast (Saccharomyces cerevisiae). The data suggest that the genomic organization of the cpn gene complex has been conserved across the vertebrates.

Amino Acid Sequence↗

PubMed1997

Genetics of erythropoiesis: induced mutations in mice and zebrafish.

Production of red blood cells (erythropoiesis) in the vertebrate embryo is critical to its survival and subsequent development. As red cells are the first blood cells to appear in embryogenesis, their origin reflects commitment of mesoderm to an hematopoietic fate and provides an avenue by which to examine the development of the hematopoietic system, including the hematopoietic stem cell (HSC). We discuss the genetics of erythropoiesis as studied in two systems: the mouse and zebrafish (Danio rerio). In the mouse, targeted disruption has established several genes as essential at different stages of hematopoiesis or erythroid precursor cell maturation. In the zebrafish, numerous mutants displaying a wide range of phenotypes have been isolated, although the affected genes are unknown. In comparing mouse knockout and zebrafish mutant phenotypes, we propose a pathway for erythropoiesis that emphasizes the apparent similarity of the mutants and the complementary nature of investigation in the two species. We speculate that further genetic studies in mouse and zebrafish will identify the majority of essential genes and define a regulatory network for hematopoiesis in vertebrates.

Animals↗

PubMed2003

Functional and morphological evidence for a ventricular conduction system in zebrafish and Xenopus hearts.

Zebrafish and Xenopus have become popular model organisms for studying vertebrate development of many organ systems, including the heart. However, it is not clear whether the single ventricular hearts of these species possess any equivalent of the specialized ventricular conduction system found in higher vertebrates. Isolated hearts of adult zebrafish (Danio rerio) and African toads (Xenopus laevis) were stained with voltage-sensitive dye and optically mapped in spontaneous and paced rhythms followed by histological examination focusing on myocardial continuity between the atrium and the ventricle. Spread of the excitation wave through the atria was uniform with average activation times of 20 +/- 2 and 50 +/- 2 ms for zebrafish and Xenopus toads, respectively. After a delay of 47 +/- 8 and 414 +/- 16 ms, the ventricle became activated first in the apical region. Ectopic ventricular activation was propagated significantly more slowly (total ventricular activation times: 24 +/- 3 vs. 14 +/- 2 ms in zebrafish and 74 +/- 14 vs. 35 +/- 9 ms in Xenopus). Although we did not observe any histologically defined tracts of specialized conduction cells within the ventricle, there were trabecular bands with prominent polysialic acid-neural cell adhesion molecule staining forming direct myocardial continuity between the atrioventricular canal and the apex of the ventricle; i.e., the site of the epicardial breakthrough. We thus conclude that these hearts are able to achieve the apex-to-base ventricular activation pattern observed in higher vertebrates in the apparent absence of differentiated conduction fascicles, suggesting that the ventricular trabeculae serve as a functional equivalent of the His-Purkinje system.

Animals↗

PubMed2002

The zebrafish: a new model organism for integrative physiology.

This brief review summarizes features of the zebrafish, Danio rerio, that make it a suitable model organism for studies of regulatory physiology. The review presents the argument that random mutagenesis screens are a valuable gene-finding strategy to identify genes of functional importance and that their utility, although well established for developmental issues, will extend to a variety of topics of interest to the regulatory physiologist. Particular attention is drawn to the range of functional responses amenable to mutagenesis screens in larval zebrafish. Other virtues of the organism, the range of genomic tools, the potential for innovative optical methods, and the tractability for genetic and other experimental manipulations, are also described. Finally, the review provides examples of functional studies in zebrafish, including studies in sensory neurons, cardiac rhythm disturbances, gastrointestinal function, and studies of the developing kidney, that illustrate potential applications. Because of the relative ease with which combinatorial studies can be performed, the zebrafish may eventually be particularly valuable in understanding the functional interaction between subtle gene defects that cause polygenic disorders.

Animals↗