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PubMed2006

Functional analysis of zebrafish microfibril-associated glycoprotein-1 (Magp1) in vivo reveals roles for microfibrils in vascular development and function.

Mutations in fibrillin-1 (FBN1) result in Marfan syndrome, demonstrating a critical requirement for microfibrils in vessel structure and function. However, the identity and function of many microfibril-associated molecules essential for vascular development and function have yet to be characterized. In our morpholino-based screen for members of the secretome required for vascular development, we identified a key player in microfibril formation in zebrafish embryogenesis. Microfibril-associated glycoprotein-1 (MAGP1) is a conserved protein found in mammalian and zebrafish microfibrils. Expression of magp1 mRNA is detected in microfibril-producing cells. Analysis of a functional Magp1-mRFP fusion protein reveals localization along the midline and in the vasculature during embryogenesis. Underexpression and overexpression analyses demonstrate that specific Magp1 protein levels are critical for vascular development. Integrin function is compromised in magp1 morphant embryos, suggesting that reduced integrin-matrix interaction is the main mechanism for the vascular defects in magp1 morphants. We further show that Magp1 and fibrillin-1 interact in vivo. This study implicates MAGP1 as a key player in microfibril formation and integrity during development. The essential role for MAGP1 in vascular morphogenesis and function also supports a wide range of clinical applications, including therapeutic targets in vascular disease and cardiovascular tissue engineering.

Animals↗

PubMed1999

Development of the retina.

As in other vertebrate species, the zebrafish retina is simpler than other regions of the central nervous system. This relative simplicity along with rapid development, and accessibility to genetic analysis make the zebrafish retina an excellent model system for studies of neurogenesis in the vertebrate CNS. Several genetic screens have led to the isolation of an impressive collection of mutants affecting the retina and the retinotectal projections in zebrafish. A variety of techniques and markers are available to study the isolated mutants. These include several antigen- and transcript-detection methods, retrograde and anterograde labeling of neurons, blastomere transplantations, H3 labeling, and others. As past genetic screens have achieved a rather low level of saturation, the current collection of mutants can only grow in the future. Morphological and behavioral criteria have been successfully applied in zebrafish to search for defects in spinal development. In future genetic screens, progressively more sophisticated screening approaches will make it possible to detect very subtle changes in the retinal development. The remarkable evolutionary conservation of the vertebrate eye provides the basis for using the zebrafish as a model system for the detection and analysis of genetic defects potentially related to human eye disorders. Some of the genetic defects of the zebrafish retina indeed resemble human retinopathies. As the genetic analysis of the vertebrate visual system is far from being complete and new techniques are being introduced at a rapid pace, the zebrafish embryo will become increasingly useful as a model for studies of the vertebrate retina.

Animals↗

PubMed2005

Analysis of cell movements in zebrafish embryos: morphometrics and measuring movement of labeled cell populations in vivo.

Cell movements occur in all phases of animal life from embryogenesis, to maintaining adult organs, to comprising a critical component of pathology. During gastrulation, cells demonstrate a repertoire of morphogenetic movements coordinated with fate inductions to sculpt the embryonic body. The morphogenetic behaviors, underlying mechanisms, and their control, are the subject of much current study. External development of the transparent zebrafish embryo, the abundance of mutations influencing cell movements, as well as a range of observation and manipulation methods, make the zebrafish valuable for cell movement studies. This chapter offers a conceptual background for analysis of gastrulation cell movements by reviewing how region specific cell movements shape the wild-type zebrafish embryo, and how defective morphogenetic movements alone or in combination with altered cell fate specification distort the body plans of known zebrafish mutants. We furnish methods for the morphometric analysis of embryonic shape and organ rudiments in live and fixed embryos, and present data collected from live wild-type, dorsoventral patterning (somitabun and chordino) and convergence and extension (knypek and trilobite) classes of mutants. We provide a method for quantitative assessment of the movements of cell populations in vivo, and a method for determining whether cell fate and/or movement are disturbed.

Animals↗

PubMed1994

A genetic linkage map for the zebrafish.

To facilitate molecular genetic analysis of vertebrate development, haploid genetics was used to construct a recombination map for the zebrafish Danio (Brachydanio) rerio. The map consists of 401 random amplified polymorphic DNAs (RAPDs) and 13 simple sequence repeats spaced at an average interval of 5.8 centimorgans. Strategies that exploit the advantages of haploid genetics and RAPD markers were developed that quickly mapped lethal and visible mutations and that placed cloned genes on the map. This map is useful for the position-based cloning of mutant genes, the characterization of chromosome rearrangements, and the investigation of evolution in vertebrate genomes.

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PubMed2004

The T box transcription factor no tail in ciliated cells controls zebrafish left-right asymmetry.

The heart, brain, and gut develop essential left-right (LR) asymmetries. Specialized groups of ciliated cells have been implicated in LR patterning in mouse, chick, frog, and zebrafish embryos. In zebrafish, these ciliated cells are found in Kupffer's vesicle (KV) and are progeny of dorsal forerunner cells (DFCs). However, there is no direct evidence in any vertebrate that the genes involved in LR development are specifically required in ciliated cells. By using a novel method in zebrafish, we knocked down the function of no tail (ntl, homologous to mouse brachyury) in DFCs without affecting its expression in other cells in the embryo. We find that the Ntl transcription factor functions cell autonomously in DFCs to regulate KV morphogenesis and LR determination. This is the first evidence that loss-of-gene function exclusively in ciliated cells perturbs vertebrate LR patterning. Our results demonstrate that the ciliated KV, a transient embryonic organ of previously unknown function, is involved in the earliest known step in zebrafish LR development, suggesting that a ciliary-based mechanism establishes the LR axis in all vertebrate embryos.

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PubMed2006

Resolution of inflammation by retrograde chemotaxis of neutrophils in transgenic zebrafish.

Neutrophil chemotaxis to sites of inflammation is a critical process during normal immune responses to tissue injury and infection and pathological immune responses leading to chronic inflammation. Although progress has been made in understanding the mechanisms that promote neutrophil recruitment to inflamed tissue, the mechanisms that regulate the resolution phase of the inflammatory response have remained relatively elusive. To define the mechanisms that regulate neutrophil-mediated inflammation in vivo, we have developed a novel transgenic zebrafish in which the neutrophils express GFP under control of the myeloperoxidase promoter (zMPO:GFP). Tissue injury induces a robust, inflammatory response, which is characterized by the rapid chemotaxis of neutrophils to the wound site. In vivo time-lapse imaging shows that neutrophils subsequently display directed retrograde chemotaxis back toward the vasculature. These findings implicate retrograde chemotaxis as a novel mechanism that regulates the resolution phase of the inflammatory response. The zMPO:GFP zebrafish provides unique insight into the mechanisms of neutrophil-mediated inflammation and thereby offers opportunities to identify new regulators of the inflammatory response in vivo.

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PubMed1995

Caudal fin regeneration in wild type and long-fin mutant zebrafish is affected by retinoic acid.

Zebrafish (Danio rerio) represents an ideal experimental model to tackle fundamental issues concerned with organogenesis during development and regeneration of complex body structures. We discuss here the development of the skeleton in zebrafish caudal fins, their regenerative ability in wild type and long-fin mutant adult fish, and how retinoic acid (RA), which induces duplications along the proximodistal axis in regenerating limbs, affects regeneration of the caudal fin. The dorsal and ventral lobes of zebrafish caudal fins are apparently symmetrical along the dorsoventral axis, but all of the skeletal elements and most of the soft tissues of both lobes originate from the ventral part of the embryo, as demonstrated by whole-mount staining of developing fish. Analysis of caudal fin regenerates in wild type adults does not reveal any difference in the regenerative ability of the two lobes, and in the length of the regenerate in comparison with the amputated part. In contrast, in the long-fin mutant the regenerated caudal fin is always somehow defective in that the original asymmetry in the length of the two lobes observed in this mutant is not reproduced in the regenerate. Furthermore, in the majority of the batches studied the regenerate is much smaller in size than the amputated part. This suggests that this mutant may be valuable to further our understanding of the mechanisms underlying growth control and patterning during regeneration. Finally, we show that the regenerating caudal fin is sensitive to RA-treatment, and clear teratogenic effects on the dorso-ventral axis are observed under many of the experimental conditions investigated both in wild type and long-fin mutants.(ABSTRACT TRUNCATED AT 250 WORDS)

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PubMed2006

The transcription factor ZBP-89 controls generation of the hematopoietic lineage in zebrafish and mouse embryonic stem cells.

Hematopoietic development is closely linked to that of blood vessels and the two processes are regulated in large part by transcription factors that control cell fate decisions and cellular differentiation. Both blood and blood vessels derive from a common progenitor, termed the hemangioblast, but the factor(s) specifying the development and differentiation of this stem cell population into the hematopoietic and vascular lineages remain ill defined. Here, we report that knockdown of the Krüppel-like transcription factor ZBP-89 in zebrafish embryos results in a bloodless phenotype, caused by disruption of both primitive and definitive hematopoiesis, while leaving primary blood vessel formation intact. Injection of ZBP-89 mRNA into cloche zebrafish embryos, which lack both the hematopoietic and endothelial lineages, rescues hematopoiesis but not vasculogenesis. Injection of mRNA for Stem Cell Leukemia (SCL), a transcription factor that directs hemangioblast development into blood cell precursors, rescues the bloodless phenotype in ZBP-89 zebrafish morphants. Forced expression of ZBP-89 induces the expansion of hematopoietic progenitors in wild-type zebrafish and in mouse embryonic stem cell cultures but inhibits angiogenesis in vivo and in vitro. These findings establish a unique regulatory role for ZBP-89, positioned at the interface between early blood and blood vessel development.

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↗

PubMed2003

DNA delivery into anterior neural tube of zebrafish embryos by electroporation.

The zebrafish is widely used for functional studies of vertebrate genes. It is accessible to manipulations during all stages of embryogenesis because the embryo develops externally and is optically transparent. However, functional studies conducted on the zebrafish have been generally limited to the earliest phase of activity of the gene of interest, which is a limitation in studies of genes that are expressed at various stages of embryonic development. It is therefore necessary to develop methods that allow for the modulation of gene activity during later stages of zebrafish development while leaving earlier functions intact. We have successfully electroporated the green fluorescent protein (GFP) reporter gene into the neural tube of the zebrafish embryo in a unidirectional or bilateral manner. This approach can be used for the functional analysis of the late role of developmental genes in the neural tube of zebrafish embryo and larvae.

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PubMed2005

Investigating chorion softening of zebrafish embryos with a microrobotic force sensing system.

The zebrafish is a model organism for addressing questions of vertebrate embryo development. In this paper, the softening phenomenon of the chorion envelope of zebrafish embryos at different developmental stages was mechanically quantitated by using a microrobotic force sensing system. The microrobotic system integrates a piezoelectric cellular force sensor to measure the required forces for penetrating the chorion envelope. Magnitude of penetration forces was found to decrease as an embryo develops. The results mechanically quantitate "chorion softening" in zebrafish embryos due to protease activities subtly modifying the chorion structure, providing an understanding of zebrafish embryo development.

Animals↗

PubMed2003

Effects of 17a-ethinylestradiol on the expression of three estrogen-responsive genes and cellular ultrastructure of liver and testes in male zebrafish.

In order to monitor the influence of estrogenic compounds on the reproductive physiology of fish, molecular markers for zebrafish vitellogenin, estrogen receptor and ZP2 were developed. For this purpose, sequence information about the zebrafish estrogen receptor and vitellogenin had to be obtained. By means of RT-PCR, a sequence fragment of the zebrafish estrogen receptor alpha was cloned and sequenced. Continuous cDNAs of two zebrafish vitellogenin-like gene products (zfvg1 and zfvg3) were constructed by the help of expressed sequence tags of zebrafish and completely sequenced. The sequences of the estrogen receptor and of the vitellogenins showed significant similarities to corresponding cDNAs of other fish species. Expression of these gene products was measured following exposure to 17alpha-ethinylestradiol and compared with histological endpoints. RT-PCR was used as a semiquantitative technique to record gene expression in adult male zebrafish, which were exposed to 17alpha-ethinylestradiol in time-and dose-response experiments. As for time-dependent expression, all hepatic genes investigated were expressed at considerable amounts from 24 h after onset of exposure to 50 ng/l 17alpha-ethinylestradiol to the end of experiment (17 days). In testes, expression of the estrogen receptor- as well as ZP2-mRNA remained unchanged for the entire experiment, except for the individuals exposed for 17 days, which displayed elevated expression levels of ZP2. In the dose-response experiment, male zebrafish were exposed to 17alpha-ethinylestradiol in concentrations from 0.25-85 ng/l for 4 and 21 days. LOECs for vitellogenin as well as estrogen receptor alpha expression were found to be 2.5 ng/l already after 4 d of exposure. Extension of the exposure time to 21 days resulted in enhanced transcription of vitellogenin-mRNAs at 2.5 ng/l 17alpha-ethinylestradiol, whereas the detection limit could not be lowered. In contrast, in testes no induction of both ZP2 as well as estrogen receptor expression was detected at any concentration tested. To examine estrogen-caused alterations at the ultrastructural level, liver and testes of males exposed to 25 ng/l 17alpha-ethinylestradiol were analysed. Male livers responded with a feminisation reflected by the proliferation of rough endoplasmatic reticulum and Golgi apparatus typical of female hepatocytes during vitellogenesis. However, in testes no signs of feminisation were detectable; rather, destructive phenomena like phagocytosis of sperm cells by Sertoli cells were observed. Thus, in sexually differentiated males no reorganisation of the gonadal tissue towards an ovary could be definitely detected at any level investigated.

Animals↗

PubMed2004

Zebrafish heparin-binding neurotrophic factor enhances neurite outgrowth during its development.

Heparin-binding neurotrophic factor (HBNF) is a secreted heparin-binding protein containing highly basic and cysteine-rich amino acid residues. In this study, we cloned the full-length HBNF cDNA from zebrafish and determined its genomic structure by bioinformatics analysis. Zebrafish HBNF gene is composed of five exons and four introns spanning approximately 82kb. RT-PCR analysis revealed that zebrafish HBNF transcript was highly expressed in adult brain and intestine tissues while less in other tissues. During embryogenesis, zebrafish HBNF transcript was observed to be moderately expressed at earlier stages with a gradual decline. Higher expression level was observed after hatching and maintaining this level into adulthood. The overall amino acid sequence of zebrafish HBNF shows 60% identity to human HBNF, but with approximately 40% identity to other midkine proteins. Like mammalian homolog, zebrafish HBNF could induce significant neurite outgrowth in PC12 cells without NGF stimulation. In addition, zebrafish HBNF was able to enhance extensive neurite outgrowth in zebrafish embryos during embryogenesis. In summary, a feasible in vivo assay for neurite outgrowth was established in zebrafish.

Amino Acid Sequence↗

PubMed2002

Caspy, a zebrafish caspase, activated by ASC oligomerization is required for pharyngeal arch development.

The pyrin domain was identified recently in multiple proteins that are associated with apoptosis and/or inflammation, but the physiological and molecular function of these proteins remain poorly understood. We have identified Caspy and Caspy2, two zebrafish caspases containing N-terminal pyrin domains. Expression of Caspy and Caspy2 induced apoptosis in mammalian cells that were inhibited by general caspase inhibitors. Biochemical analysis revealed that both Caspy and Caspy2 are active caspases, but they exhibit different substrate specificity. Caspy, but not Caspy2, interacted with the zebrafish orthologue of ASC (zAsc), a pyrin- and caspase recruitment domain-containing protein identified previously in mammals. The pyrin domains of both Caspy and zAsc were required for their interaction. Furthermore, zAsc and Caspy co-localized to the "speck" when co-transfected into mammalian cells. Enforced oligomerization of zAsc, but not simple interaction with zAsc, induced specific proteolytic activation of Caspy and enhanced Caspy-dependent apoptosis. Injection of zebrafish embryos with a morpholino antisense oligonucleotide corresponding to caspy resulted in an "open mouth" phenotype associated with defective formation of the cartilaginous pharyngeal skeleton. These studies suggest that zAsc mediates the activation of Caspy, a caspase that plays an important role in the morphogenesis of the jaw and gill-bearing arches.

Amino Acid Sequence↗

PubMed1996

Specification of cell fates at the dorsal margin of the zebrafish gastrula.

Using fate mapping techniques, we have analyzed development of cells of the dorsal marginal region in wild-type and mutant zebrafish. We define a domain in the early gastrula that is located just at the margin and centered on the dorsal midline, in which most cells generate clones that develop exclusively as notochord. The borders of the notochord domain are sharp at the level of single cells, and coincide almost exactly with the border of the expression domain of the homeobox gene floating head (flh; zebrafish homologue of Xnot), a gene essential for notochord development. In flh mutants, cells in the notochord domain generate clones of muscle cells. In contrast, notochord domain cells form mesenchyme in embryos mutant for no tail (ntl; zebrafish homologue of Brachyury). A minority of cells in the notochord domain in wild-type embryos develop as unrestricted mesoderm, invariably located in the tail, suggesting that early gastrula expression of flh does not restrict cellular potential to the notochord fate. The unrestricted tail mesodermal fate is also expressed by the forerunner cells, a cluster of cells located outside the blastoderm, adjacent to the notochord domain. We show that cells can leave the dorsal blastoderm to join the forerunners, suggesting that relocation between fate map domains might respecify notochord domain cells to the tail mesodermal fate. An intermediate fate of the forerunners is to form the epithelial lining of Kupffer's vesicle, a transient structure of the teleost tailbud. The forerunners appear to generate the entire structure of Kupffer's vesicle, which also develops in most flh mutants. Although forerunner cells are present in ntl mutants, Kupffer's vesicle never appears, which is correlated with the later severe disruption of tail development.

Animals↗

PubMed2005

Zebrafish as a model for caveolin-associated muscle disease; caveolin-3 is required for myofibril organization and muscle cell patterning.

Caveolae are an abundant feature of many animal cells. However, the exact function of caveolae remains unclear. We have used the zebrafish, Danio rerio, as a system to understand caveolae function focusing on the muscle-specific caveolar protein, caveolin-3 (Cav3). We have identified caveolin-1 (alpha and beta), caveolin-2 and Cav3 in the zebrafish. Zebrafish Cav3 has 72% identity to human CAV3, and the amino acids altered in human muscle diseases are conserved in the zebrafish protein. During embryonic development, cav3 expression is apparent by early segmentation stages in the first differentiating muscle precursors, the adaxial cells and slightly later in the notochord. cav3 expression appears in the somites during mid-segmentation stages and then later in the pectoral fins and facial muscles. Cav3 and caveolae are located along the entire sarcolemma of late stage embryonic muscle fibers, whereas beta-dystroglycan is restricted to the muscle fiber ends. Down-regulation of Cav3 expression causes gross muscle abnormalities and uncoordinated movement. Ultrastructural analysis of isolated muscle fibers reveals defects in myoblast fusion and disorganized myofibril and membrane systems. Expression of the zebrafish equivalent to a human muscular dystrophy mutant, CAV3P104L, causes severe disruption of muscle differentiation. In addition, knockdown of Cav3 resulted in a dramatic up-regulation of eng1a expression resulting in an increase in the number of muscle pioneer-like cells adjacent to the notochord. These studies provide new insights into the role of Cav3 in muscle development and demonstrate its requirement for correct intracellular organization and myoblast fusion.

Amino Acid Sequence↗

PubMed2001

Zebrafish acetylcholinesterase is encoded by a single gene localized on linkage group 7. Gene structure and polymorphism; molecular forms and expression pattern during development.

We cloned and sequenced the acetylcholinesterase gene and cDNA of zebrafish, Danio rerio. We found a single gene (ache) located on linkage group LG7. The relative organization of ache, eng2, and shh genes is conserved between zebrafish and mammals and defines a synteny. Restriction fragment length polymorphism analysis was allowed to identify several allelic variations. We also identified two transposable elements in non-coding regions of the gene. Compared with other vertebrate acetylcholinesterase genes, ache gene contains no alternative splicing at 5' or 3' ends where only a T exon is present. The translated sequence is 60-80% identical to acetylcholinesterases of the vertebrates and exhibits an extra loop specific to teleosts. Analysis of molecular forms showed a transition, at the time of hatching, from the globular G4 form to asymmetric A12 form that becomes prominent in adults. In situ hybridization and enzymatic activity detection on whole embryos confirmed early expression of the acetylcholinesterase gene in nervous and muscular tissues. We found no butyrylcholinesterase gene or activity in Danio. These findings make zebrafish a promising model to study function of acetylcholinesterase during development and regulation of molecular forms assembly in vivo.

Acetylcholinesterase↗

PubMed2001

Zebrafish mutants: behavioral genetic studies of visual system defects.

Zebrafish are a promising model for behavioral and genetic studies of vertebrate visual system development and retinal degeneration. In the past few years, numerous studies on zebrafish vision have been published. While most of the studies focus on the molecular and cellular characterization of mutations that disrupt zebrafish visual system structure in early development, others examine the mechanisms that underlie inherited visual system disorders in adults. Behavioral assays, along with morphologic and electrophysiological methods, are powerful tools for functional analyses of zebrafish visual development and performance.

Animals↗