Zebrafish models of cerebrovascular disease

Perturbations in cerebral blood flow and abnormalities in blood vessel structure are the hallmarks of cerebrovascular disease. While there are many genetic and environmental factors that affect these entities through a heterogeneous group of disease processes, the ultimate final pathologic insult in humans is defined as a stroke, or damage to brain parenchyma. In the case of ischemic stroke, blood fails to reach its target destination whereas in hemorrhagic stroke, extravasation of blood occurs outside of the blood vessel lumen, resulting in direct damage to brain parenchyma. As these acute events can be neurologically devastating, if not fatal, development of novel therapeutics are urgently needed. The zebrafish (Danio rerio) is an attractive model for the study of cerebrovascular disease because of its morphological and physiological similarity to human cerebral vasculature, its ability to be genetically manipulated, and its fecundity allowing for large-scale, phenotype-based screens

Zebrafish-Based Small Molecule Discovery

AbstractThe earliest examples of small molecule discovery involved serendipitous phenotypic observations in whole organisms, but this organism-based process has given way in recent decades to systematic, high-throughput assays using purified proteins, cells, or cell extracts. In vitro screens have been successful at identifying modifiers of well-understood biological processes, but they are limited in their ability to discover modifiers of processes that are poorly understood or occur only in an integrated physiological context. Small model organisms, especially the zebrafish, make it possible to combine the advantages of organism-based small molecule discovery with the technologies and throughput of modern screening. The combination of model organisms with high-throughput screening is likely to extend small molecule discovery efforts to fields of study such as developmental biology and to broaden the range of diseases for which drug screening can be performed

Isonitrile-responsive and bioorthogonally removable tetrazine protecting groups.

In vivo compatible reactions have a broad range of possible applications in chemical biology and the pharmaceutical sciences. Here we report tetrazines that can be removed by exposure to isonitriles under very mild conditions. Tetrazylmethyl derivatives are easily accessible protecting groups for amines and phenols. The isonitrile-induced removal is rapid and near-quantitative. Intriguingly, the deprotection is especially effective with (trimethylsilyl)methyl isocyanide, and serum albumin can catalyze the elimination under physiological conditions. NMR and computational studies revealed that an imine-tautomerization step is often rate limiting, and the unexpected cleavage of the Si-C bond accelerates this step in the case with (trimethylsilyl)methyl isocyanide. Tetrazylmethyl-removal is compatible with use on biomacromolecules, in cellular environments, and in living organisms as demonstrated by cytotoxicity experiments and fluorophore-release studies on proteins and in zebrafish embryos. By combining tetrazylmethyl derivatives with previously reported tetrazine-responsive 3-isocyanopropyl groups, it was possible to liberate two fluorophores in vertebrates from a single bioorthogonal reaction. This chemistry will open new opportunities towards applications involving multiplexed release schemes and is a valuable asset to the growing toolbox of bioorthogonal dissociative reactions

Heating the hot atmospheres of galaxy groups and clusters with cavities: the relationship between jet power and low-frequency radio emission

We present scaling relations between jet power and radio power measured using the Giant Metrewave Radio Telescope (GMRT), Chandra and XMM-Newton, for a sample of 9 galaxy groups combined with the Birzan et al. sample of clusters. Cavity power is used as a proxy for mechanical jet power. Radio power is measured at 235 MHz and 1.4 GHz, and the integrated 10 MHz-10 GHz radio luminosity is estimated from the GMRT 610-235 MHz spectral index. The use of consistently analysed, high resolution low-frequency radio data from a single observatory makes the radio powers for the groups more reliable than those used by previous studies, and the combined sample covers 6-7 decades in radio power and 5 decades in cavity power. We find a relation of the form Pjet proportional to Lradio^~0.7 for integrated radio luminosity, with a total scatter of sigma_Lrad=0.63 and an intrinsic scatter of sigma_i,Lrad=0.59. A similar relation is found for 235 MHz power, but a slightly flatter relation with greater scatter is found for 1.4 GHz power, suggesting that low-frequency or broad band radio measurements are superior jet power indicators. We find our low-frequency relations to be in good agreement with previous observational results. Comparison with jet models shows reasonable agreement, which may be improved if radio sources have a significant low-energy electron population. We consider possible factors which could bias our results or render them more uncertain, and find that correcting for such factors in those groups we are able to study in detail leads to a flattening of the Pjet:Lradio relation.Comment: Accepted for publication in ApJ, 7 pages, 3 figure

Zebrafish behavioural profiling identifies GABA and serotonin receptor ligands related to sedation and paradoxical excitation.

Anesthetics are generally associated with sedation, but some anesthetics can also increase brain and motor activity-a phenomenon known as paradoxical excitation. Previous studies have identified GABAA receptors as the primary targets of most anesthetic drugs, but how these compounds produce paradoxical excitation is poorly understood. To identify and understand such compounds, we applied a behavior-based drug profiling approach. Here, we show that a subset of central nervous system depressants cause paradoxical excitation in zebrafish. Using this behavior as a readout, we screened thousands of compounds and identified dozens of hits that caused paradoxical excitation. Many hit compounds modulated human GABAA receptors, while others appeared to modulate different neuronal targets, including the human serotonin-6 receptor. Ligands at these receptors generally decreased neuronal activity, but paradoxically increased activity in the caudal hindbrain. Together, these studies identify ligands, targets, and neurons affecting sedation and paradoxical excitation in vivo in zebrafish

Rapid Mutation of Endogenous Zebrafish Genes Using Zinc Finger Nucleases Made by Oligomerized Pool ENgineering (OPEN)

Background: Customized zinc finger nucleases (ZFNs) form the basis of a broadly applicable tool for highly efficient genome modification. ZFNs are artificial restriction endonucleases consisting of a non-specific nuclease domain fused to a zinc finger array which can be engineered to recognize specific DNA sequences of interest. Recent proof-of-principle experiments have shown that targeted knockout mutations can be efficiently generated in endogenous zebrafish genes via non-homologous end-joining-mediated repair of ZFN-induced DNA double-stranded breaks. The Zinc Finger Consortium, a group of academic laboratories committed to the development of engineered zinc finger technology, recently described the first rapid, highly effective, and publicly available method for engineering zinc finger arrays. The Consortium has previously used this new method (known as OPEN for Oligomerized Pool ENgineering) to generate high quality ZFN pairs that function in human and plant cells. Methodology/Principal Findings: Here we show that OPEN can also be used to generate ZFNs that function efficiently in zebrafish. Using OPEN, we successfully engineered ZFN pairs for five endogenous zebrafish genes: tfr2, dopamine transporter, telomerase, hif1aa, and gridlock. Each of these ZFN pairs induces targeted insertions and deletions with high efficiency at its endogenous gene target in somatic zebrafish cells. In addition, these mutations are transmitted through th

Heritable and Precise Zebrafish Genome Editing Using a CRISPR-Cas System

We have previously reported a simple and customizable CRISPR (clustered regularly interspaced short palindromic repeats) RNA-guided Cas9 nuclease (RGN) system that can be used to efficiently and robustly introduce somatic indel mutations in endogenous zebrafish genes. Here we demonstrate that RGN-induced mutations are heritable, with efficiencies of germline transmission reaching as high as 100%. In addition, we extend the power of the RGN system by showing that these nucleases can be used with single-stranded oligodeoxynucleotides (ssODNs) to create precise intended sequence modifications, including single nucleotide substitutions. Finally, we describe and validate simple strategies that improve the targeting range of RGNs from 1 in every 128 basepairs (bps) of random DNA sequence to 1 in every 8 bps. Together, these advances expand the utility of the CRISPR-Cas system in the zebrafish beyond somatic indel formation to heritable and precise genome modifications

Efficient genome editing in zebrafish using a CRISPR-Cas system

In bacteria, foreign nucleic acids are silenced by clustered, regularly interspaced, short palindromic repeats (CRISPR)--CRISPR-associated (Cas) systems. Bacterial type II CRISPR systems have been adapted to create guide RNAs that direct site-specific DNA cleavage by the Cas9 endonuclease in cultured cells. Here we show that the CRISPR-Cas system functions in vivo to induce targeted genetic modifications in zebrafish embryos with efficiencies similar to those obtained using zinc finger nucleases and transcription activator-like effector nucleases