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At least 559 records · Page 31Linked to original sources

PubMed1982

Effects of immobilization and partial denervation on the differentiation of muscle fiber types in the Zebrafish, Brachydanio rerio.

In this study we tested the effects of immobilization and of spinal cord lesion on the differentiation of muscle fiber types in the myotomal muscle of the zebrafish. We found that the differentiation of embryonic red and white muscle fibers occurs in immobilized as well as in denervated myotomes of zebrafish embryos. Intermediate and adult red muscle fibers are not formed in denervated myotomes of larvae. Adult zebrafishes with spinal cord lesions in the anal region show abnormal undulating movements in the rostral part of the body. This change in muscle activity correlates with an increase in the number of intermediate scattered fibers. We conclude that direct contact with spinal cord motorneurons is not necessary for the differentiation of embryonic red and white fibers. The differentiation of intermediate muscle fibers is under extrinsic control. The factors that determine the formation of adult red fibers are not yet clear.

Animals↗

PubMed2006

Nuclear translocation of the calcium-binding protein ALG-2 induced by the RNA-binding protein RBM22.

By yeast two-hybrid screening using the calcium-binding protein ALG-2 as bait a new target of ALG-2 was identified, the RNA-binding protein RBM22. In order to confirm these interactions in vivo we prepared fluorescent constructs by using the monomeric red fluorescent protein to label ALG-2 and the enhanced green fluorescent protein to label RBM22. Confocal microscopy of NIH 3T3 cells transfected with either ALG-2 or RBM22 expression constructs encoding fluorescent fusion proteins alone revealed that the majority of ALG-2 was localized in the cytoplasm whereas RBM22 was located in the nucleus. When cells were co-transfected with expression vectors encoding both fusion proteins ALG-2 was found in the nucleus indicating that RBM22 which can shuttle between the cytoplasm and the nucleus may play a role in nuclear translocation of ALG-2. Using zebrafish as a model mRNA homologues of ALG-2 and RBM22 were microinjected into the blastodisc-yolk margin of zebrafish embryos at the 1-cell stage followed by monitoring the fusion proteins during development of the zebrafish. Hereby, we observed that ALG-2 alone evenly distributed within the cell, whereas in the presence of RBM22 the two proteins co-localized within the nucleus. More than 95% of the two proteins co-localized within the same area in the nucleus suggesting a functional interaction between the Ca(2+)-signaling protein ALG-2 and the RNA-binding protein RBM22.

Active Transport, Cell Nucleus↗

PubMed2001

Caged Q-rhodamine dextran: a new photoactivated fluorescent tracer.

An amine-reactive caged rhodamine was synthesized and conjugated to aminodextran. The resulting tracer was injected into a single cell zebrafish embryo, and a portion of the tracer was photolyzed in a single cell after development. The resulting fluorescent cell was imaged by fluorescence microscopy through a single round of cell division.

Animals↗

PubMed2003

Induction of cytochrome P450 1A is required for circulation failure and edema by 2,3,7,8-tetrachlorodibenzo-p-dioxin in zebrafish.

The mechanism of toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is thought to result from changes in gene expression via the aryl hydrocarbon receptor (AHR). The induction of cytochrome P450 1A (CYP1A) in various organs is a cardinal effect of TCDD. However, whether CYP1A is involved in endpoints of TCDD toxicity is controversial. We investigated the role of CYP1A in TCDD-induced developmental toxicities using gene knock-down with morpholino antisense oligos. Exposure of zebrafish embryos to TCDD, at concentrations eliciting the hallmark endpoints of developmental toxicity, induced CYP1A in the heart and vascular endothelium throughout the body. This induction by TCDD was markedly inhibited by morpholinos to zebrafish arylhydrocarbon receptor 2 (zfAHR2-MO) and to zebrafish CYP1A (zfCYP1A-MO). The zfAHR2-MO but not the zfCYP1A-MO inhibited zfCYP1A mRNA expression, indicating the specificities of these morpholinos. Injection of either zfAHR2-MO or zfCYP1A-MO blocked the representative signs of TCDD developmental toxicity in zebrafish, pericardial edema and trunk circulation failure. The morpholinos appeared do not affect normal development in TCDD-untreated embryos. These results suggest a mediatory role of zfCYP1A induction through zfAHR2 activation in causing circulation failure by TCDD in zebrafish. This is the first molecular evidence demonstrating an essential requirement for CYP1A induction in TCDD-evoked developmental toxicities in any vertebrate species.

Animals↗

PubMed2004

Lipid metabolism in zebrafish.

Forward genetics is an unbiased methodology to discover new genes or functions of genes. At the present, the zebrafish is one of the few vertebrate systems where large-scale forward genetic studies are practical. Fluorescent lipid labeling of zebrafish larvae derived from families created from ENU-mutagenized fish enabled us to perform a large scale in vivo screen to identify mutants with perturbed lipid processing. With the aid of the zebrafish genome project, positional cloning of mutated genes with abnormal lipid metabolism can be accelerated. MO- and gripNA-based transient gene silencing is feasible in zebrafish embryos and provides a reverse genetic screening strategy to search for important lipid regulators. The advantages of using zebrafish as a vertebrate model to study lipid metabolism include its rapid external development and its optical clarity that enables the monitoring of biological processes. Large scale, high-throughput drug screening in vivo, especially for drugs that inhibit lipid absorption, can be easily achieved in this model. These zebrafish-based assays are important tools to understand aspects of lipid biology with significant clinical implications.

4-Chloro-7-nitrobenzofurazan↗

PubMed2027

Genomic Profiling of Chromatin State Using CUT&Tag.

Alterations in chromatin state, mediated through histone modifications and the incorporation of histone variants, are fundamental to establishing transcriptional networks and cell identity. Recent advances in low-input epigenome profiling methods, such as CUT&Tag and CUT&RUN, have enabled the study of chromatin states from very limited starting materials. In this chapter, we describe procedures for generating CUT&Tag libraries to profile histone modifications and histone variants in early-developing zebrafish embryos.

Animals↗

PubMed2006

AlphaA-crystallin expression prevents gamma-crystallin insolubility and cataract formation in the zebrafish cloche mutant lens.

Cataracts, the loss of lens transparency, are the leading cause of human blindness. The zebrafish embryo, with its transparency and relatively large eyes, is an excellent model for studying ocular disease in vivo. We found that the zebrafish cloche mutant, both the cloche(m39) and cloche(S5) alleles, which have defects in hematopoiesis and blood vessel development, also have lens cataracts. Quantitative examination of the living zebrafish lens by confocal microscopy showed significant increases in lens reflectance. Histological analysis revealed retention of lens fiber cell nuclei owing to impeded terminal differentiation. Proteomics identified gamma-crystallin as a protein that was substantially diminished in cloche mutants. Crystallins are the major structural proteins in mouse, human and zebrafish lens. Defects in crystallins have previously been shown in mice and humans to contribute to cataracts. The loss of gamma-crystallin protein in cloche was not due to lowered mRNA levels but rather to gamma-crystallin protein insolubility. AlphaA-crystallin is a chaperone that protects proteins from misfolding and becoming insoluble. The cloche lens is deficient in both alphaA-crystallin mRNA and protein during development from 2-5 dpf. Overexpression of exogenous alphaA-crystallin rescued the cloche lens phenotype, including solubilization of gamma-crystallin, increased lens transparency and induction of lens fiber cell differentiation. Taken together, these results indicate that alphaA-crystallin expression is required for normal lens development and demonstrate that cataract formation can be prevented in vivo. In addition, these results show that proteomics is a valuable tool for detecting protein alterations in zebrafish.

Amino Acid Sequence↗

PubMed2005

Perp is required for tissue-specific cell survival during zebrafish development.

The tumor suppressor p53 has two alternative effects, causing either cell cycle arrest or apoptosis. These different effects are supposed to be mediated by the transcriptional activation of different target genes. perp, encoding a transmembrane protein of the Pmp22 family, is a transcriptional p53 target exclusively upregulated in apoptotic cells. However, its role during normal development had remained largely unclear. Here, we report the isolation and characterization of a zebrafish perp homolog. Upon overexpression in early zebrafish embryos, perp induces apoptosis. In addition, it contributes to p53-dependent and UV-induced cell death. However, during normal zebrafish development, perp displays a p53-independent and spatially restricted expression in specific cell types and tissues. Antisense-mediated loss of Perp function leads to increased apoptosis in perp-expressing cells of the developing skin and notochord. We conclude that, in contrast to its proapoptotic function in stressed cells, Perp plays an antiapoptotic role during normal zebrafish development to regulate tissue-specific cell survival.

Animals↗

PubMed1997

Both apolipoprotein E and A-I genes are present in a nonmammalian vertebrate and are highly expressed during embryonic development.

Apolipoprotein E (apoE) is associated with several classes of plasma lipoproteins and mediates uptake of lipoproteins through its ability to interact with specific cell surface receptors. Besides its role in cardiovascular diseases, accumulating evidence has suggested that apoE could play a role in neurodegenerative diseases, such as Alzheimer disease. In vertebrates, apoA-I is the major protein of high-density lipoprotein. ApoA-I may play an important role in regulating the cholesterol content of peripheral tissues through the reverse cholesterol transport pathway. We have isolated cDNA clones that code for apoE and apoA-I from a zebrafish embryo library. Analysis of the deduced amino acid sequences showed the presence of a region enriched in basic amino acids in zebrafish apoE similar to the lipoprotein receptor-binding region of human apoE. We demonstrated by whole-mount in situ hybridization that apoE and apoA-I genes are highly expressed in the yolk syncytial layer, an extraembryonic structure implicated in embryonic and larval nutrition. ApoE transcripts were also observed in the deep cell layer during blastula stage, in numerous ectodermal derivatives after gastrulation, and after 3 days of development in a limited number of cells both in brain and in the eyes. Our data indicate that apoE can be found in a nonmammalian vertebrate and that the duplication events, from which apoE and apoA-I genes arose, occurred before the divergence of the tetrapod and teleost ancestors. Zebrafish can be used as a simple and useful model for studying the role of apolipoproteins in embryonic and larval nutrition and of apoE in brain morphogenesis and regeneration.

Amino Acid Sequence↗

PubMed2001

Functional analysis of zebrafish GDNF.

We have identified zebrafish orthologues of glial cell line-derived neurotrophic factor (GDNF) and the ligand-binding component of its receptor GFRalpha1. We examined the mRNA expression pattern of these genes in the developing spinal cord primary motor neurons (PMN), kidney, and enteric nervous systems (ENS) and have identified areas of correlated expression of the ligand and the receptor that suggest functional significance. Many aspects of zebrafish GDNF expression appear conserved with those reported in mouse, rat, and avian systems. In the zebrafish PMN, GFRalpha1 is only expressed in the CaP motor neuron while GDNF is expressed in the ventral somitic muscle that it innervates. To test the functional significance of this correlated expression pattern, we ectopically overexpressed GDNF in somitic muscle during the period of motor axon outgrowth and found specific perturbations in the pattern of CaP axon growth. We also depleted GDNF protein in zebrafish embryos using morpholino antisense oligos and found that GDNF protein is critical for the development of the zebrafish ENS but appears dispensable for the development of the kidney and PMN.

Amino Acid Sequence↗

PubMed1997

A ribozyme-mediated, gene "knockdown" strategy for the identification of gene function in zebrafish.

The zebrafish system offers many unique opportunities for the study of molecular biology. To date, only random mutagenesis, and not directed gene knockouts, have been demonstrated in this system. To more fully develop the potential of the zebrafish system, an approach to effectively inhibit the expression of any targeted gene in the developing zebrafish embryo has been developed. This approach uses a transient, cytoplasmic, T7 expression system, injected into the fertilized zebrafish egg to rapidly produce high levels of a ribozyme directed against the mRNA encoded by the targeted gene to inhibit its expression. In a demonstration of this strategy, expression of the recessive dominant zebrafish no tail gene was effectively inhibited by using this strategy to yield a phenotype identical to that resulting from a known defective mutation in this same gene. This, ribozyme-mediated, message deletion strategy may have use in determining the function of genetic coding sequences of unknown function.

Animals↗

PubMed2005

Computerized image analysis for quantitative neuronal phenotyping in zebrafish.

An integrated microscope image analysis pipeline is developed for automatic analysis and quantification of phenotypes in zebrafish with altered expression of Alzheimer's disease (AD)-linked genes. We hypothesize that a slight impairment of neuronal integrity in a large number of zebrafish carrying the mutant genotype can be detected through the computerized image analysis method. Key functionalities of our zebrafish image processing pipeline include quantification of neuron loss in zebrafish embryos due to knockdown of AD-linked genes, automatic detection of defective somites, and quantitative measurement of gene expression levels in zebrafish with altered expression of AD-linked genes or treatment with a chemical compound. These quantitative measurements enable the archival of analyzed results and relevant meta-data. The structured database is organized for statistical analysis and data modeling to better understand neuronal integrity and phenotypic changes of zebrafish under different perturbations. Our results show that the computerized analysis is comparable to manual counting with equivalent accuracy and improved efficacy and consistency. Development of such an automated data analysis pipeline represents a significant step forward to achieve accurate and reproducible quantification of neuronal phenotypes in large scale or high-throughput zebrafish imaging studies.

Animals↗

PubMed1996

Specific teratogenic effects of different retinoic acid isomers and analogs in the developing anterior central nervous system of zebrafish.

Vertebrate embryos are sensitive to retinoic acid, either in deficiency or in excess. Although all-trans retinoic acid (RA) and 9-cis RA are known to have distinct but overlapping activities in higher organisms, only the all-trans isomer has been investigated in detail as a teratogen in zebrafish. We have identified profound and specific effects of 9-cis RA when administered to zebrafish embryos, and have confirmed the results of prior studies on the teratogenic effects of exogenous all-trans RA. Moreover, we have identified a 1-hr period during gastrulation in which embryos are particularly sensitive to the teratogenic effects of RA. In the course of these investigations, we have also studied the effects of two synthetic retinoids-a 9-cis RA analog, SR11217, and an all-trans RA analog, TTAB. An application of all-trans RA to the early zebrafish gastrula leads to defects that are limited to the caudal midbrain and rostral hindbrain. Our experiments show that an application of exogenous 9-cis RA for a period as short as 1 hr and at a concentration as low as 0.1 mu M can block differentiation of the rostral CNS. We have observed abnormal phenotypes using DIC optics, and have demonstrated further abnormalities using whole-mount immunocytochemical staining with antibodies to HNK-1 and acetylated alpha-tubulin. Major axon tract formation in the anterior CNS is unambiguously disrupted by the administration of 9-cis RA but not all-trans RA. Furthermore, exogenous 9-cis RA produces a qualitative alteration in the multiple-site expression pattern of the hlx-1 gene within the rostral CNS, while treatment with all-trans RA leads only to a weakened expression signal. The administration of TTAB and SR11217 result in distinctive inhibitions of hlx-1 expression. Unlike all-trans RA, which causes premature par-2 expression in the posterior midbrains of a majority of embryos, 9-cis RA leads to a complete deletion of this domain throughout development. These results suggest that 9-cis RA is a more active teratogen than all-trans RA in rostral CNS structures of the zebrafish embryo.

Animals↗

PubMed2004

Zebrafish as a genomics research model.

The zebrafish (Danio rerio) is a recent addition to the genomic scientists' repertoire of vertebrate animal model systems. Unlike simple invertebrates such as the fly or the nematode, this teleost maintains the biological and genomic complexity found in higher vertebrates. Furthermore, the zebrafish has many advantageous technical and genomic properties that open the door to experimental approaches not practical using more classical models. The zebrafish genome can be functionally accessed using both forward and reverse genetics based approaches. A notable recent addition to the zebrafish genomics toolbox is the development of morpholino-based antisense gene inhibition for sequence-based 'knockdown' screening. This method offers the opportunity to examine the role of significant subsets of the vertebrate genome for specific gene function in vivo. The zebrafish embryo can rapidly provide critical information for drug target discovery purposes when examined with an emphasis on clinically-relevant biological processes. Finally, the advent of chemical genetics in zebrafish suggests that, in addition to the identification and understanding of drug targets and their biology, this system will be a powerful tool in the direct development of novel pharmaceuticals in the near future.

Animals↗

PubMed2004

Constitutive and xenobiotics-induced expression of a novel CYP3A gene from zebrafish larva.

In mammals, CYP3A isozymes collectively comprise the largest portion of the liver and small intestinal CYP protein. They are involved in the metabolism of an extensive range of endogenous substrates and xenobiotics and make a significant contribution to the termination of the action of steroid hormones. A full-length cDNA of CYP3A gene, named CYP3A65, was cloned from zebrafish by RT-PCR. The CYP3A65 mRNA was initially transcribed only in the liver and intestine upon hatching of the zebrafish embryos. Like the human CYP3A genes, CYP3A65 transcription in the foregut region was enhanced by treatment of the zebrafish larvae with the steroid dexamethasone and the macrocyclic antibiotic rifampicin. Differing from mammalian CYP3A genes, CYP3A65 transcription was also elicited by 2,3,7,8-tetrachloro-dibenzo-p-dioxin (TCDD) during early larval stages. Repression of AHR2 translation by antisense morpholino oligonucleotides abrogated both of constitutive and TCDD-stimulated CYP3A65 transcription in larval intestine. These findings suggested that the AHR2 signaling pathway plays an essential role in CYP3A65 transcription.

Animals↗

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↗

PubMed2001

Hedgehog signaling is required for primary motoneuron induction in zebrafish.

Sonic hedgehog (Shh) is crucial for motoneuron development in chick and mouse. However, zebrafish embryos homozygous for a deletion of the shh locus have normal numbers of motoneurons, raising the possibility that zebrafish motoneurons may be specified differently. Unlike other vertebrates, zebrafish express three hh genes in the embryonic midline: shh, echidna hedgehog (ehh) and tiggywinkle hedgehog (twhh). Therefore, it is possible that Twhh and Ehh are sufficient for motoneuron formation in the absence of Shh. To test this hypothesis we have eliminated, or severely reduced, all three Hh signals using mutations that directly or indirectly reduce Hh signaling and antisense morpholinos. Our analysis shows that Hh signals are required for zebrafish motoneuron induction. However, each of the three zebrafish Hhs is individually dispensable for motoneuron development because the other two can compensate for its loss. Our results also suggest that Twhh and Shh are more important for motoneuron development than Ehh.

Animals↗

PubMed2006

Cloning, expression and functional study of translation elongation factor 2 (EF-2) in zebrafish.

We have identified translation elongation factor 2 (EF-2) in zebrafish (GenBank Accession No. AAQ91234). Analysis of the DNA sequence of zebrafish EF-2 shows that the 2826 bp cDNA spans an open reading frame between nucleotide 55 to 2631 and encodes a protein of 858 amino acids. Zebrafish EF-2 protein shares 92%, 93%, 93% and 92% identity with the corresponding amino acid sequence in human, mouse, Chinese hamster and Gallus EF-2, respectively. Whole-mount in situ hybridization showed that zebrafish EF-2 was a developmentally regulated gene and might play important roles during the early development of zebrafish embryos. Therefore, we further studied the function of EF-2 during early embryogenesis. Using morpholino antisense oligo knockdown assays, anti-MO injected embryos were found to display abnormal development. The yolk balls were larger than normal and the melanophores spreading on their bodies became fewer. Furthermore, their tails were incurvate and their lenses were much smaller than those of the normal embryos. However the EF-2 overexpression data showed that extra EF-2 protein had no obvious effect on zebrafish embryonic development.

Amino Acid Sequence↗