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.The cell-surface glycome is a rich source of information that reports on the cell's physiological state. For example, changes in glycan structures serve as markers of altered gene expression during development (1) and disease progression (2). The dynamics of glycans at the plasma membrane reflect the activity of the cell's secretory machinery (3), and their relative abundances report on flux in metabolic pathways inside the cell (4). Glycans are therefore attractive targets for in vivo imaging but have been inaccessible because of their incompatibility with genetically encoded reporters (5).To image glycans in vivo, we employed a strategy in which an azide is introduced into target biomolecules, priming them for selective covalent reaction with fluorescent probes (5). The azide is small, stable in biological systems, and selectively reactive with phosphines or activated alkynes. Previously, the Staudinger ligation (6,7) or copper-catalyzed click chemistry (8,9) have been used to detect azide-labeled biomolecules on cells ex vivo. However, in vivo †To whom correspondence should be addressed. E-mail: E-mail: crb@berkeley.edu. * These authors contributed equally to this work. imaging of dynamic biological processes using these chemistries could be complicated by slow reaction kinetics or reagent toxicity. The copper-free click reaction of azides with difluorinated cyclooctyne (DIFO) reagents (10) overcomes these limitations, suggesting its potential application to in vivo imaging.We chose zebrafish as a model organism because of their well-defined developmental program (11), emerging disease models (12), and amenability to optical imaging. The metabolic substrate peracetylated N-azidoacetylgalactosamine (Ac 4 GalNAz) was selected on the basis of its known incorporation into mucin-type O-linked glycoproteins in mammalian cells and mice via the N-acetylgalactosamine (GalNAc) salvage pathway (13,14) ( fig. S1). We envisioned an imaging experiment ( Before performing imaging experiments, we confirmed that the zebrafish glycan biosynthetic enzymes are permissive of the unnatural sugar. The...

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.

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Summary 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.

We have used transient transfections in MM14 skeletal muscle cells, newborn rat primary ventricular myocardiocytes, and nonmuscle cells to characterize regulatory elements of the mouse muscle creatine kinase (MCK) gene. Deletion analysis of MCK 5'-flanking sequence reveals a striated muscle-specific, positive regulatory region between-1256 and-1020. A 206-bp fragment from this region acts as a skeletal muscle enhancer and confers orientation-dependent activity in myocardiocytes. A 110-bp enhancer subfragment confers high-level expression in skeletal myocytes but is inactive in myocardiocytes, indicating that skeletal and cardiac muscle MCK regulatory sites are distinguishable. To further delineate muscle regulatory sequences, we tested six sites within the MCK enhancer for their functional importance. Mutations at five sites decrease expression in skeletal muscle, cardiac muscle, and nonmuscle cells. Mutations at two of these sites, Left E box and MEF2, cause similar decreases in all three cell types. Mutations at three sites have larger effects in muscle than nonmuscle cells; an AlT-rich site mutation has a pronounced effect in both striated muscle types, mutations at the MEF1 (Right E-box) site are relatively specific to expression in skeletal muscle, and mutations at the CArG site are relatively specific to expression in cardiac muscle. Changes at the AP2 site tend to increase expression in muscle cells but decrease it in nonmuscle cells. In contrast to reports involving cotransfection of 1OT1/2 cells with plasmids expressing the myogenic determination factor MyoD, we show that the skeletal myocyte activity of multimerized MEF1 sites is 30-fold lower than that of the 206-bp enhancer. Thus, MyoD binding sites alone are not sufficient for high-level expression in skeletal myocytes containing endogenous levels of MyoD and other myogenic determination factors. The mammalian muscle creatine kinase (MCK) gene is expressed primarily in skeletal and cardiac muscle. The mouse MCK gene contains an enhancer located approximately 1,100 bp 5' of the transcription start site. A 206-bp enhancer-containing fragment of the mouse gene and analogous regions of other mammalian MCK genes confer muscle-specific expression in cultured cells and transgenic mice (30, 36-38, 76, 81, 92). In vitro and in vivo footprinting have identified a variety of binding sites within the rat and mouse MCK enhancers (10, 28, 30,31, 57). MEF1, a skeletal myocyte-specific nuclear factor, was identified by gel shift assays and DNase footprinting using the MCK enhancer (10). The MEF1 or Right site contains the E-box sequence, CAnnTG (16, 23), characteristic of the recognition sites of the helix-loop-helix (HLH) family of DNA-binding proteins (58). The MEF1 complex contains the HLH myogenic determination factor, MyoD (41). MyoD, as well as the skeletal muscle determination factors myogenin, Myf5, and MRF4/herculin/Myf6, can bind the MEF1 site in vitro with various affinities, either alone or in combination with other HLH partners (2, 6, 9, 11, 12, 43, 59...