Screen In Zebrafish Research Articles

High-content screening in zebrafish identifies perfluorooctanesulfonamide as a potent developmental toxicant

Per- and polyfluoroalkyl substances (PFASs) have been used for decades within industrial processes and consumer products, resulting in frequent detection within the environment. Using zebrafish embryos, we screened 38 PFASs for developmental toxicity and revealed that perfluorooctanesulfonamide (PFOSA) was the most potent developmental toxicant, resulting in elevated mortality and developmental abnormalities following exposure from 6 to 24 h post fertilization (hpf) and 6 to 72 hpf. PFOSA resulted in a concentration-dependent increase in mortality and abnormalities, with surviving embryos exhibiting a >12-h delay in development at 24 hpf. Exposures initiated at 0.75 hpf also resulted in a concentration-dependent delay in epiboly, although these effects were not driven by a specific sensitive window of development. We relied on mRNA-sequencing to identify the potential association of PFOSA-induced developmental delays with impacts on the embryonic transcriptome. Relative to stage-matched vehicle controls, these data revealed that pathways related to hepatotoxicity and lipid transport were disrupted in embryos exposed to PFOSA from 0.75 to 14 hpf and 0.75 to 24 hpf. Therefore, we measured liver area as well as neutral lipids in 128-hpf embryos exposed to vehicle (0.1% DMSO) or PFOSA from 0.75 to 24 hpf and clean water from 24 to 128 hpf, and showed that PFOSA exposure from 0.75 to 24 hpf resulted in a decrease in liver area and increase in yolk sac neutral lipids at 128 hpf. Overall, our findings show that early exposure to PFOSA adversely impacts embryogenesis, an effect that may lead to altered lipid transport and liver development.

Characterization of immune response against Mycobacterium marinum infection in the main hematopoietic organ of adult zebrafish (Danio rerio)

Tuberculosis remains a major global health challenge. To gain information about genes important for defense against tuberculosis, we used a well-established tuberculosis model; Mycobacterium marinum infection in adult zebrafish. To characterize the immunological response to mycobacterial infection at 14 days post infection, we performed a whole-genome level transcriptome analysis using cells from kidney, the main hematopoietic organ of adult zebrafish. Among the upregulated genes, those associated with immune signaling and regulation formed the largest category, whereas the largest group of downregulated genes had a metabolic role. We also performed a forward genetic screen in adult zebrafish and identified a fish line with severely impaired survival during chronic mycobacterial infection. Based on transcriptome analysis, these fish have decreased expression of several immunological genes. Taken together, these results give new information about the genes involved in the defense against mycobacterial infection in zebrafish.

Identification of Two Novel Small Compounds that Inhibit Liver Cancer Formation in Zebrafish and Analysis of Their Conjugation to Nanodiamonds to Further Reduce Toxicity

AbstractLiver cancer, which is ranked fourth in cancer‐related mortality worldwide, lacks effective therapeutic treatments. The development of new targeted therapies for liver cancer is urgently needed. The zebrafish is an excellent preclinical model organism for drug screening. Therefore, in a zebrafish model, hundreds of small molecules are screened, and two compounds (LIB1O0078 and LIB1O0144) are identified as the strongest inducers of antiangiogenic effects without side effects. LIB1O0078 exhibits better antiproliferation ability, and LIB1O0144 has better antimigration ability, as shown by xenotransplantation assays. Furthermore, LIB1O0078 and LIB1O0144 exhibit anti‐HCC effects after retro‐orbital injection into adult Tg(fabp10a:HBx,Src, p53‐) triple transgenic fish with obesity and liver cancer. Because of the embryonic toxicity induced by these compounds, they are conjugated with nanodiamonds (ND), which are highly biocompatible function‐based carriers. ND‐coated small molecules not only reduce the embryonic toxicity and hepatotoxicity of the compounds, also exhibit better anti‐HCC effects when administered by oral gavage in adult Tg(fabp10a:HBx,Src, p53‐) fish with obesity and liver cancer. This study integrates nanotechnology and biomedical technology, identifies new potential anticancer drugs, and demonstrates the effectiveness of coating them on nanodiamonds in vivo. Facilitated by such a rapid zebrafish screening system, new anticancer drugs can be identified in a personalized and timely manner.

Activity of lumacaftor is not conserved in zebrafish Cftr bearing the major cystic fibrosis-causing mutation.

F508del‐cystic fibrosis transmembrane conductance regulator (CFTR) is the major mutant responsible for cystic fibrosis (CF). ORKAMBI®, approved for patients bearing this mutant, contains lumacaftor (VX‐809) that partially corrects F508del‐CFTR's processing defect and ivacaftor (VX‐770) that potentiates its defective channel activity. Unfortunately, the clinical efficacy of ORKAMBI® is modest, highlighting the need to understand how the small molecules work so that superior compounds can be developed. Because, human CFTR (hCFTR) and zebrafish Cftr (zCftr) are structurally conserved as determined in recent cryo‐EM structural models, we hypothesized that the consequences of the major mutation and small molecule modulators would be similar for the two species of protein. As expected, like the F508del mutation in hCFTR, the homologous mutation in zCftr (F507del) is misprocessed, yet not as severely as the human mutant and this defect was restored by low‐temperature (27°C) culture conditions. After rescue to the cell surface, F507del‐zCftr exhibited regulated channel activity that was potentiated by ivacaftor. Surprisingly, lumacaftor failed to rescue misprocessing of the F507del‐zCftr at either 37 or 27°C suggesting that future comparative studies with F508del‐hCFTR would provide insight into its structure: function relationships. Interestingly, the robust rescue of F508del‐zCftr at 27°C and availability of methods for in vivo screening in zebrafish present the opportunity to define the cellular pathways underlying rescue.

Phenotypical microRNA screen reveals a noncanonical role of CDK2 in regulating neutrophil migration

Neutrophil migration is essential for inflammatory responses to kill pathogens; however, excessive neutrophilic inflammation also leads to tissue injury and adverse effects. To discover novel therapeutic targets that modulate neutrophil migration, we performed a neutrophil-specific microRNA (miRNA) overexpression screen in zebrafish and identified 8 miRNAs as potent suppressors of neutrophil migration. Among those, miR-199 decreases neutrophil chemotaxis in zebrafish and human neutrophil-like cells. Intriguingly, in terminally differentiated neutrophils, miR-199 alters the cell cycle-related pathways and directly suppresses cyclin-dependent kinase 2 (Cdk2), whose known activity is restricted to cell cycle progression and cell differentiation. Inhibiting Cdk2, but not DNA replication, disrupts cell polarity and chemotaxis of zebrafish neutrophils without inducing cell death. Human neutrophil-like cells deficient in CDK2 fail to polarize and display altered signaling downstream of the formyl peptide receptor. Chemotaxis of primary human neutrophils is also reduced upon CDK2 inhibition. Furthermore, miR-199 overexpression or CDK2 inhibition significantly improves the outcome of lethal systemic inflammation challenges in zebrafish. Our results therefore reveal previously unknown functions of miR-199 and CDK2 in regulating neutrophil migration and provide directions in alleviating systemic inflammation.

Inhibition of COX-2, mPGES-1 and CYP4A by isoliquiritigenin blocks the angiogenic Akt signaling in glioma through ceRNA effect of miR-194-5p and lncRNA NEAT1

BackgroundArachidonic acid (AA) metabolic enzymes including cyclooxygenase-2 (COX-2), microsomal prostaglandin E synthase-1 (mPGES-1) and cytochrome P450 (CYP) 4A11 play important roles in glioma angiogenesis. Thus, there is an urgent need to identify the underlying mechanisms and develop strategies to overcome them.MethodsA homology model of human CYP4A11 was constructed using SYBYL-X 2.0. Structure-based virtual screening against COX-2, mPGES-1 and CYP4A11was performed using the Surflex-Dock of the SYBYL suite. The candidates were further evaluated their antiangiogenic activities in a zebrafish embryo and rabbit corneal angiogenesis model. Laser doppler analysis was used to measure tumor perfusion. The expression of CD31 and α-SMA was measured by immunofluorescence. Western blot was used to measure the expression of HIF-1, Akt and p-Akt. The gene expression of FGF-2, G-CSF, PDGF, TGF-β, Tie-2, VEGF, lncRNA NEAT1 and miR-194-5p were determined using qPCR. The production of FGF-2, TGF-β and VEGF were analyzed using ELISA. Bioinformatic analysis and luciferase reporter assays confirmed the interaction between lncRNA NEAT1 and miR-194-5p.ResultsThe nearly 36,043 compounds from the Traditional Chinese Medicine (TCM) database were screened against COX-2, mPGES-1 and CYP4A11 3D models, and the 17 top flavonoids were identified. In zebrafish screening, isoliquiritigenin (ISL) exhibited the most potent antiangiogenic activities with the EC50 values of 5.9 μM. Conversely, the antiangiogenic effects of ISL in the zebrafish and rabbit corneal models were partly reversed by 20-hydroxyeicosatetraenoic acid (20-HETE) or prostaglandin E2 (PGE2). ISL normalized glioma vasculature and improved the efficacy of temozolomide therapy in the rat C6 glioma model. Inhibition of COX-2, mPGES-1 and CYP4A by ISL decreased FGF-2, TGF-β and VEGF production in the C6 and U87 glioma cells with p-Akt downregulation, which was reversed by Akt overexpression. Furthermore, ISL downregulated lncRNA NEAT1 but upregulated miR-194-5p in the U87 glioma cell. Importantly, lncRNA NEAT1 overexpression reversed ISL-mediated increase in miR-194-5p expression, and thereby attenuated FGF-2, TGF-β and VEGF production.ConclusionsReprogramming COX-2, mPGES-1 and CYP4A mediated-AA metabolism in glioma by flavonoid ISL inhibits the angiogenic Akt- FGF-2/TGF-β/VEGF signaling through ceRNA effect of miR-194-5p and lncRNA NEAT1, and may serve as a novel therapeutic strategy for human glioma.

Therapeutic Silencing of Centromere Protein X Ameliorates Hyperglycemia in Zebrafish and Mouse Models of Type 2 Diabetes Mellitus.

Type 2 diabetes mellitus (T2DM) is characterized by persistent hyperglycemia and is influenced by genetic and environmental factors. Optimum T2DM management involves early diagnosis and effective glucose-lowering therapies. Further research is warranted to improve our understanding of T2DM pathophysiology and reveal potential roles of genetic predisposition. We have previously developed an obesity-induced diabetic zebrafish model that shares common pathological pathways with humans and may be used to identify putative pharmacological targets of diabetes. Additionally, we have previously identified several candidate genes with altered expression in T2DM zebrafish. Here, we performed a small-scale zebrafish screening for these genes and discovered a new therapeutic target, centromere protein X (CENPX), which was further validated in a T2DM mouse model. In zebrafish, cenpx knockdown by morpholino or knockout by CRISPR/Cas9 system ameliorated overfeeding-induced hyperglycemia and upregulated insulin level. In T2DM mice, small-interfering RNA-mediated Cenpx knockdown decreased hyperglycemia and upregulated insulin synthesis in the pancreas. Gene expression analysis revealed insulin, mechanistic target of rapamycin, leptin, and insulin-like growth factor 1 pathway activation following Cenpx silencing in pancreas tissues. Thus, CENPX inhibition exerted antidiabetic effects via increased insulin expression and related pathways. Therefore, T2DM zebrafish may serve as a powerful tool in the discovery of new therapeutic gene targets.

Optimization-by-design of hepatotropic lipid nanoparticles targeting the sodium-taurocholate cotransporting polypeptide.

Active targeting and specific drug delivery to parenchymal liver cells is a promising strategy to treat various liver disorders. Here, we modified synthetic lipid-based nanoparticles with targeting peptides derived from the hepatitis B virus large envelope protein (HBVpreS) to specifically target the sodium-taurocholate cotransporting polypeptide (NTCP; SLC10A1) on the sinusoidal membrane of hepatocytes. Physicochemical properties of targeted nanoparticles were optimized and NTCP-specific, ligand-dependent binding and internalization was confirmed in vitro. The pharmacokinetics and targeting capacity of selected lead formulations was investigated in vivo using the emerging zebrafish screening model. Liposomal nanoparticles modified with 0.25 mol% of a short myristoylated HBV derived peptide, that is Myr-HBVpreS2-31, showed an optimal balance between systemic circulation, avoidance of blood clearance, and targeting capacity. Pronounced liver enrichment, active NTCP-mediated targeting of hepatocytes and efficient cellular internalization were confirmed in mice by 111In gamma scintigraphy and fluorescence microscopy demonstrating the potential use of our hepatotropic, ligand-modified nanoparticles.

Identification of a novel turn-on albumin binding small-molecule bioprobe in live zebrafish and its potential application in drug discovery

A novel small-molecule fluorescent probe (4f) for staining blood in live zebrafish was identified. Compound 4f turns on the fluorescent property in the presence of albumin in the blood and it mainly binds on the site II of human serum albumin. 4f efficiently visualized the blood vessels in live zebrafish, Hence, it could be used as an alternative blood vessel imaging tool. Compound 4f applied to test for anti-cancer drug screening in live zebrafish, and showed a potential as an effective in vivo drug screening agent.

Abstract 1789: The importance of the phosphatase PRL family sub-cellular localization during the cellular cycle

Abstract Acute lymphoblastic leukemia (ALL) is one of the most prevalent cancers diagnosed in adolescents. The survival rate for ALL is good during the initial diagnosis but is much less optimistic for patients who have relapsed ALL, due to limited treatment options. Additionally, the high dose cytotoxic chemotherapies given to ALL patients have long term adverse consequences. New and less toxic targeted therapies are needed for the treatment of ALL. We previously performed a large scale transplantation screen in zebrafish to identify genes associated with high relapse potential in ALL, and a top hit from this screen was the protein tyrosine phosphatase 4A3, PTP4A3 or PRL3. PRL3 overexpression in zebrafish enhances ALL progression and penetrance, and small molecule targeting of PRL3 in human cells rapidly induces apoptosis. The mechanisms through which PRL3 drive ALL progression and survival are unknown. PRL3 has a predicted nuclear localization site as well as a predicted prenylation motif, causing its localization to the plasma membrane. My preliminary data suggest that PRL3 shifts its localization between the plasma membrane and nucleus during the cell cycle. I am currently examining the localization of PRL3 and other PRL family members during different phases of the cell cycle and identifying signaling pathways that they may be involved with at these distinct sites to promote ALL progression, using CLIP-tag and SNAP-tag technologies that will allow me to image directly PRL localization in vitro. Biotin ligase approaches (Turbo-ID) will be used to determine the substrates of PRL activity and PRL interacting partners at these distinct locations. My project will provide scientific data that supports our hypothesis that PRL3 can transition between the membrane and nucleus, and plays a role in cell cycle. Identification of substrates of PRL3 that promote ALL progression may provide novel drug targets for this disease. Citation Format: Anna K. O'Leary, Jessica Blackburn. The importance of the phosphatase PRL family sub-cellular localization during the cellular cycle [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1789.

Orthogonal Drug Pooling Enhances Phenotype-Based Discovery of Ocular Antiangiogenic Drugs in Zebrafish Larvae.

Unbiased screening of large randomized chemical libraries in vivo is a powerful tool to find new drugs and targets. However, forward chemical screens in zebrafish can be time consuming and usually >99% of test compounds have no significant effect on the desired phenotype. Here, we sought to find bioactive drugs more efficiently and to comply with the 3R principles of replacement, reduction, and refinement of animals in research. We investigated if pooling of drugs to simultaneously test 8–10 compounds in zebrafish larvae can increase the screening efficiency of an established assay that identifies drugs inhibiting developmental angiogenesis in the eye. In a phenotype-based screen, we tested 1,760 small molecule compounds from the ChemBridge DIVERSet™ chemical library for their ability to inhibit the formation of distinct primary hyaloid vessels in the eye. Applying orthogonal pooling of the chemical library, we treated zebrafish embryos from 3 to 5 days post fertilization with pools of 8 or 10 compounds at 10 μM each. This reduced the number of tests from 1,760 to 396. In 63% of cases, treatment showed sub-threshold effects of <40% reduction of primary hyaloid vessels. From 18 pool hits, we identified eight compounds that reduce hyaloid vessels in the larval zebrafish eye by at least 40%. Compound 4-[4-(1H-benzimidazol-2-yl)phenoxy]aniline ranked as the most promising candidate with reproducible and dose-dependent effects. To our knowledge, this is the first report of a self-deconvoluting matrix strategy applied to drug screening in zebrafish. We conclude that the orthogonal drug pooling strategy is a cost-effective, time-saving, and unbiased approach to discover novel inhibitors of developmental angiogenesis in the eye. Ultimately, this approach may identify new drugs or targets to mitigate disease caused by pathological angiogenesis in the eye, e.g., diabetic retinopathy or age-related macular degeneration, wherein blood vessel growth and leaky vessels lead to vision impairment or clinical blindness.

Automated in vivo screen in zebrafish identifies Clotrimazole as targeting a metabolic vulnerability in a melanoma model

Therapeutic approaches for cutaneous melanoma are flourishing, but despite promising results, there is an increasing number of reported primary or secondary resistance to the growing sets of drugs approved for therapy in the clinics. Combinatorial approaches may overcome resistance, as they may tackle specific weaknesses of melanoma cells, not sufficient on their own, but effective in combination with other therapies.The transgenic zebrafish line kita:ras develops melanoma with high frequency. At 3 dpf, transgenic kita:ras larvae show a hyperpigmentation phenotype as earliest evidence of abnormal melanocyte growth. Using this model, we performed a chemical screen based on automated detection of a reduction of melanocyte number caused by any of 1280 FDA or EMA approved drugs of the library. The analysis showed that 55 molecules were able to reduce by 60% or more the number of melanocytes per embryo. We further tested two compounds for each of the 5 classes, and a farnesyltransferase inhibitor (Lonafarnib), that inhibits an essential post-translational modification of HRAS and suppresses the hyperpigmentation phenotype. Combinations of Clotrimazole and Lonafarnib showed the most promising results in zebrafish embryos, allowing a dose reduction of both drugs. We performed validation of these observations in the metastatic human melanoma cell line A375M, and in normal human epithelial melanocytes (NHEM) in order to investigate the mechanism of action of Clotrimazole in blocking the proliferation of transformed melanocytes. Viability assay and analysis of energy metabolism in Clotrimazole treated cells show that this drug specifically affects melanoma cells in vitro and transformed melanocytes in vivo, having no effects on NHEM or wild type larvae. Similar effects were observed with another hit of the same class, Miconazole. Furthermore, we show that the effects of Clotrimazole are mediated by the inhibition of hexokinase activity, which is lethal to the abnormal metabolic profile of melanoma cells in vitro and in vivo. Thus, our study shows that the zebrafish can provide a phenotype-rich assay for fully automated screening approaches to identify drugs for synthetic lethal treatment in melanoma and suggest further testing of Clotrimazole in combinatorial treatments.

ZF-Mapper: Simple and Complete Freeware for Fluorescence Quantification in Zebrafish Images.

Zebrafish embryos and larvae have become popular vertebrate models because their body walls are transparent, which enables live imaging of target organs using fluorescent protein transgenes or dye staining. Software packages for the quantification of these fluorescent signals are available from both commercial and noncommercial sources; however, their algorithms are complicated and their resources (code) have mostly not been openly shared. In this study, we developed a simple and robust open-source software tool named "ZF-Mapper" for the quantification of the fluorescence intensity of each pixel in zebrafish images with batch image file processing capability. Using this software, we can evaluate the three-dimensional (3D) distribution of fluorescence intensity among zebrafish cells by analyzing each image pixel. We tested ZF-Mapper for the analysis of zebrafish with macrophage-specific enhanced green fluorescent protein (EGFP) and obtained results that were equivalent to those acquired using the conventional image analysis software ImageJ. We further applied ZF-Mapper to the analysis of zebrafish with cancer cell xenografts and quantified the amount of implanted melanoma cells labeled with a tdTomato red fluorescent protein in the whole body and the tail region. In addition, by combining ZF-Mapper with R freeware, we created an interactive 3D scatter plot of the fluorescence intensities of macrophage-EGFPs in zebrafish. In summary, we developed the Python-based freeware ZF-Mapper for the quantification of fluorescent signals in multiple zebrafish images, which enables fluorescence-based zebrafish screening. We provide the source code and the executable application software for Windows (.exe) and macOS (.app).

Vitamin D Stimulates Cardiomyocyte Proliferation and Controls Organ Size and Regeneration in Zebrafish

Attaining proper organ size during development and regeneration hinges on the activity of mitogenic factors. Here, we performed a large-scale chemical screen in embryonic zebrafish to identify cardiomyocyte mitogens. Although commonly considered anti-proliferative, vitamin D analogs like alfacalcidol had rapid, potent mitogenic effects on embryonic and adult cardiomyocytes invivo. Moreover, pharmacologic or genetic manipulation of vitamin D signaling controlled proliferation in multiple adult cell types and dictated growth rates in embryonic and juvenile zebrafish. Tissue-specific modulation of vitamin D receptor (VDR) signaling had organ-restricted effects, with cardiac VDR activation causing cardiomegaly. Alfacalcidol enhanced the regenerative response of injured zebrafish hearts, whereas VDR blockade inhibited regeneration. Alfacalcidol activated cardiac expression of genes associated with ErbB2 signaling, while ErbB2 inhibition blunted its effects on cell proliferation. Our findings identify vitamin D as mitogenic for cardiomyocytes and other cell types in zebrafish and indicate a mechanism to regulate organ size and regeneration.

Forward Genetic Screen Using Zebrafish to Identify New Genes Involved in Myelination.

Zebrafish are now well established as the preeminent vertebrate model with which to carry out gene discovery/forward genetic screens to identify the molecular genetic basis of biological processes. Gene discovery screens in zebrafish have already provided novel insight into mechanisms of glial cell development and function. The vast majority of genetic screens in zebrafish are based around a three generation screen that starts with the random induction of mutations in adult males using the chemical mutagen ENU. Here we outline the methods that underlie this type of screen, detailing each step, from ENU mutagenesis, through the breeding schemes required to recover homozygous mutant animals in subsequent generations, the screening procedure itself, with a focus on the analysis of myelinating glia, and the subsequent confirmation of mutant phenotypes.

Identifying Toxicant-Interacting Genes Using Forward Genetic Screening in Zebrafish.

Forward genetic screening is an extremely powerful method for identifying novel genes driving a broad range of phenotypes. This protocol describes the complete process for conducting a forward genetic screen in zebrafish, including mutagenesis with N-ethyl-N-nitrosourea (ENU), mating, phenotypic screening, and genetic mapping.

Zebrafish studies identify serotonin receptors mediating antiepileptic activity in Dravet syndrome.

Dravet syndrome is a life-threatening early-onset epilepsy not well controlled by antiepileptic drugs. Drugs that modulate serotonin (5-HT) signalling, including clemizole, locaserin, trazodone and fenfluramine, have recently emerged as potential treatment options for Dravet syndrome. To investigate the serotonin receptors that could moderate this antiepileptic activity, we designed and synthesized 28 novel analogues of clemizole, obtained receptor binding affinity profiles, and performed in vivo screening in a scn1lab mutant zebrafish (Danio rerio) model which recapitulates critical clinical features of Dravet syndrome. We discovered three clemizole analogues with 5-HT receptor binding that exert powerful antiepileptic activity. Based on structure-activity relationships and medicinal chemistry-based analysis, we then screened an additional set of known 5-HT receptor specific drug candidates. Integrating our in vitro and in vivo data implicates 5-HT2B receptors as a critical mediator in the mechanism of seizure suppression observed in Dravet syndrome patients treated with 5-HT modulating drugs.

Endothelial Mitochondrial Preprotein Translocase Tomm7-Rac1 Signaling Axis Dominates Cerebrovascular Network Homeostasis.

Objective- Mitochondria are the important yet most underutilized target for cardio-cerebrovascular function integrity and disorders. The Tom (translocases of outer membrane) complex are the critical determinant of mitochondrial homeostasis for making organs acclimate physiological and pathological insults; however, their roles in the vascular system remain unknown. Approach and Results- A combination of studies in the vascular-specific transgenic zebrafish and genetically engineered mice was conducted. Vascular casting and imaging, endothelial angiogenesis, and mitochondrial protein import were performed to dissect potential mechanisms. A loss-of-function genetic screening in zebrafish identified that selective inactivation of the tomm7 (translocase of outer mitochondrial membrane 7) gene, which encodes a small subunit of the Tom complex, specially impaired cerebrovascular network formation. Ablation of the ortholog Tomm7 in mice recapitulated cerebrovascular abnormalities. Restoration of the cerebrovascular anomaly by an endothelial-specific transgenesis of tomm7 further indicated a defect in endothelial function. Mechanistically, Tomm7 deficit in endothelial cells induced an increased import of Rac1 (Ras-related C3 botulinum toxin substrate 1) protein into mitochondria and facilitated the mitochondrial Rac1-coupled redox signaling, which incurred angiogenic impairment that underlies cerebrovascular network malformation. Conclusions- Tomm7 drives brain angiogenesis and cerebrovascular network formation through modulating mitochondrial Rac1 signaling within the endothelium.

High-throughput Screening in Larval Zebrafish Identifies Novel Potent Sedative-hypnotics.

WHAT THIS ARTICLE TELLS US THAT IS NEW: BACKGROUND:: Many general anesthetics were discovered empirically, but primary screens to find new sedative-hypnotics in drug libraries have not used animals, limiting the types of drugs discovered. The authors hypothesized that a sedative-hypnotic screening approach using zebrafish larvae responses to sensory stimuli would perform comparably to standard assays, and efficiently identify new active compounds. The authors developed a binary outcome photomotor response assay for zebrafish larvae using a computerized system that tracked individual motions of up to 96 animals simultaneously. The assay was validated against tadpole loss of righting reflexes, using sedative-hypnotics of widely varying potencies that affect various molecular targets. A total of 374 representative compounds from a larger library were screened in zebrafish larvae for hypnotic activity at 10 µM. Molecular mechanisms of hits were explored in anesthetic-sensitive ion channels using electrophysiology, or in zebrafish using a specific reversal agent. Zebrafish larvae assays required far less drug, time, and effort than tadpoles. In validation experiments, zebrafish and tadpole screening for hypnotic activity agreed 100% (n = 11; P = 0.002), and potencies were very similar (Pearson correlation, r > 0.999). Two reversible and potent sedative-hypnotics were discovered in the library subset. CMLD003237 (EC50, ~11 µM) weakly modulated γ-aminobutyric acid type A receptors and inhibited neuronal nicotinic receptors. CMLD006025 (EC50, ~13 µM) inhibited both N-methyl-D-aspartate and neuronal nicotinic receptors. Photomotor response assays in zebrafish larvae are a mechanism-independent platform for high-throughput screening to identify novel sedative-hypnotics. The variety of chemotypes producing hypnosis is likely much larger than currently known.

Mutations in dock1 disrupt early Schwann cell development

BackgroundIn the peripheral nervous system (PNS), specialized glial cells called Schwann cells produce myelin, a lipid-rich insulating sheath that surrounds axons and promotes rapid action potential propagation. During development, Schwann cells must undergo extensive cytoskeletal rearrangements in order to become mature, myelinating Schwann cells. The intracellular mechanisms that drive Schwann cell development, myelination, and accompanying cell shape changes are poorly understood.MethodsThrough a forward genetic screen in zebrafish, we identified a mutation in the atypical guanine nucleotide exchange factor, dock1, that results in decreased myelination of peripheral axons. Rescue experiments and complementation tests with newly engineered alleles confirmed that mutations in dock1 cause defects in myelination of the PNS. Whole mount in situ hybridization, transmission electron microscopy, and live imaging were used to fully define mutant phenotypes.ResultsWe show that Schwann cells in dock1 mutants can appropriately migrate and are not decreased in number, but exhibit delayed radial sorting and decreased myelination during early stages of development.ConclusionsTogether, our results demonstrate that mutations in dock1 result in defects in Schwann cell development and myelination. Specifically, loss of dock1 delays radial sorting and myelination of peripheral axons in zebrafish.