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PubMed1996

Highly efficient germ-line transmission of proviral insertions in zebrafish.

An important technology in model organisms is the ability to make transgenic animals. In the past, transgenic technology in zebrafish has been limited by the relatively low efficiency with which transgenes could be generated using either DNA microinjection or retroviral infection. Previous efforts to generate transgenic zebrafish with retroviral vectors used a pseudotyped virus with a genome based on the Moloney murine leukemia virus and the envelope protein of the vesicular stomatitis virus. This virus was injected into blastula-stage zebrafish, and 16% of the injected embryos transmitted proviral insertions to their offspring, with most founders transmitting a single insertion to approximately 2% of their progeny. In an effort to improve this transgenic frequency, we have generated pseudotyped viral stocks of two new Moloney-based genomes. These viral stocks have titers up to two orders of magnitude higher than that used previously. Injection of these viruses resulted in a dramatic increase in transgenic efficiency; over three different experiments, 83% (110/133) of the injected embryos transmitted proviral insertions to 24% of their offspring. Furthermore, founders made with one of the viruses transmitted an average of 11 different insertions through their germ line. These results represent a 50- to 100-fold improvement in the efficiency of generating transgenic zebrafish, making it now feasible for a single lab to rapidly generate tens to hundreds of thousands of transgenes. Consequently, large-scale insertional mutagenesis strategies, previously limited to invertebrates, may now be possible in a vertebrate.

3T3 Cells↗

PubMed1996

DANA elements: a family of composite, tRNA-derived short interspersed DNA elements associated with mutational activities in zebrafish (Danio rerio).

DNA is the first SINE isolated from zebrafish (Danio rerio) exhibiting all the hallmarks of these tRNA-derived elements. DANA is unique in its clearly defined substructure of distinct cassettes. In contrast to generic SINE elements, DANA appears to have been assembled by insertions of short sequences into a progenitor, tRNA-derived element. Once associated with each other, these subunits were amplified as a new transposable element with such a remarkable success that DANA-related sequences comprise approximately 10% of the modern zebrafish genome. At least some of the sequences comprised by the full-length element were capable of movement, forming a new group of mobile, composite transposons, one of which caused an insertional mutation in the zebrafish no tail gene. Being present only in the genus Danio, and estimated to be as old as the genus itself, DANA may have played a role in Danio speciation by massive amplification and genome-wide dispersion. There are extensive DNA polymorphisms between zebrafish populations and strains detected by PCR amplification using primers specific to DANA, suggesting that the DANA element will be useful as a molecular tool for genetic and phylogenetic analyses.

Animals↗

PubMed1997

Promoter analysis in living zebrafish embryos identifies a cis-acting motif required for neuronal expression of GATA-2.

We have used zebrafish embryos to dissect the promoter activity of a gene with a complex expression pattern during embryogenesis. GATA-2 is a transcription factor required for hematopoiesis and is dynamically expressed in hematopoietic tissues and in the central nervous system. Using constructs containing zebrafish GATA-2 genomic flanking sequences and the green fluorescent protein (GFP) reporter gene, we demonstrate that distinct regulatory domains are required for hematopoietic, enveloping layer (EVL), and neuronal expression of GATA-2. During gastrulation, GFP expression is confined to the ventral ectoderm and lateral mesoderm and is lacking in the dorsal shield. Cells derived from the regions expressing GFP give rise to hematopoietic progenitors, EVL cells, and neurons. Deletion analysis of the 7.3-kb GATA-2 promoter region revealed that a 1.1-kb DNA sequence is critical for expression of GATA-2 in neurons. Fine mapping revealed that a 31-bp region is required for neuron enhancer activity, and mutagenesis showed that the DNA motif CCCTCCT is essential for GATA-2 promoter activity in the central nervous system of zebrafish. Our use of zebrafish embryos can be exploited as a whole animal system for the dissection of any developmentally regulated vertebrate promoter.

Animals↗

PubMed1998

Transposition of the mariner element from Drosophila mauritiana in zebrafish.

With the increased popularity of zebrafish (Danio rerio) for mutagenesis studies, efficient methods for manipulation of its genome are needed. One approach is the use of a transposable element as a vector for gene transfer in this species. We report here the transformation of zebrafish and germ-line transmission of the mariner element from Drosophila mauritiana. The mariner element was selected because its transposition is independent of host-specific factors. One- to two-cell-stage zebrafish embryos were coinjected with a supercoiled plasmid carrying the nonautonomous mariner element peach and mRNA encoding the transposase. Surviving larvae were reared to adulthood, and the transmission of peach to the F1 generation was tested by PCR. Four of the 12 founders, following plasmid injections on 2 different days, transmitted the element to their progeny. Inheritance of the transgene from the F1 to the F2 generation showed a Mendelian pattern. No plasmid sequences were detected by PCR or Southern blot analysis, indicating transposition of peach rather than random integration of the plasmid DNA. These data provide evidence of transformation of a vertebrate with a transposable element and support the host-independent mechanism for transposition of the mariner element. We suggest this system could be used for insertional mutagenesis or for identifying active regions of the genome in the zebrafish.

Animals↗

PubMed2006

Identification of anti-prion compounds as efficient inhibitors of polyglutamine protein aggregation in a zebrafish model.

Several neurodegenerative diseases, including Huntington disease (HD), are associated with aberrant folding and aggregation of polyglutamine (polyQ) expansion proteins. Here we established the zebrafish, Danio rerio, as a vertebrate HD model permitting the screening for chemical suppressors of polyQ aggregation and toxicity. Upon expression in zebrafish embryos, polyQ-expanded fragments of huntingtin (htt) accumulated in large SDS-insoluble inclusions, reproducing a key feature of HD pathology. Real time monitoring of inclusion formation in the living zebrafish indicated that inclusions grow by rapid incorporation of soluble htt species. Expression of mutant htt increased the frequency of embryos with abnormal morphology and the occurrence of apoptosis. Strikingly, apoptotic cells were largely devoid of visible aggregates, suggesting that soluble oligomeric precursors may instead be responsible for toxicity. As in nonvertebrate polyQ disease models, the molecular chaperones, Hsp40 and Hsp70, suppressed both polyQ aggregation and toxicity. Using the newly established zebrafish model, two compounds of the N'-benzylidene-benzohydrazide class directed against mammalian prion proved to be potent inhibitors of polyQ aggregation, consistent with a common structural mechanism of aggregation for prion and polyQ disease proteins.

Animals↗

PubMed2006

Light reaches the very heart of the zebrafish clock.

Zebrafish are typically used as a model system to study various aspects of developmental biology, largely as a consequence of their ex vivo development, high degree of transparency, and, of course, ability to perform forward genetic mutant screens. More recently, zebrafish have been developed as a model system with which to study circadian clocks. Cell lines generated from early-stage zebrafish embryos contain clocks that are directly light-responsive. We describe recent experiments using single-cell luminescent imaging approaches to study clock function in this novel cell line system. Furthermore, studies examining the process of entrainment to light pulses within this cell population are described in this review, as are experiments examining light-responsiveness of early-stage zebrafish embryos.

Animals↗

PubMed2005

A three-dimensional functional assessment of heart and vessel development in the larva of the zebrafish (Danio rerio).

There has been considerable recent interest in the development of the circulation in the zebrafish. Optical techniques typically used to visualize changes in heart size allow measurement of stroke volume during early vertebrate development, but this approach is complicated in zebrafish larvae because of the heart's irregular shape and its significant change in morphology during the first 6 d of development. By use of a three-dimensional integration of the early zebrafish heart and vessels, we have greatly reduced measurement error of stroke volume and cardiac output and have determined the cross-sectional growth of major vessels in the developing zebrafish larvae. A dramatic 500%-600% increase in cardiac output (from 10 to 50-60 nL min(-1)) occurs on days 5 and 6 postfertilization in Danio rerio. Cross-sectional area of key vessels (dorsal artery, caudal artery, dorsal vein) as well as between-individual variation significantly decreased over the first 6 d of development. Associated with the decrease in cross-sectional area is a significant increase in red blood cell velocity on days 5 and 6 postfertilization. Together, the three-dimensional data of the cardiac and vascular systems have shown that the most profound physiological and developmental changes occur in days 5 and 6, which corresponds with the appearance of the adult form of the heart and the transition from diffusive to convective O2 supply to internal tissues.

Animals↗

PubMed2005

A 23-amino acid motif spanning the basic domain targets zebrafish myogenic regulatory factor myf5 into nucleolus.

Myf5 is a nuclear protein and one of the basic helix-loop-helix (bHLH) myogenic factors that play an important role in muscle specification and differentiation. The motif responsible for the nuclear translocation of Myf5 was unknown. Using on-line monitoring of EGFP (enhanced green fluorescent protein)-tagged zebrafish Myf5 translocation, we demonstrated that Myf5-EGFP protein resided in the nucleoplasm and nucleolus of zebrafish fibroblast cell lines (ZEM2S and ZF4), mammalian nonmuscle cell line (COS1), and muscle cell lines (RD and C2C12). In contrast, zebrafish MyoD-EGFP was localized in the nucleus but did not condense in the nucleolus. Using indirect immunofluorescent staining, we determined that zebrafish Myf5 was colocalized with nucleophosmin/B23, a nucleolus protein. Deletion analysis revealed that amino acid residues 60 to 82 (60KRKASTVDRRRAATMRERRRLKK82) of Myf5 were sufficient and necessary for nucleolus targeting. A GST pulldown assay followed by Western analysis showed that nucleolin/C23 could be pulled down specifically by GST-Myf5, but not by GST-MyoD. Based on these findings, we propose that the distinct functions of Myf5 and MyoD may result from their differential binding affinity to nucleolin/C23.

Animals↗

PubMed2003

Coupled mutagenesis screens and genetic mapping in zebrafish.

Forward genetic analysis is one of the principal advantages of the zebrafish model system. However, managing zebrafish mutant lines derived from mutagenesis screens and mapping the corresponding mutations and integrating them into the larger collection of mutations remain arduous tasks. To simplify and focus these endeavors, we developed an approach that facilitates the rapid mapping of new zebrafish mutations as they are generated through mutagenesis screens. We selected a minimal panel of 149 simple sequence length polymorphism markers for a first-pass genome scan in crosses involving C32 and SJD inbred lines. We also conducted a small chemical mutagenesis screen that identified several new mutations affecting zebrafish embryonic melanocyte development. Using our first-pass marker panel in bulked-segregant analysis, we were able to identify the genetic map positions of these mutations as they were isolated in our screen. Rapid mapping of the mutations facilitated stock management, helped direct allelism tests, and should accelerate identification of the affected genes. These results demonstrate the efficacy of coupling mutagenesis screens with genetic mapping.

Animals↗

PubMed2003

Biochemical and molecular characterization of galectins from zebrafish (Danio rerio): notochord-specific expression of a prototype galectin during early embryogenesis.

Galectins are a family of beta-galactoside-binding lectins that on synthesis are either translocated into the nucleus or released to the extracellular space. Their developmentally regulated expression, extracellular location, and affinity for extracellular components (such as laminin and fibronectin) suggest a role in embryonic development, but so far this has not been unequivocally established. Zebrafish constitute an ideal model for developmental studies because of their external fertilization, transparent embryos, rapid growth, and availability of a large collection of mutants. As a first step in addressing the biological roles in zebrafish embryogenesis, we identified and characterized members of the three galectin types: three protogalectins (Drgal1-L1, Drgal1-L2, Drgal1-L3), one chimera galectin (Drgal3), and one tandem-repeat galectin (Drgal9-L1). Like mammalian prototype galectin-1, Drgal1-L2 preferentially binds to N-acetyllactosamine. Genomic structure of Drgal1-L2 revealed four exons, with the exon-intron boundaries conserved with the mammalian galectin-1. Interestingly, this gene also encodes an alternatively spliced form of Drgal1-L2 that lacks eight amino acids near the carbohydrate-binding domain. Zebrafish galectins exhibited distinct patterns of temporal expression during embryo development. Drgal1-L2 is expressed postbud stage, and its expression is strikingly specific to the notochord. In contrast, Drgal1-L1 is expressed maternally in the oocytes. Drgal1-L3, Drgal3, and Drgal9-L1 are expressed both maternally and zygotically, ubiquitously in the adult tissues. The distinct temporal and spatial patterns of expression of members of the zebrafish galectin repertoire suggest that each may play distinct biological roles during early embryogenesis.

Amino Acid Sequence↗

PubMed2005

Glycomic survey mapping of zebrafish identifies unique sialylation pattern.

Functional genomics and proteomics studies of the developmental glycobiology of zebrafish are greatly hampered by the current lack of knowledge on its glycosylation profile. To furnish the requisite structural basis for a more insightful functional delineation and genetic manipulation, we have initiated a survey mapping of the possible expression of stage-specific glycoconjugates in zebrafish. High-sensitivity mass spectrometry (MS) analysis in conjunction with the usual array of enzymatic and chemical derivatization was employed as the principal method for rapid differential mapping of the glycolipids and sequentially liberated N- and O-glycans from the total extracts. We demonstrated that all developmental stages of the zebrafish under investigation, from fertilized eggs to hatched embryos, synthesize oligomannosyl types of N-glycans, as well as complex types with additionally beta4-galactosylated, Neu5Ac/Neu5Gc monosialylated Lewis x termini. A combination of collision-induced dissociation (CID)-MS/MS and nuclear magnetic resonance (NMR) analyses led to the identification of an abundant and unusual mucin-type O-glycosylation, based on a novel sequence Fucalpha1-3GalNAcbeta1-4(Neu5Ac/Neu5Gcalpha2-3)Galbeta1-3GalNAc. This core structure may be further oligosialylated, but exclusively in the earlier development stages. Similarly, MS and MS/MS analyses of the extracted glycolipid fraction revealed the presence of a heterogeneous family of oligosialylated lactosylceramide compounds. In contrast to the O-glycans, these glycolipids only appear in the later development stages, suggesting a complex pattern of regulation for sialyltransferase activities during zebrafish embryogenesis.

Animals↗

PubMed2005

Reconstitution of ancestral green visual pigments of zebrafish and molecular mechanism of their spectral differentiation.

We previously reported that zebrafish have four tandemly duplicated green (RH2) opsin genes (RH2-1, RH2-2, RH2-3, and RH2-4). Absorption spectra vary widely among the four photopigments reconstituted with 11-cis retinal, with their peak absorption spectra (lambda(max)) being 467, 476, 488, and 505 nm, respectively. In this study, we inferred the ancestral amino acid (aa) sequences of the zebrafish RH2 opsins by likelihood-based Bayesian statistics and reconstituted the ancestral opsins by site-directed mutagenesis. The ancestral pigment (A1) to the four zebrafish RH2 pigments and that (A3) to RH2-3 and RH2-4 showed lambda(max) at 506 nm, while that (A2) to RH2-1 and RH2-2 showed a lambda(max) at 474 nm, indicating that a spectral shift had occurred toward the shorter wavelength on the evolutionary lineages A1 to A2 by 32 nm, A2 to RH2-1 by 7 nm, and A3 to RH2-3 by 18 nm. Pigment chimeras and site-directed mutagenesis revealed a large contribution (approximately 15 nm) of glutamic acid to glutamine substitution at residue 122 (E122Q) to the A1 to A2 and A3 to RH2-3 spectral shifts. However, the remaining spectral differences appeared to result from complex interactive effects of a number of aa replacements, each of which has only a minor spectral contribution (1-3 nm). The four zebrafish RH2 pigments cover nearly an entire range of lambda(max) distribution among vertebrate RH2 pigments and provide an excellent model to study spectral tuning mechanisms of RH2 in vertebrates.

Amino Acid Sequence↗

PubMed2001

The Zebrafish Information Network (ZFIN): a resource for genetic, genomic and developmental research.

The Zebrafish Information Network, ZFIN, is a WWW community resource of zebrafish genetic, genomic and developmental research information (http://zfin.org). ZFIN provides an anatomical atlas and dictionary, developmental staging criteria, research methods, pathology information and a link to the ZFIN relational database (http://zfin. org/ZFIN/). The database, built on a relational, object-oriented model, provides integrated information about mutants, genes, genetic markers, mapping panels, publications and contact information for the zebrafish research community. The database is populated with curated published data, user submitted data and large dataset uploads. A broad range of data types including text, images, graphical representations and genetic maps supports the data. ZFIN incorporates links to other genomic resources that provide sequence and ortholog data. Zebrafish nomenclature guidelines and an automated registration mechanism for new names are provided. Extensive usability testing has resulted in an easy to learn and use forms interface with complex searching capabilities.

Animals↗

PubMed2005

Heart malformation is an early response to TCDD in embryonic zebrafish.

The zebrafish (Danio rerio) has become an attractive vertebrate model for studying developmental processes, and is emerging as a model system for studying the mechanisms by which toxic compounds perturb normal development. When exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) shortly after fertilization, zebrafish embryos exhibit pericardial edema and reduced blood flow by 72 h post fertilization (hpf). To better understand the progression of dioxin toxicity in zebrafish, we have examined the effects of TCDD on heart development. At 72 hpf, TCDD-treated embryos exhibited altered looping, with the atria positioned distinctly posterior to the ventricles, contrary to the looping of control hearts, where the two chambers lied side by side. Moreover, the ventricles in dioxin-exposed hearts became more compact, and the atria elongated in comparison to controls. These defects are not secondary to pericardial edema because they were observed when edema formation was suppressed with osmotic support. In addition to morphological changes, TCDD produced functional deficits in the developing hearts, including blood regurgitation and a striking ventricular standstill that became prevalent by 120 hpf. We also assessed the effect of TCDD on the heart size using stereological measurements, which demonstrated significant reduction in heart tissue volume at 72 hpf. Perhaps our most significant finding was a decrease in the total number of cardiomyocytes in TCDD-exposed embryos by 48 hpf, one day prior to observable effects on peripheral blood flow. We conclude that the developing heart is an important target for TCDD in zebrafish.

Abnormalities, Drug-Induced↗

PubMed2004

Developmental profiles of activin betaA, betaB, and follistatin expression in the zebrafish ovary: evidence for their differential roles during sexual maturation and ovulatory cycle.

Our recent experiments showed that gonadotropin(s) stimulated activin betaA and follistatin expression through the cAMP-PKA pathway but suppressed betaB via a cAMP-dependent but PKA-independent pathway in cultured zebrafish follicle cells. Given that pituitary gonadotropins are the major hormones controlling the development and function of the ovary, the differential expression of activin betaA and betaB as well as follistatin in response to gonadotropin(s) raises an interesting question about the temporal expression patterns of these molecules in vivo during sexual maturation and ovulatory cycle. Three experiments were performed in the present study. In the first experiment using sexually immature zebrafish, we followed the expression of activin betaA, betaB, and follistatin at the whole ovary level during a 10-day period in which the ovary developed from the primary growth stage to the one with nearly full-grown follicles. Activin betaA expression was very low at the primary growth stage but significantly increased with the growth of the ovary, and its rise was accompanied by an increase in follistatin expression. In contrast, the expression of activin betaB could be easily detected in the ovary of all stages; however, it did not exhibit an obvious trend of variation during the development. The second experiment examined the stage-dependent expression of activin betaA, betaB, and follistatin at the follicle level in the adult mature zebrafish. The expression of activin betaA was again low in the follicles during the primary growth stage, but exhibited a phenomenal increase after the follicles entered vitellogenesis with the peak level reached at midvitellogenic stage; in contrast, activin betaB mRNA could be easily detected at all stages with a slight increase during follicle growth. The expression of follistatin, on the other hand, also increased significantly during vitellogenesis; however, its level dropped sharply after reaching the peak at the midvitellogenic stage. In the third experiment, we investigated the dynamic changes of the ovarian activin betaA, betaB, and follistatin expression during the daily ovulatory cycle. The expression of activin betaA and follistatin gradually increased from 1800 h onward and reached the peak level around 0400 h when the germinal vesicles had migrated to the periphery in the full-grown oocytes. In contrast, activin betaB expression steadily declined, although not statistically significant, during the same period, but increased sharply at 0700 h when mature oocytes started to appear in most of the ovaries collected. In conclusion, activin betaA and betaB exhibit distinct expression patterns during the development of the ovary and the daily ovarian cycle of the zebrafish. It seems that activin betaA is involved in promoting ovary and follicle growth, whereas activin betaB may have a tonic role throughout follicle development but becomes critical at the late stage of oocyte maturation and/or ovulation.

Activins↗

PubMed2001

Isolation of gonadal mutations in adult zebrafish from a chemical mutagenesis screen.

A mutagenesis screen was conducted on zebrafish using N:-ethyl N:-nitrosourea as a mutagen and an F2 crossing scheme to obtain homozygous mutants in the F3 generation. Whole abdomens of 3-mo-old F3 zebrafish progeny were fixed and mass-embedded in paraffin blocks. Blocks were cut with a microtome to obtain cross-sections of the entire body cavity that included the ovaries and testes. Slides of the cross-sections were analyzed for alterations in gonadal structure and gametogenesis and were compared with gonads of wild-type fish. A total of 125 mutagenized genomes in 81 families were screened and 11 mutations were observed that produced visible phenotypes in only one sex per family. Male mutations included testes without mature sperm that contained either predominantly spermatocytes or spermatogonia. Female mutations included ovaries containing 1) degenerating oocytes surrounded by hypertrophied follicle walls or stroma, 2) extrafollicular tissue proliferation, 3) proliferating postovulatory follicle walls, and 4) large numbers of degenerating preovulatory and postovulatory oocytes. While past screens on zebrafish have concentrated on early developmental mutations, the results of this study demonstrate for the first time that mutagenesis can be used with zebrafish to study reproduction in adult animals.

Animals↗

PubMed2001

Fertilization of eggs of zebrafish, Danio rerio, by intracytoplasmic sperm injection.

To evaluate the potential for fertilization by sperm injection into fish eggs, sperm from zebrafish, Danio rerio, were microinjected directly into egg cytoplasm of two different zebrafish lines. To evaluate physiological changes of gametes on the possible performance of intracytoplasmic sperm injection (ICSI), four different combinations of injection conditions were conducted using activated or nonactivated gametes. From a total of 188 zebrafish eggs injected with sperm in all treatments, 31 (16%) developed to blastula, 28 (15%) developed to gastrula, 10 (5%) developed abnormally to larval stages, and another 3 (2%) developed normally and hatched. The highest fertilization rate (blastodisc formation) was achieved by injection of activated spermatozoa into nonactivated eggs (35%). Injections were most effective when performed within the first hour after egg collection. Flow cytometric analysis of the DNA content of the developing ICSI embryos revealed diploidy, and the use of a dominant pigment marker confirmed paternal inheritance. Our study indicates that injection of a single sperm cell into the cytoplasm of zebrafish eggs allows fertilization and subsequent development of normal larvae to hatching and beyond.

Animals↗

PubMed2000

Zebrafish: a genetic approach in studying hematopoiesis.

The zebrafish (Danio rerio) has emerged in recent years as an exciting animal model system for studying vertebrate organ development and, in particular, the development of the hematopoietic system. The combined advantages of developmental biology and genetic screens for mutations in zebrafish have provided insights into early events in hematopoiesis and identified several genes required for normal blood development in vertebrates. As a result of the large-scale mutagenesis screens for developmental mutants, several zebrafish mutants with defects in blood development have been recovered. This review discusses how these blood mutations in zebrafish have given new perspectives on hematopoietic development.

Animals↗