Search tips
Search criteria

Results 1-13 (13)

Clipboard (0)

Select a Filter Below

more »
Year of Publication
Document Types
1.  A Streamlined CRISPR Pipeline to Reliably Generate Zebrafish Frameshifting Alleles 
Zebrafish  2014;11(6):583-585.
PMCID: PMC4248249  PMID: 25470533
2.  Emerging gene knockout technology in zebrafish: zinc-finger nucleases 
One advantage of the zebrafish model system is the ability to use forward genetics to reveal critical gene functions by their mutant phenotype. Reverse genetic tools are available, although it is more challenging and time-consuming to identify mutations in specific genes of interest and virtually impossible to induce mutations in a targeted manner. Two recent papers have shown that locus-specific zinc-finger nucleases (ZFNs) can be used to create mutations in investigator-specified loci at high frequency, generating considerable enthusiasm that the technology may be generally applicable to many zebrafish genes. The rate-limiting step in ZFN application is typically the zinc-finger protein (ZFP) design phase, partly because ZFPs that bind to intended target sequences in naked DNA may not recognize the target within chromatin, or may recognize cryptic sites. Importantly, both papers also provide new tools to validate or pre-select ZFNs that work well in vivo and thus greatly facilitate the identification of active ZFNs. Finally, work in other model systems and in cultured cells show that ZFNs can facilitate homology-directed repair, raising the exciting possibility that ZFNs may facilitate homologous recombination in zebrafish, allowing site-specific modification of endogenous genes via a method that does not require embryonic stem cell technology.
PMCID: PMC2722258  PMID: 19109309
gene targeting; targeted mutagenesis; zebrafish; zinc-finger nuclease; non-homologous end joining; double-strand break repair
3.  Control of morphogenetic cell movements in the early zebrafish myotome 
Developmental biology  2007;309(2):169-179.
As the vertebrate myotome is generated, myogenic precursor cells undergo extensive and coordinated movements as they differentiate into properly positioned embryonic muscle fibers. In the zebrafish, the “adaxial” cells adjacent to the notochord are the first muscle precursors to be specified. After initially differentiating into slow-twitch myosin-expressing muscle fibers, these cells have been shown to undergo a remarkable radial migration through the lateral somite, to populate the superficial layer of slow-twitch muscle of the mature myotome. Here we characterize an earlier set of adaxial cell behaviors; the transition from a roughly 4 × 5 array of cuboidal cells to a 1 × 20 stack of elongated cells, prior to the migration event. We find that adaxial cells display a highly stereotypical series of behaviors as they undergo this rearrangement. Further, we show that the actin regulatory molecule, Cap1, is specifically expressed in adaxial cells and is required for the progression of these behaviors. The requirement of Cap1 for a cellular apical constriction step is reminiscent of similar requirements of Cap during apical constriction in Drosophila development, suggesting a conservation of gene function for a cell biological event critical to many developmental processes.
PMCID: PMC2723113  PMID: 17689522
Myotome; Muscle; cap; Adaxial; Apical Constriction; Somite; Zebrafish
4.  Single cell resolution imaging of the impact of Notch signaling and mitosis on segmentation clock dynamics 
Developmental cell  2012;23(5):995-1005.
Vertebrate body segmentation is controlled by the segmentation clock, a molecular oscillator involving transcriptional oscillations of cyclic genes in presomitic mesoderm cells. The rapid and highly dynamic nature of this oscillating system has proved challenging for study at the single cell level. We achieved visualization of clock activity with a cellular level of resolution in living embryos, allowing direct comparison of oscillations in neighbor cells. We provide direct evidence that presomitic mesoderm cells oscillate asynchronously in zebrafish Notch pathway mutants. By tracking oscillations in mitotic cells, we reveal that a robust cell-autonomous, Notch-independent mechanism resumes oscillations after mitosis. Finally, we find that cells preferentially divide at a certain oscillation phase, likely reducing the noise generated by cell division in cell synchrony and suggesting an intriguing relationship between the mitotic cycle and clock oscillation.
PMCID: PMC3500528  PMID: 23153496
5.  Rbfox-regulated alternative splicing is critical for zebrafish cardiac and skeletal muscle function 
Developmental biology  2011;359(2):251-261.
Rbfox RNA binding proteins are implicated as regulators of phylogenetically-conserved alternative splicing events important for muscle function. To investigate the function of rbfox genes, we used morpholino-mediated knockdown of muscle-expressed rbfox1l and rbfox2 in zebrafish embryos. Single and double morphant embryos exhibited changes in splicing of overlapping sets of bioinformatically-predicted rbfox target exons, many of which exhibit a muscle-enriched splicing pattern that is conserved in vertebrates. Thus, conservation of intronic Rbfox binding motifs is a good predictor of Rbfox-regulated alternative splicing. Morphology and development of single morphant embryos was strikingly normal; however, muscle development in double morphants was severely disrupted. Defects in cardiac muscle were marked by reduced heart rate and in skeletal muscle by complete paralysis. The predominance of wavy myofibers and abnormal thick and thin filaments in skeletal muscle revealed that myofibril assembly is defective and disorganized in double morphants. Ultra-structural analysis revealed that although sarcomeres with electron dense M- and Z-bands are present in muscle fibers of rbfox1l/rbox2 morphants, they are substantially reduced in number and alignment. Importantly, splicing changes and morphological defects were rescued by expression of morpholino-resistant rbfox cDNA. Additionally, a target-blocking MO complementary to a single UGCAUG motif adjacent to an rbfox target exon of fxr1 inhibited inclusion in a similar manner to rbfox knockdown, providing evidence that Rbfox regulates the splicing of target exons via direct binding to intronic regulatory motifs. We conclude that Rbfox proteins regulate an alternative splicing program essential for vertebrate heart and skeletal muscle function.
PMCID: PMC3521583  PMID: 21925157
alternative splicing; Zebrafish; muscle; rbfox; a2bp1l; rbm9
6.  Deep Intron Elements Mediate Nested Splicing Events at Consecutive AG Dinucleotides To Regulate Alternative 3′ Splice Site Choice in Vertebrate 4.1 Genes 
Molecular and Cellular Biology  2012;32(11):2044-2053.
Distal intraexon (iE) regulatory elements in 4.1R pre-mRNA govern 3′ splice site choice at exon 2 (E2) via nested splicing events, ultimately modulating expression of N-terminal isoforms of cytoskeletal 4.1R protein. Here we explored intrasplicing in other normal and disease gene contexts and found conservation of intrasplicing through vertebrate evolution. In the paralogous 4.1B gene, we identified ∼120 kb upstream of E2 an ultradistal intraexon, iEB, that mediates intrasplicing by promoting two intricately coupled splicing events that ensure selection of a weak distal acceptor at E2 (E2dis) by prior excision of the competing proximal acceptor (E2prox). Mutating iEB in minigene splicing reporters abrogated intrasplicing, as did blocking endogenous iEB function with antisense morpholinos in live mouse and zebrafish animal models. In a human elliptocytosis patient with a mutant 4.1R gene lacking E2 through E4, we showed that aberrant splicing is consistent with iER-mediated intrasplicing at the first available exons downstream of iER, namely, alternative E5 and constitutive E6. Finally, analysis of heterologous acceptor contexts revealed a strong preference for nested 3′ splice events at consecutive pairs of AG dinucleotides. Distal regulatory elements may control intrasplicing at a subset of alternative 3′ splice sites in vertebrate pre-mRNAs to generate proteins with functional diversity.
PMCID: PMC3372232  PMID: 22473990
7.  Imaging the Sialome during Zebrafish Development with Copper-Free Click Chemistry 
PMCID: PMC3385855  PMID: 22262667
bioorthogonal chemistry; embryonic development; fluorescence imaging; glycosylation; sialic acids
8.  In Vivo Imaging of Membrane-Associated Glycans in Developing Zebrafish 
Science (New York, N.Y.)  2008;320(5876):664-667.
Glycans are attractive targets for molecular imaging but have been inaccessible because of their incompatibility with genetically encoded reporters. We demonstrated the noninvasive imaging of glycans in live developing zebrafish, using a chemical reporter strategy. Zebrafish embryos were treated with an unnatural sugar to metabolically label their cell-surface glycans with azides. Subsequently, the embryos were reacted with fluorophore conjugates by means of copper-free click chemistry, enabling the visualization of glycans in vivo at subcellular resolution during development. At 60 hours after fertilization, we observed an increase in de novo glycan biosynthesis in the jaw region, pectoral fins, and olfactory organs. Using a multicolor detection strategy, we performed a spatiotemporal analysis of glycan expression and trafficking and identified patterns that would be undetectable with conventional molecular imaging approaches.
PMCID: PMC2701225  PMID: 18451302
9.  Heritable Targeted Gene Disruption in Zebrafish Using Designed Zinc Finger Nucleases 
Nature biotechnology  2008;26(6):702-708.
We describe here the use of zinc finger nucleases (ZFNs) for somatic and germline disruption of genes in zebrafish (Danio rerio), where targeted mutagenesis was previously intractable. ZFNs induce a targeted double-strand break in the genome that is repaired to generate small insertions and deletions. We designed ZFNs targeting the zebrafish golden and no tail/Brachyury genes. In both cases, injection of ZFN-encoding mRNA into 1-cell embryos yielded a high percentage of animals carrying distinct mutations at the ZFN-specified position and exhibiting expected loss-of-function phenotypes. Disrupted ntl alleles were transmitted from ZFN mRNA-injected founder animals in over half the adults tested at frequencies averaging 20%. The frequency and precision of gene disruption events observed, in combination with the ability to design ZFNs against any locus, open fundamentally novel avenues of experimentation, and suggest that ZFN technology may be widely applied to many organisms that allow mRNA delivery into the fertilized egg.
PMCID: PMC2674762  PMID: 18500334
10.  A Web Based Resource Characterizing the Zebrafish Developmental Profile of over 16,000 transcripts 
Gene expression patterns : GEP  2007;8(3):171-180.
Using a spotted 65-mer oligonucleotide microarray, we have characterized the developmental expression profile from mid-gastrulation (75% epiboly) to 5 days post-fertilization (dpf) for >16,000 unique transcripts in the zebrafish genome. Microarray profiling data sets are often immense, and one challenge is validating the results and prioritizing genes for further study. The purpose of the current study was to address such issues, as well as to generate a publicly available resource for investigators to examine the developmental expression profile of any of the over 16,000 zebrafish genes on the array. On the chips, there are 16,459 printed spots corresponding to 16,288 unique transcripts and 172 β-actin (AF025305) spots spatially distributed throughout the chip as a positive control. We have collected 55 microarray gene expression profiling results from various zebrafish laboratories and created a Perl/CGI-based software tool ( for researchers to look for the expression patterns of their gene of interest. Users can search for their genes of interest by entering the accession numbers or the nucleotide sequences and the expression profiling will be reported in the form of expression intensities versus time-course graphical displays. In order to validate this web tool, we compared seventy-four genes’ expression results between our web tool and the in situ hybridization results from Thisse et al. (2004) as well as those reported by Mathavan et al. (2005). The comparison indicates the expression patterns are 80% and 75% in agreement between our web resource with the in situ database (Thisse et al. 2004) and with those reported by Mathavan et al. (2005), respectively. Those genes that conflict between our web tool and the in situ database either have high sequence similarity with other genes or the in situ probes are not reliable. Among those genes that disagree between our web tool and those reported by Mathavan et al. (2005), 93% of the genes are in agreement between our web tool and the in situ database, indicating our web tool results are quite reliable. Thus, this resource provides a user-friendly web based platform for researchers to determine the developmental profile of their gene of interest and to prioritize genes identified in microarray analyses by their developmental expression profile.
PMCID: PMC2253684  PMID: 18068546
11.  Zebrafish paraxial protocadherin is a downstream target of spadetail involved in morphogenesis of gastrula mesoderm 
Development (Cambridge, England)  1998;125(17):3389-3397.
Zebrafish paraxial protocadherin (papc) encodes a transmembrane cell adhesion molecule (PAPC) expressed in trunk mesoderm undergoing morphogenesis. Microinjection studies with a dominant-negative secreted construct suggest that papc is required for proper dorsal convergence movements during gastrulation. Genetic studies show that papc is a close downstream target of spadetail, gene encoding a transcription factor required for mesodermal morphogenetic movements. Further, we show that the floating head homeobox gene is required in axial mesoderm to repress the expression of both spadetail and papc, promoting notochord and blocking differentiation of paraxial mesoderm. The PAPC structural cell-surface protein may provide a link between regulatory transcription factors and the actual cell biological behaviors that execute morphogenesis during gastrulation.
PMCID: PMC2280034  PMID: 9693142
Protocadherin; spadetail; floating head; Gastrulation; Convergence movements; Somitogenesis; Zebrafish
12.  Metabolic Labeling of Fucosylated Glycans in Developing Zebrafish 
ACS Chemical Biology  2011;6(6):547-552.
Many developmental processes depend on proper fucosylation, but this post-translational modification is difficult to monitor in vivo. Here we applied a chemical reporter strategy to visualize fucosylated glycans in developing zebrafish. Using azide-derivatized analogues of fucose, we metabolically labeled cell-surface glycans and then detected the incorporated azides via copper-free click chemistry with a difluorinated cyclooctyne probe. We found that the fucose salvage pathway enzymes are expressed during zebrafish embryogenesis but that they process the azide-modified substrates inefficiently. We were able to bypass the salvage pathway by using an azide-functionalized analogue of GDP-fucose. This nucleotide sugar was readily accepted by fucosyltransferases and provided robust cell-surface labeling of fucosylated glycans, as determined by flow cytometry and confocal microscopy analysis. We used this technique to image fucosylated glycans in the enveloping layer of zebrafish embryos during the first 5 days of development. This work provides a method to study the biosynthesis of fucosylated glycans in vivo.
PMCID: PMC3117394  PMID: 21425872
13.  Imaging the Sialome during Zebrafish Development with Copper-Free Click Chemistry 
Chembiochem  2012;13(3):353-357.
PMCID: PMC3385855  PMID: 22262667
bioorthogonal chemistry; embryonic development; fluorescence imaging; glycosylation; sialic acids

Results 1-13 (13)