Combination of the ALCR/alcA ethanol switch and GAL4/VP16-UAS enhancer trap system enables spatial and temporal control of transgene expression in Arabidopsis

The experimental control of gene expression in specific tissues or cells at defined time points is a useful tool for the analysis of gene function. GAL4/VP16-UAS enhancer trap lines can be used to selectively express genes in specific tissues or cells, and an ethanol-inducible system can help to control the time of expression. In this study, the combination of the two methods allowed the successful regulation of gene expression in both time and space. For this purpose, a binary vector, 962-UAS::GUS, was constructed in which the ALCR activator and β-glucuronidase (GUS) reporter gene were placed under the control of upstream activator sequence (UAS) elements and the alcA response element, respectively. Three different GAL4/VP16-UAS enhancer trap lines of Arabidopsis were transformed, resulting in transgenic plants in which GUS activity was detected only on ethanol induction and exclusively in the predicted tissues of the enhancer trap lines. As a library of different enhancer trap lines with distinct green fluorescent protein (GFP) patterns exist, transformation with a similar vector, in which GUS is replaced by another gene, would enable the control of the time and place of transgene expression. We have constructed two vectors for easy cloning of the gene of interest, one with a polylinker site and one that is compatible with the GATEWAY™ vector conversion system. The method can be extended to other species when enhancer trap lines become available.     

A versatile Multisite Gateway‐compatible promoter and transgenic line collection for cell type‐specific functional genomics in Arabidopsis

Multicellular organisms are composed of many cell types that acquire their specific fate through a precisely controlled pattern of gene expression in time and space dictated in part by cell type‐specific promoter activity. Understanding the contribution of highly specialized cell types in the development of a whole organism requires the ability to isolate or analyze different cell types separately. We have characterized and validated a large collection of root cell type‐specific promoters and have generated cell type‐specific marker lines. These benchmarked promoters can be readily used to evaluate cell type‐specific complementation of mutant phenotypes, or to knockdown gene expression using targeted expression of artificial miRNA. We also generated vectors and characterized transgenic lines for cell type‐specific induction of gene expression and cell type‐specific isolation of nuclei for RNA and chromatin profiling. Vectors and seeds from transgenic Arabidopsis plants will be freely available, and will promote rapid progress in cell type‐specific functional genomics. We demonstrate the power of this promoter set for analysis of complex biological processes by investigating the contribution of root cell types in the IRT1‐dependent root iron uptake. Our findings revealed the complex spatial expression pattern of IRT1 in both root epidermis and phloem companion cells and the requirement for IRT1 to be expressed in both cell types for proper iron homeostasis.     

Genetic analysis of the Drosophila ellipsoid body neuropil: Organization and development of the central complex

The central complex is an important center for higher‐order brain function in insects. It is an intricate neuropil composed of four substructures. Each substructure contains repeated neuronal elements which are connected by processes such that topography is maintained. Although the neuronal architecture has been described in several insects and the behavioral role investigated in various experiments, the exact function of this neuropil has proven elusive. To describe the architecture of the central complex, we study 15 enhancer‐trap lines that label various ellipsoid body neuron types. We find evidence for restriction of gene expression that is correlated with specific neuronal types: such correlations suggest functional classifications as well. We show that some enhancer‐trap patterns reveal a single ellipsoid body neuron type, while others label multiple types. We describe the development of the ellipsoid body neuropil in wild‐type animals and propose developmental mechanisms based on animals displaying structural mutations of this neuropil. The experiments performed here demonstrate the degree of resolution possible from the analysis of enhancer‐trap lines and form a useful library of tools for future structure/function studies of the ellipsoid body. © 1999 John Wiley & Sons, Inc. J Neurobiol 41: 189–207, 1999     

Functional independence of circadian clocks that regulate plant gene expression

BACKGROUND: Circadian clocks regulate the gene expression, metabolism and behaviour of most eukaryotes, controlling an orderly succession of physiological processes that are synchronised with the environmental day/night cycle. Central circadian pacemakers that control animal behaviour are located in the brains of insects and rodents, but the location of such a pacemaker has not been determined in plants. Peripheral plant and animal tissues also maintain circadian rhythms when isolated in culture, indicating that these tissues contain circadian clocks. The degree of autonomy that the multiple, peripheral circadian clocks have in the intact organism is unclear. RESULTS: We used the bioluminescent luciferase reporter gene to monitor rhythmic expression from three promoters in transgenic Arabidopsis and tobacco plants. The rhythmic expression of a single gene could be set at up to three phases in different anatomical locations of a single plant, by applying light/dark treatments to restricted tissue areas. The initial phases were stably maintained after the entraining treatments ended, indicating that the circadian oscillators in intact plants are autonomous. This result held for all the vegetative plant organs and for promoters expressed in all major cell types. The rhythms of one organ were unaffected by entrainment of the rest of the plant, indicating that phase-resetting signals are also autonomous. CONCLUSIONS: Higher plants contain a spatial array of autonomous circadian clocks that regulate gene expression without a localised pacemaker. Circadian timing in plants might be less accurate but more flexible than the vertebrate circadian system.     

What's new: Conditional cell ablation in Drosophila

Targeting of cell ablation agents under the control of tissue-specific promoters promises to be an important tool for studies of development and function in higher organisms. Temperature-sensitive cell ablation agents, recently developed for Drosophila, extend control to temporal as well as spatial aspects of toxin expression. Here we discuss achievements to date, together with a novel form of enhancer trap technology with the potential for driving toxin expression in a large range of cell types.     

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.     

Tissue‐specific inactivation by cytosine deaminase/uracil phosphoribosyl transferase as a tool to study plant biology

Recent advances in the study of plant developmental and physiological responses have benefited from tissue‐specific approaches, revealing the role of some cell types in these processes. Such approaches have relied on the inactivation of target cells using either toxic compounds or deleterious genes; however, both tissue‐specific and truly inducible tools are lacking in order to precisely target a developmental window or specific growth response. We engineered the yeast fluorocytosine deaminase (FCY1) gene by creating a fusion with the bacterial uracil phosphoribosyl transferase (UPP) gene. The recombinant protein converts the precursor 5‐fluorocytosine (5‐FC) into 5‐fluorouracyl, a drug used in the treatment of a range of cancers, which triggers DNA and RNA damage. We expressed the FCY‐UPP gene construct in specific cell types using enhancer trap lines and promoters, demonstrating that this marker acts in a cell‐autonomous manner. We also showed that it can inactivate slow developmental processes like lateral root formation by targeting pericycle cells. It also revealed a role for the lateral root cap and the epidermis in controlling root growth, a faster response. The 5‐FC precursor acts systemically, as demonstrated by its ability to inhibit stomatal movements when supplied to the roots in combination with a guard cell‐specific promoter. Finally, we demonstrate that the tissular inactivation is reversible, and can therefore be used to synchronize plant responses or to determine cell type‐specific functions during different developmental stages. This tool will greatly enhance our capacity to understand the respective role of each cell type in plant physiology and development.     

Enhancer trap expression patterns provide a novel teaching resource

A collection of Arabidopsis enhancer trap transposants has been identified for use as a teaching tool. This collection serves to assist students in understanding the patterning and organization of plant tissues and cells, and will be useful in plant anatomy, morphology, and developmental biology courses. Each transposant exhibits reporter gene expression in a specific tissue, cell type, or domain, and these lines collectively offer a glimpse of compartments of gene expression. Some compartments correspond to classical definitions of botanical anatomy and can assist in anatomical identification. Other patterns of reporter gene expression are more complex and do not necessarily correspond to known anatomical features. The sensitivity of the beta-glucuronidase histochemical stain provides the student with a colorful and direct way to visualize difficult aspects of plant development and anatomy, and provides the teacher with an invaluable tool for a practical laboratory session.     

An autoregulatory element of the murine Hox-4.2 gene.

Hox-4.2 promoter activity was assayed by transient expression assays in P19 embryonal carcinoma (EC) cells. Cotransfection of a luciferase reporter gene construct driven by Hox-4.2 upstream sequences with an expression vector for the Hox-4.2 gene product resulted in a 20-fold increase in luciferase activity. This activity was specific in that the Hox-1.6 gene product had no effect in the same assay. Mutational analysis defined a cis-acting element with enhancer function which conferred most of this increase. Activation was largely dependent on two TAAT/ATTA motifs within this 217 bp fragment and HOX-4.2 bound specifically to both of these motifs. The 217 bp element maps within a highly conserved region of the human Hox-4.2 gene (HOX4B) which has been shown to display spatial enhancer activity in mice and flies. These findings suggest a conserved autoregulatory mechanism for the control of Hox-4.2 expression.     

Generation of a conditional lima1a allele in zebrafish using the FLEx switch technology

Gene trapping has emerged as a valuable tool to create conditional alleles in various model organisms. Here we report the FLEx‐based gene trap vector SAGFLEx that allows the generation of conditional mutations in zebrafish by gene‐trap mutagenesis. The SAGFLEx gene‐trap cassette comprises the rabbit β‐globin splice acceptor and the coding sequence of GFP, flanked by pairs of inversely oriented heterotypic target sites for the site‐specific recombinases Cre and Flp. Insertion of the gene‐trap cassette into endogenous genes can result in conditional mutations that are stably inverted by Cre and Flp, respectively. To test the functionality of this system we performed a pilot screen and analyzed the insertion of the gene‐trap cassette into the lima1a gene locus. In this lima1a allele, GFP expression faithfully recapitulated the endogenous lima1a expression and resulted in a complete knockout of the gene in homozygosity. Application of either Cre or Flp was able to mediate the stable inversion of the gene trap cassette and showed the ability to conditionally rescue or reintroduce the gene inactivation. Combined with pharmacologically inducible site specific recombinases the SAGFLEx vector insertions will enable precise conditional knockout studies in a spatial‐ and temporal‐controlled manner. genesis 54:19–28, 2016. © 2015 Wiley Periodicals, Inc.     

Inducible reporter/driver lines for the Arabidopsis root with intrinsic reporting of activity state

Cell‐, tissue‐ or organ‐specific inducible expression systems are powerful tools for functional analysis of changes to the pattern, level or timing of gene expression. However, plant researchers lack standardised reagents that promote reproducibility across the community. Here, we report the development and functional testing of a Gateway‐based system for quantitatively, spatially and temporally controlling inducible gene expression in Arabidopsis that overcomes several drawbacks of the legacy systems. We used this modular driver/effector system with intrinsic reporting of spatio‐temporal promoter activity to generate 18 well‐characterised homozygous transformed lines showing the expected expression patterns specific for the major cell types of the Arabidopsis root; seed and plasmid vectors are available through the Arabidopsis stock centre. The system's tight regulation was validated by assessing the effects of diphtheria toxin A chain expression. We assessed the utility of Production of Anthocyanin Pigment 1 (PAP1) as an encoded effector mediating cell‐autonomous marks. With this shared resource of characterised reference driver lines, which can be expanded with additional promoters and the use of other fluorescent proteins, we aim to contribute towards enhancing reproducibility of qualitative and quantitative analyses.     

Activity of a novel PDGF beta-receptor enhancer during the cell cycle and upon differentiation of neuroblastoma

PDGF acts as an autocrine and paracrine factor in certain tumors through upregulation of the PDGF beta-receptor expression. In order to elucidate the control mechanism for the receptor expression, we have isolated an enhancer from two P1 clones that together contain a 102 kb NotI region covering the entire human PDGFRB gene. They were partially digested with TspI and cloned into the PDGFRB enhancer trap vector to make a library for identification of enhancers. The digested DNA containing enhancer was identified by expression of GFP when transfected in PDGF beta-receptor expressing cells. One of the enhancer clones was further examined by making several deletion mutants in a luciferase vector. This enhancer was most active in neuroblastoma cells, IMR32 and BE2, but less active in hemangioma and in smooth muscle cell lines. Chip assay revealed that SP1, AP2, and GATA2 bound the enhancer in BE2 cells. Their interaction occurred dependently of the cell cycle and synchronously with their binding to the promoter. Transfection of GATA2 alone or with Ets, which binds adjacent to GATA, resulted in differentiation of BE2 cells in parallel with increased PDGF beta-receptor expression. Furthermore, over-expression of the PDGF beta-receptor in BE2 cells induced neurite extension.     

Infrared laser‐mediated local gene induction in medaka,zebrafish and Arabidopsis thaliana

Heat shock promoters are powerful tools for the precise control of exogenous gene induction in living organisms. In addition to the temporal control of gene expression, the analysis of gene function can also require spatial restriction. Recently, we reported a new method for in vivo, single‐cell gene induction using an infrared laser‐evoked gene operator (IR‐LEGO) system in living nematodes (Caenorhabditis elegans). It was demonstrated that infrared (IR) irradiation could induce gene expression in single cells without incurring cellular damage. Here, we report the application of IR‐LEGO to the small fish, medaka (Japanese killifish; Oryzias latipes) and zebrafish (Danio rerio), and a higher plant (Arabidopsis thaliana). Using easily observable reporter genes, we successfully induced gene expression in various tissues in these living organisms. IR‐LEGO has the potential to be a useful tool in extensive research fields for cell/tissue marking or targeted gene expression in local tissues of small fish and plants.     

GAL4-GFP enhancer trap lines for genetic manipulation of lateral root development in Arabidopsis thaliana

Lateral root development occurs throughout the life of the plant and is responsible for the plasticity of the root system. In Arabidopsis thaliana, lateral root founder cells originate from pericycle cells adjacent to xylem poles. In order to study the mechanisms of lateral root development, a population of Arabidopsis GAL4-GFP enhancer trap lines were screened and two lines were isolated with GAL4 expression in root xylem-pole pericycle cells (J0121), i.e. in cells competent to become lateral root founder cells, and in young lateral root primordia (J0192). These two enhancer trap lines are very useful tools with which to study the molecular and cellular bases of lateral root development using targeted gene expression. These lines were used for genetic ablation experiments by targeting the expression of a toxin-encoding gene. Moreover, the molecular bases of the enhancer trap expression pattern were characterized. These results suggest that the lateral-root-specific GAL4 expression pattern in J0192 is due to a strong enhancer in the promoter of the LOB-domain protein gene LBD16.