The Tol2-mediated Gal4-UAS method for gene and enhancer trapping in zebrafish

The Gal4-UAS system provides powerful tools to analyze the function of genes and cells in vivo and has been extensively employed in Drosophila. The usefulness of this approach relies on the P element-mediated Gal4 enhancer trapping, which can efficiently generate transgenic fly lines expressing Gal4 in specific cells. Similar approaches, however, had not been developed in vertebrate systems due to the lack of an efficient transgenesis method. We have been developing transposon techniques by using the madaka fish Tol2 element. Taking advantage of its ability to generate genome-wide insertions, we developed the Gal4 gene trap and enhancer trap methods in zebrafish that enabled us to create various transgenic fish expressing Gal4 in specific cells. The Gal4-expressing cells can be visualized and manipulated in vivo by crossing the transgenic Gal4 lines with transgenic lines carrying various reporter and effector genes downstream of UAS (upstream activating sequence). Thus, the Gal4 gene trap and enhancer trap methods together with UAS lines now make detailed analyses of genes and cells in zebrafish feasible. Here, we describe the protocols to perform Gal4 gene trap and enhancer trap screens in zebrafish and their application to the studies of vertebrate neural circuits.     

Targeted gene expression by the Gal4-UAS system in zebrafish

Targeted gene expression by the Gal4-UAS system is a powerful methodology for analyzing function of genes and cells in vivo and has been extensively used in genetic studies in Drosophila . On the other hand, the Gal4-UAS system had not been applied effectively to vertebrate systems for a long time mainly due to the lack of an efficient transgenesis method. Recently, a highly efficient transgenesis method using the medaka fish Tol2 transposable element was developed in zebrafish. Taking advantage of the Tol2 transposon system, we and other groups developed the Gal4 gene trap and enhancer trap methods and established various transgenic fish expressing Gal4 in specific cells. By crossing such Gal4 lines with transgenic fish lines harboring various reporter genes and effector genes downstream of UAS (upstream activating sequence), specific cells can be visualized and manipulated in vivo by targeted gene expression. Thus, the Gal4 gene trap and enhancer trap approaches together with various UAS lines should be important tools for investigating roles of genes and cells in vertebrates.     

Characterization of transgenic zebrafish lines that express GFP in the retina,pineal gland,olfactory bulb,hatching gland,and optic tectum

Transgenic animals are powerful tools to study gene function invivo. Here we characterize several transgenic zebrafish lines that express green fluorescent protein (GFP) under the control of the LCR^RH2-RH2-1 or LCR^RH2-RH2-2 green opsin regulatory elements. Using confocal immunomicroscopy, stereo-fluorescence microscopy, and Western blotting, we show that the Tg(LCR^RH2-RH2-1:GFP)^pt112 and Tg(LCR^RH2-RH2-2:GFP)^pt115 transgenic zebrafish lines express GFP in the pineal gland and certain types of photoreceptors. In addition, some of these lines also express GFP in the hatching gland, optic tectum, or olfactory bulb. Some of the expression patterns differ significantly from previously published similar transgenic fish lines, making them useful tools for studying the development of the corresponding tissues and organs. In addition, the variations of GFP expression among different lines corroborate the notion that transgenic expression is often subjected to position effect, thus emphasizing the need for careful verification of expression patterns when transgenic animal models are utilized for research.     

The effect of excess expression of GFP in a novel heart-specific green fluorescence zebrafish regulated by nppa enhancer at early embryonic development

In order to study the impalpable effect of GFP in homozygous heart-specific GFP-positive zebrafish during the early stage, the researchers analyzed the heart function of morphology and physiology at the first 3 days after fertilization. This zebrafish line was produced by a large-scale Tol2 transposon mediated enhancer trap screen that generated a transgenic zebrafish with a heart-specific expression of green fluorescent protein (GFP)-tagged under control of the nppa enhancer. In situ hybridization experiments showed that the nppa:GFP line faithfully recapitulated both the spatial and temporal expressions of the endogenous nppa. Green fluorescence was intensively and specifically expressed in the myocardial cells located both in the heart chambers and in the atrioventricular canal. The embryonic heart of nppa:GFP line developed normally compared with those in the wild type. There was no difference between the nappa:GFP and wild type lines with respect to heart rate, overall size, ejection volume, and fractional shortening. Thus the excess expression of GFP in this transgenic line seemed to exert no detrimental effects on zebrafish hearts during the early stages.     

Development and evaluation of a Gal4-mediated LUC/GFP/GUS enhancer trap system in Arabidopsis

Background  

Gal4 enhancer trap systems driving expression of LacZ and GFP reporters have been characterized and widely used in Drosophila. However, a Gal4 enhancer trap system in Arabidopsis has not been described in the primary literature. In Drosophila, the reporters possess a Gal4 upstream activation sequence (UAS) as five repeats (5XUAS) and lines that express Gal4 from tissue specific enhancers have also been used for the ectopic expression of any transgene (driven by a 5XUAS). While Gal4 transactivation has been demonstrated in Arabidopsis, wide use of a trap has not emerged in part because of the lack of detailed analysis, which is the purpose of the present study.     

The Annotation of Zebrafish Enhancer Trap Lines Generated with PB Transposon

An enhancer trap (ET) mediated by a transposon is an effective method for functional gene research. Here, an ET system based on a PB transposon that carries a mini Krt4 promoter (the keratin4 minimal promoter from zebrafish) and the green fluorescent protein gene (GFP) has been used to produce zebrafish ET lines. One enhancer trap line with eye-specific expression GFP named EYE was used to identify the trapped enhancers and genes. Firstly, GFP showed a temporal and spatial expression pattern with whole-embryo expression at 6, 12, and 24 hpf stages and eye-specific expression from 2 to 7 dpf. Then, the genome insertion sites were detected by splinkerette PCR (spPCR). The Krt4-GFP was inserted into the fourth intron of the gene itgav (integrin, alpha V) in chromosome 9 of the zebrafish genome, with the GFP direction the same as that of the itgav gene. By the alignment of homologous gene sequences in different species, three predicted endogenous enhancers were obtained. The trapped endogenous gene itgav, whose overexpression is related to hepatocellular carcinoma, showed a similar expression pattern as GFP detected by in situ hybridization, which suggested that GFP and itgav were possibly regulated by the same enhancers. In short, the zebrafish enhancer trap lines generated by the PB transposon-mediated enhancer trap technology in this study were valuable resources as visual markers to study the regulators and genes. This work provides an efficient method to identify and isolate tissue-specific enhancer sequences.     

Visualization of monoaminergic neurons and neurotoxicity of MPTP in live transgenic zebrafish

We describe an enhancer trap transgenic zebrafish line, ETvmat2:GFP, in which most monoaminergic neurons are labeled by green fluorescent protein (GFP) during embryonic development. The reporter gene of ETvmat2:GFP was inserted into the second intron of vesicular monoamine transporter 2 (vmat2) gene, and the GFP expression pattern recapitulates that of the vmat2 gene. The GFP positive neurons include the large and pear-shaped tyrosine hydroxylase positive neurons (TH populations 2 and 4) in the posterior tuberculum of ventral diencephalon (PT neurons), which are thought to be equivalent to the midbrain dopamine neurons in mammals. We found that these PT neurons and two other GFP labeled non-TH type neuronal groups, one in the paraventricular organ of the posterior tuberculum and the other in the hypothalamus, were significantly reduced after exposure to MPTP, while the rest of GFP-positive neuronal clusters, including those in telencephalon, pretectum, raphe nuclei and locus coeruleus, remain largely unchanged. Furthermore, we showed that the effects of hedgehog signaling pathway inhibition on the development of monoaminergic neurons can be easily visualized in individual living ETvmat2:GFP embryos. This enhancer trap line should be useful for genetic and pharmacological analyses of monoaminergic neuron development and processes underlying Parkinson's disease.     

Tracking Cells in GFP-transgenic Zebrafish Using the Photoconvertible PSmOrange System

The rapid development of transparent zebrafish embryos (Danio rerio) in combination with fluorescent labelings of cells and tissues allows visualizing developmental processes as they happen in the living animal. Cells of interest can be labeled by using a tissue specific promoter to drive the expression of a fluorescent protein (FP) for the generation of transgenic lines. Using fluorescent photoconvertible proteins for this purpose additionally allows to precisely follow defined structures within the expression domain. Illuminating the protein in the region of interest, changes its emission spectrum and highlights a particular cell or cell cluster leaving other transgenic cells in their original color. A major limitation is the lack of known promoters for a large number of tissues in the zebrafish. Conversely, gene- and enhancer trap screens have generated enormous transgenic resources discretely labeling literally all embryonic structures mostly with GFP or to a lesser extend red or yellow FPs. An approach to follow defined structures in such transgenic backgrounds would be to additionally introduce a ubiquitous photoconvertible protein, which could be converted in the cell(s) of interest. However, the photoconvertible proteins available involve a green and/or less frequently a red emission state¹ and can therefore often not be used to track cells in the FP-background of existing transgenic lines. To circumvent this problem, we have established the PSmOrange system for the zebrafish^2,3. Simple microinjection of synthetic mRNA encoding a nuclear form of this protein labels all cell nuclei with orange/red fluorescence. Upon targeted photoconversion of the protein, it switches its emission spectrum to far red. The quantum efficiency and stability of the protein makes PSmOrange a superb cell-tracking tool for zebrafish and possibly other teleost species.     

Conditional UAS-targeted repression in Drosophila

The Gal4–UAS enhancer trap system is useful for driving gene expression in various tissues. A new tool that extends Gal4 technology is described here. A fusion protein containing the Gal4 binding domain and the repression domain of the isolator suppressor of hairy wing was placed under the control of a heat shock-inducible promoter. The construct mediates the conditional repression of genes located downstream of a UAS sequence. The repressive effects of the chimeric protein on fasII gene expression were tested by western-blot analysis and in brain sections of adult Drosophila. Owing to the increasing number of Gal4 and UAS transgenic lines, this versatile system will facilitate the study of gene function.     

Hedgehog-PKA Signaling and gnrh3 Regulate the Development of Zebrafish gnrh3 Neurons

GnRH neurons secrete GnRH that controls the development of the reproduction system. Despite many studies, the signals controlling the development of GnRH neurons from its progenitors have not been fully established. To understand the development of GnRH neurons, we examined the development of gnrh3-expressing cells using a transgenic zebrafish line that expresses green fluorescent protein (GFP) and LacZ driven by the gnrh3 promoter. GFP and LacZ expression recapitulated that of gnrh3 in the olfactory region, olfactory bulb and telencephalon. Depletion of gnrh3 by morpholinos led to a reduction of GFP- and gnrh3-expressing cells, while over-expression of gnrh3 mRNA increased the number of these cells. This result indicates a positive feed-forward regulation of gnrh3 cells by gnrh3. The gnrh3 cells were absent in embryos that lack Hedgehog signaling, but their numbers were increased in embryos overexpressing shhb. We manipulated the amounts of kinase that antagonizes the Hedgehog signaling pathway, protein kinase A (PKA), by treating embryos with PKA activator forskolin or by injecting mRNAs encoding its constitutively active catalytic subunit (PKA*) and dominant negative regulatory subunit (PKI) into zebrafish embryos. PKA* misexpression or forskolin treatment decreased GFP cell numbers, while PKI misexpression led to ectopic production of GFP cells. Our data indicate that the Hedgehog-PKA pathway participates in the development of gnrh3-expressing neurons during embryogenesis.     

Live Imaging of Innate Immune and Preneoplastic Cell Interactions Using an Inducible Gal4/UAS Expression System in Larval Zebrafish Skin

Here we describe a method to conditionally induce epithelial cell transformation by the use of the 4-Hydroxytamoxifen (4-OHT) inducible KalTA4-ER^T2/UAS expression system¹ in zebrafish larvae, and the subsequent live imaging of innate immune cell interaction with HRAS^G12V expressing skin cells. The KalTA4-ER^T2/UAS system is both inducible and reversible which allows us to induce cell transformation with precise temporal/spatial resolution in vivo. This provides us with a unique opportunity to live image how individual preneoplastic cells interact with host tissues as soon as they emerge, then follow their progression as well as regression. Recent studies in zebrafish larvae have shown a trophic function of innate immunity in the earliest stages of tumorigenesis^2,3. Our inducible system would allow us to live image the onset of cellular transformation and the subsequent host response, which may lead to important insights on the underlying mechanisms for the regulation of oncogenic trophic inflammatory responses. We also discuss how one might adapt our protocol to achieve temporal and spatial control of ectopic gene expression in any tissue of interest.     

<Emphasis Type="Italic">In silico</Emphasis> and <Emphasis Type="Italic">in situ</Emphasis> characterization of the zebrafish (<Emphasis Type="Italic">Danio rerio</Emphasis>) <Emphasis Type="Italic">gnrh3</Emphasis> (sGnRH) gene

Background

Gonadotropin releasing hormone (GnRH) is responsible for stimulation of gonadotropic hormone (GtH) in the hypothalamus-pituitary-gonadal axis (HPG). The regulatory mechanisms responsible for brain specificity make the promoter attractive for in silico analysis and reporter gene studies in zebrafish (Danio rerio).

Results

We have characterized a zebrafish [Trp⁷, Leu⁸] or salmon (s) GnRH variant, gnrh 3. The gene includes a 1.6 Kb upstream regulatory region and displays the conserved structure of 4 exons and 3 introns, as seen in other species. An in silico defined enhancer at -976 in the zebrafish promoter, containing adjacent binding sites for Oct-1, CREB and Sp1, was predicted in 2 mammalian and 5 teleost GnRH promoters. Reporter gene studies confirmed the importance of this enhancer for cell specific expression in zebrafish. Interestingly the promoter of human GnRH-I, known as mammalian GnRH (mGnRH), was shown capable of driving cell specific reporter gene expression in transgenic zebrafish.

Conclusions

The characterized zebrafish Gnrh3 decapeptide exhibits complete homology to the Atlantic salmon (Salmo salar) GnRH-III variant. In silico analysis of mammalian and teleost GnRH promoters revealed a conserved enhancer possessing binding sites for Oct-1, CREB and Sp1. Transgenic and transient reporter gene expression in zebrafish larvae, confirmed the importance of the in silico defined zebrafish enhancer at -976. The capability of the human GnRH-I promoter of directing cell specific reporter gene expression in zebrafish supports orthology between GnRH-I and GnRH-III.

     

Enhancer trap infidelity in Drosophila optomotor-blind

Reporter gene activity in enhancer trap lines is often implicitly assumed to mirror quite faithfully the endogenous expression of the “trapped” gene, even though there are numerous examples of enhancer trap infidelity. optomotor-blind (omb) is a 160 kb gene in which 16 independent P-element enhancer trap insertions of three different types have been mapped in a range of more than 60 kb. We have determined the expression pattern of these elements in wing, eye-antennal and leg imaginal discs as well as in the pupal tergites. We noted that one pGawB insertion (omb^P4) selectively failed to report parts of the omb pattern even though the missing pattern elements were apparent in all other 15 lines. We ruled out that omb^P4 was defective in the Gal4 promoter region or had inactivated genomic enhancers in the integration process. We propose that the Gal4 reporter gene in pGawB may be sensitive to orientation or promoter proximity effects.     

Green fluorescent protein marks skeletal muscle in murine cell lines and zebrafish

The green fluorescent protein (GFP) acts as a vital dye upon the absorption of blue light. When the gfp gene is expressed in bacteria, flies or nematodes, green fluorescence can be directly observed in the living organism. We inserted the cDNA encoding this 238-amino-acid (aa) jellyfish protein into an expression vector containing the rat myosin light-chain enhancer (MLC-GFP) to evaluate its ability to serve as a muscle-specific marker. Transiently, as well as stably, transfected C2C12 cell lines produced high levels of GFP distributed homogeneously throughout the cytoplasm and was not toxic through several cell passages. Expression of MLC-GFP was strictly muscle-specific, since Cos 7 fibroblasts transfected with MLC-GFP did not fluoresce. When GFP and βGal markers were compared, the GFP signal was visible in the cytoplasm of the living cell, whereas visualization of βGal required fixation and resulted in deformation of the cells. When the MLC-GFP construct was injected into zebrafish embryos, muscle-specific gfp expression was apparent within 24 h of development, gfp expression was never observed in non-muscle tissues using the MLC-GFP construct. Transgenic fish continued to express high levels of gfp in skeletal muscle at 1.5 months, demonstrating that GFP is an effective marker of muscle cells in vivo.