Clemens Grabher

The Tol2kit: a multisite gateway-based construction kit for Tol2 transposon transgenesis constructs.

Transgenesis is an important tool for assessing gene function. In zebrafish, transgenesis has suffered from three problems: the labor of building complex expression constructs using conventional subcloning; low transgenesis efficiency, leading to mosaicism in transient transgenics and infrequent germline incorporation; and difficulty in identifying germline integrations unless using a fluorescent marker transgene. The Tol2kit system uses site‐specific recombination‐based cloning (multisite Gateway technology) to allow quick, modular assembly of [promoter]–[coding sequence]–[3′ tag] constructs in a Tol2 transposon backbone. It includes a destination vector with a cmlc2:EGFP (enhanced green fluorescent protein) transgenesis marker and a variety of widely useful entry clones, including hsp70 and beta‐actin promoters; cytoplasmic, nuclear, and membrane‐localized fluorescent proteins; and internal ribosome entry sequence–driven EGFP cassettes for bicistronic expression. The Tol2kit greatly facilitates zebrafish transgenesis, simplifies the sharing of clones, and enables large‐scale projects testing the functions of libraries of regulatory or coding sequences. Developmental Dynamics 236:3088–3099, 2007.

A tissue-scale gradient of hydrogen peroxide mediates rapid wound detection in zebrafish

Barrier structures (for example, epithelia around tissues and plasma membranes around cells) are required for internal homeostasis and protection from pathogens. Wound detection and healing represent a dormant morphogenetic program that can be rapidly executed to restore barrier integrity and tissue homeostasis. In animals, initial steps include recruitment of leukocytes to the site of injury across distances of hundreds of micrometres within minutes of wounding. The spatial signals that direct this immediate tissue response are unknown. Owing to their fast diffusion and versatile biological activities, reactive oxygen species, including hydrogen peroxide (H2O2), are interesting candidates for wound-to-leukocyte signalling. Here we probe the role of H2O2 during the early events of wound responses in zebrafish larvae expressing a genetically encoded H2O2 sensor. This reporter revealed a sustained rise in H2O2 concentration at the wound margin, starting ∼3 min after wounding and peaking at ∼20 min, which extended ∼100–200 μm into the tail-fin epithelium as a decreasing concentration gradient. Using pharmacological and genetic inhibition, we show that this gradient is created by dual oxidase (Duox), and that it is required for rapid recruitment of leukocytes to the wound. This is the first observation, to our knowledge, of a tissue-scale H2O2 pattern, and the first evidence that H2O2 signals to leukocytes in tissues, in addition to its known antiseptic role.

I-SceI meganuclease mediates highly efficient transgenesis in fish

The widespread use of fish as model systems is still limited by the mosaic distribution of cells transiently expressing transgenes leading to a low frequency of transgenic fish. Here we present a strategy that overcomes this problem. Transgenes of interest were flanked by two I-SceI meganuclease recognition sites, and co-injected together with the I-SceI meganuclease enzyme into medaka embryos (Oryzias latipes) at the one-cell stage. First, the promoter dependent expression was strongly enhanced. Already in F0, 76% of the embryos exhibited uniform promoter dependent expression compared to 26% when injections were performed without meganuclease. Second, the transgenesis frequency was raised to 30.5%. Even more striking was the increase in the germline transmission rate. Whereas in standard protocols it does not exceed a few percent, the number of transgenic F1 offspring of an identified founder fish reached the optimum of 50% in most lines resulting from meganuclease co-injection. Southern blot analysis showed that the individual integration loci contain only one or few copies of the transgene in tandem. At a lower rate this method also leads to enhancer trapping effects, novel patterns that are likely due to the integration of the transgene in the vicinity of enhancer elements. Meganuclease co-injection thus provides a simple and highly efficient tool to improve transgenesis by microinjection.

Notch 1 activation in the molecular pathogenesis of T-cell acute lymphoblastic leukaemia.

The chromosomal translocation t(7;9) in human T-cell acute lymphoblastic leukaemia (T-ALL) results in deregulated expression of a truncated, activated form of Notch 1 (TAN1) under the control of the T-cell receptor-β (TCRB) locus. Although TAN1 efficiently induces T-ALL in mouse models, t(7;9) is present in less than 1% of human T-ALL cases. The recent discovery of novel activating mutations in NOTCH1 in more than 50% of human T-ALL samples has made it clear that Notch 1 is far more important in human T-ALL pathogenesis than previously suspected.

A systematic genome-wide screen for mutations affecting organogenesis in Medaka, Oryzias latipes.

A large-scale mutagenesis screen was performed in Medaka to identify genes acting in diverse developmental processes. Mutations were identified in homozygous F3 progeny derived from ENU-treated founder males. In addition to the morphological inspection of live embryos, other approaches were used to detect abnormalities in organogenesis and in specific cellular processes, including germ cell migration, nerve tract formation, sensory organ differentiation and DNA repair. Among 2031 embryonic lethal mutations identified, 312 causing defects in organogenesis were selected for further analyses. From these, 126 mutations were characterized genetically and assigned to 105 genes. The similarity of the development of Medaka and zebrafish facilitated the comparison of mutant phenotypes, which indicated that many mutations in Medaka cause unique phenotypes so far unrecorded in zebrafish. Even when mutations of the two fish species cause a similar phenotype such as one-eyed-pinhead or parachute, more genes were found in Medaka than in zebrafish that produced the same phenotype when mutated. These observations suggest that many Medaka mutants represent new genes and, therefore, are important complements to the collection of zebrafish mutants that have proven so valuable for exploring genomic function in development.

HyPer-3: A Genetically Encoded H 2 O 2 Probe with Improved Performance for Ratiometric and Fluorescence Lifetime Imaging

High-performance sensors for reactive oxygen species are instrumental to monitor dynamic events in cells and organisms. Here, we present HyPer-3, a genetically encoded fluorescent indicator for intracellular H2O2 exhibiting improved performance with respect to response time and speed. HyPer-3 has an expanded dynamic range compared to HyPer and significantly faster oxidation/reduction dynamics compared to HyPer-2. We demonstrate this performance by in vivo imaging of tissue-scale H2O2 gradients in zebrafish larvae. Moreover, HyPer-3 was successfully employed for single-wavelength fluorescent lifetime imaging of H2O2 levels both in vitro and in vivo.

A high-throughput chemically induced inflammation assay in zebrafish

BackgroundStudies on innate immunity have benefited from the introduction of zebrafish as a model system. Transgenic fish expressing fluorescent proteins in leukocyte populations allow direct, quantitative visualization of an inflammatory response in vivo. It has been proposed that this animal model can be used for high-throughput screens aimed at the identification of novel immunomodulatory lead compounds. However, current assays require invasive manipulation of fish individually, thus preventing high-content screening.ResultsHere we show that specific, noninvasive damage to lateral line neuromast cells can induce a robust acute inflammatory response. Exposure of fish larvae to sublethal concentrations of copper sulfate selectively damages the sensory hair cell population inducing infiltration of leukocytes to neuromasts within 20 minutes. Inflammation can be assayed in real time using transgenic fish expressing fluorescent proteins in leukocytes or by histochemical assays in fixed larvae. We demonstrate the usefulness of this method for chemical and genetic screens to detect the effect of immunomodulatory compounds and mutations affecting the leukocyte response. Moreover, we transformed the assay into a high-throughput screening method by using a customized automated imaging and processing system that quantifies the magnitude of the inflammatory reaction.ConclusionsThis approach allows rapid screening of thousands of compounds or mutagenized zebrafish for effects on inflammation and enables the identification of novel players in the regulation of innate immunity and potential lead compounds toward new immunomodulatory therapies. We have called this method the chemically induced inflammation assay, or ChIn assay.See Commentary article: http://www.biomedcentral.com/1741-7007/8/148.

Highly efficient zebrafish transgenesis mediated by the meganuclease I-SceI.

Publisher Summary This chapter focuses on highly efficient zebrafish transgenesis mediated by the meganuclease I-SceI. Fish are excellent candidates for the production of transgenics for two important reasons. First, fish represent the largest and most diverse group of vertebrates and provide an advantageous system for in vivo studies of developmental processes to gain knowledge of gene regulation and the action of gene products in vertebrates. Second, conventional selective breeding of fish for improved growth or other characteristics is a very slow process. Transgenic fish technology has the potential to improve genetic traits such as increased growth potential, disease resistance, improved feed conversion efficiency, or other desirable genetic traits for aquaculture in one generation. The establishment of methods for successful transgenesis is one of the basic criteria for an organism to be referred to as model organism. Several endonucleases (meganucleases) encoded by introns and inteins have been shown to promote homing (lateral transfer) of their respective genetic elements into intron- or inteinless homologous allelic sites. By introducing sitespecific double-strand breaks (DSBs) in intronless alleles, these nucleases create recombinogenic ends that engage in gene conversion, resulting in duplication of the intron. The I-SceI system has been used as a tool in mammalian cells and Drosophila. The meganuclease can also be used in fish for comparative studies of cis-acting regulatory elements and homologous recombination (HR). Meganuclease transgenesis as described in this chapter could be further improved, including insulators upstream and downstream of the DNA of interest to protect the transgene from the influences of heterochromatin and epigenetic control of surrounding genomic sequences.

Neutrophil-Delivered Myeloperoxidase Dampens the Hydrogen Peroxide Burst after Tissue Wounding in Zebrafish

Prompt neutrophil arrival is critical for host defense immediately after injury [1-3]. Following wounding, a hydrogen peroxide (H(2)O(2)) burst generated in injured tissues is the earliest known leukocyte chemoattractant [4]. Generating this tissue-scale H(2)O(2) gradient uses dual oxidase [4] and neutrophils sense H(2)O(2) by a mechanism involving the LYN Src-family kinase [5], but the molecular mechanisms responsible for H(2)O(2) clearance are unknown [6]. Neutrophils carry abundant amounts of myeloperoxidase, an enzyme catalyzing an H(2)O(2)-consuming reaction [7, 8]. We hypothesized that this neutrophil-delivered myeloperoxidase downregulates the high tissue H(2)O(2) concentrations that follow wounding. This was tested in zebrafish using simultaneous fluorophore-based imaging of H(2)O(2) concentrations and leukocytes [4, 9-11] and a new neutrophil-replete but myeloperoxidase-deficient mutant (durif). Leukocyte-depleted zebrafish had an abnormally sustained wound H(2)O(2) burst, indicating that leukocytes themselves were required for H(2)O(2) downregulation. Myeloperoxidase-deficient zebrafish also had abnormally sustained high wound H(2)O(2) concentrations despite similar numbers of arriving neutrophils. A local H(2)O(2)/myeloperoxidase interaction within wound-recruited neutrophils was demonstrated. These data demonstrate that leukocyte-delivered myeloperoxidase cell-autonomously downregulates tissue-generated wound H(2)O(2) gradients in vivo, defining a new requirement for myeloperoxidase during inflammation. Durif provides a new animal model of myeloperoxidase deficiency closely phenocopying the prevalent human disorder [7, 12, 13], offering unique possibilities for investigating its clinical consequences.

Targeting Angiogenesis via a c-Myc/Hypoxia-Inducible Factor-1α–Dependent Pathway in Multiple Myeloma

Bone marrow angiogenesis is associated with multiple myeloma (MM) progression. Here, we report high constitutive hypoxia-inducible factor-1alpha (Hif-1alpha) expression in MM cells, which is associated with oncogenic c-Myc. A drug screen for anti-MM agents that decrease Hif-1alpha and c-Myc levels identified a variety of compounds, including bortezomib, lenalidomide, enzastaurin, and adaphostin. Functionally, based on transient knockdowns and overexpression, our data delineate a c-Myc/Hif-1alpha-dependent pathway mediating vascular endothelial growth factor production and secretion. The antiangiogenic activity of our tool compound, adaphostin, was subsequently shown in a zebrafish model and translated into a preclinical in vitro and in vivo model of MM in the bone marrow milieu. Our data, therefore, identify Hif-1alpha as a novel molecular target in MM and add another facet to anti-MM drug activity.