灵活操控果蝇翅芽中wingless基因表达水平的策略

【目的】灵活操控靶基因的表达水平对于研究基因的功能十分重要。Gal4/UAS系统已被广泛应用于调控基因表达,可研究果蝇Drosophila等模式生物复杂的生物学问题。受采用载体的特性及插入位点的影响,Gal4或UAS转基因品系在构建好之后,其调控靶基因的能力基本是确定的。本研究旨在在现有Gal4/UAS系统的基础上,开发一种新的策略,实现在果蝇翅芽中灵活操控wingless(wg)基因的表达水平。【方法】用遗传学手段将黑腹果蝇Drosophila melanogaster品系的UAS-wg和UAS-wg-RNAi转基因重组到同一黑腹果蝇品系中。将该重组黑腹果蝇品系与dpp-Gal4黑腹果蝇品系杂交,同时驱动UAS-wg和UAS-wg-RNAi在果蝇幼虫翅芽中共表达。杂交子代幼虫分别放置在不同的温度(18, 25和30℃)下培养。将幼虫翅芽解剖并进行免疫组化染色,测量染色的荧光强度,分析翅芽中wg的表达水平。【结果】在低温(18℃)下,UAS-wg在基因表达调控中起主要作用,wg表现为超表达,但其超表达的效率可被UAS-wg-RNAi有效地削弱。相反,在高温(30℃)下,UAS-wg-RNAi起主导作用,wg的表达受到抑制。并且通过转换温度,可实现wg在翅芽发育的不同阶段在超表达和抑制之间相互转化,从而灵活地操控wg基因在翅芽中的表达水平。【结论】该方法可以灵活操控果蝇翅芽中wg基因的表达水平,对于调控转基因的表达有重要的意义。     

Gal80~(ts)与Gal4组合灵敏控制果蝇中UAS转基因的表达水平

【目的】Gal80~(ts)与Gal4组合驱动UAS转基因表达是黑腹果蝇Drosophila melanogaster研究中常用的转基因过表达遗传学工具,通过温度控制实现对UAS转基因表达的灵活开关。Gal80~(ts)是一种温度敏感型蛋白,低温下(18℃)与Gal4蛋白结合并抑制其转录活力,高温下(29℃)解除对Gal4的抑制,从而允许Gal4结合UAS位点,启动UAS转基因的表达。但是从18~29℃的开关只能强烈过表达UAS转基因,而不能灵活调控转基因的表达水平。本实验系统研究一系列温度下转基因的表达水平,从而实现该体系对转基因的表达水平的灵活控制。【方法】以果蝇翅芽这一常用器官组织为研究模型,以2种Gal4品系(dpp-Gal4和en-Gal4,分别由decapentaplgic和engrailed基因的启动子驱动)分别与tub-Gal80~(ts)(微管蛋白基因tubulin启动子驱动)基因重组后,再分别与UAS-wg(wingless)转基因品系杂交;在一系列温度(18,25,27.5,28,28.5和30℃)下进行子代幼虫培养,通过免疫组化染色揭示并量化分析转基因wg在3龄幼虫翅芽上的表达水平。【结果】18~25℃培养条件下,Gal80~(ts)与Gal4组合系统中的UAS转基因不能表达;30℃时培养,转基因强烈地过表达;在25~30℃区间内,随着温度升高,转基因表达水平逐渐上升。【结论】在25~30℃之间的温度调控可以实现对Gal80~(ts)与Gal4组合系统中的UAS转基因表达水平的调控。本研究结果对调控转基因表达程度有重要价值。     

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.     

Characterization of a dorsal‐eye Gal4 Line in Drosophila

In Drosophila, the Gal4‐UAS system is used to drive ectopic gene expression in a tissue‐specific manner. In this system, transgenic flies expressing tissue specific Gal4 are crossed to a line in which the gene to be expressed is under the control of a Gal4‐responsive UAS sequence. The resulting progeny express the gene of interest in the pattern of the particular Gal4 line. Since a given UAS‐transgene can be driven by any Gal4 line, this system is predominantly limited by available Gal4 lines. Here we report the characterization of a novel line, DE‐Gal4, which in the eye is expressed in the dorsal compartment for the majority of development. Furthermore, we use functional tests to show that the DE‐Gal4 line is a useful tool with which to manipulate gene expression in half of the developing eye. genesis 48:3–7, 2010. © 2009 Wiley‐Liss, Inc.     

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.     

Characterization of midgut stem cell‐ and enteroblast‐specific Gal4 lines in drosophila

The homeostasis of Drosophila midgut is maintained by multipotent intestinal stem cells (ISCs), each of which gives rise to a new ISC and an immature daughter cell, enteroblast (EB), after one asymmetric cell division. In Drosophila, the Gal4‐UAS system is widely used to manipulate gene expression in a tissue‐ or cell‐specific manner, but in Drosophila midgut, there are no ISC‐ or EB‐specific Gal4 lines available. Here we report the generation and characterization of Dl‐Gal4 and Su(H)GBE‐Gal4 lines, which are expressed specifically in the ISCs and EBs separately. Additionally, we demonstrate that Dl‐Gal4 and Su(H)GBE‐Gal4 are expressed in adult midgut progenitors (AMPs) and niche peripheral cells (PCs) separately in larval midgut. These two Gal4 lines will serve as invaluable tools for navigating ISC behaviors. genesis 48:607–611, 2010. Published 2010 Wiley‐Liss, Inc.     

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.     

Limitations of RNAi of α6 nicotinic acetylcholine receptor subunits for assessing the in vivo sensitivity to spinosad

Abstract Spinosad is a widely used insecticide that exerts its toxic effect primarily through interactions with the nicotinic acetylcholine receptor. The α6 nicotinic acetylcholine receptor subunit is involved in spinosad toxicity as demonstrated by the high levels of resistance observed in strains lacking α6. RNAi was performed against the Dα6 nicotinic acetylcholine receptor subunit in Drosophila melanogaster using the Gal4‐UAS system to examine if RNAi would yield results similar to those of Dα6 null mutants. These Dα6‐deficient flies were subject to spinosad contact bioassays to evaluate the role of the Dα6 nicotinic acetylcholine receptor subunit on spinosad sensitivity. The expression of Dα6 was reduced 60%–75% as verified by quantitative polymerase chain reaction. However, there was no change in spinosad sensitivity in D. melanogaster. We repeated RNAi experiments in Tribolium castaneum using injection of dsRNA for Tcasα6. RNAi of Tcasα6 did not result in changes in spinosad sensitivity, similar to results obtained with D. melanogaster. The lack of change in spinosad sensitivity in both D. melanogaster and T. castaneum using two routes of dsRNA administration shows that RNAi may not provide adequate conditions to study the role of nicotinic acetylcholine receptor subunits on insecticide sensitivity due to the inability to completely eliminate expression of the α6 subunit in both species. Potential causes for the lack of change in spinosad sensitivity are discussed.     

A clinally varying promoter polymorphism associated with adaptive variation in wing size in Drosophila

Body size often shows adaptive clines in many ectotherms across altitude and latitude, but little is known about the genetic basis of these adaptive clines. Here we identify a polymorphism in the Dca (Drosophila cold acclimation) gene in Drosophila melanogaster that influences wing size, affects wing:thorax allometry and also controls a substantial proportion of the clinal wing‐size variation. A polymorphism in the promoter region of Dca had two common alleles showing strong reciprocal clinal variation in frequency with latitude along the east coast of Australia. The Dca‐237 allele increased towards the tropics where wing size is smaller. A within‐population association study highlighted that an increase in the frequency of this allele decreased wing size but did not influence thorax size. A manipulated increase in the level of expression of Dca achieved through UAS‐GAL4 was associated with a decrease in wing size but had no effect on thorax size. This was consistent with higher Dca expression levels in family lines with higher frequency of the Dca‐237 allele. Genetic variation in the promoter region of the Dca gene appears to influence adaptive size variation in the eastern Australian cline of Drosophila melanogaster and accounts for more than 10% of the genetic variation in size within and between populations.     

A new brain dopamine‐deficient Drosophila and its pharmacological and genetic rescue

Dopamine (DA) is a neurotransmitter with conserved behavioral roles between invertebrate and vertebrate animals. In addition to its neural functions, in insects DA is a critical substrate for cuticle pigmentation and hardening. Drosophila tyrosine hydroxylase (DTH) is the rate limiting enzyme for DA biosynthesis. Viable brain DA‐deficient flies were previously generated using tissue‐selective GAL4‐UAS binary expression rescue of a DTH null mutation and these flies show specific behavioral impairments. To circumvent the limitations of rescue via binary expression, here we achieve rescue utilizing genomically integrated mutant DTH. As expected, our DA‐deficient flies have no detectable DTH or DA in the brain, and show reduced locomotor activity. This deficit can be rescued by l ‐DOPA/carbidopa feeding, similar to human Parkinson's disease treatment. Genetic rescue via GAL4/UAS‐DTH was also successful, although this required the generation of a new UAS‐DTH1 transgene devoid of most untranslated regions, as existing UAS‐DTH transgenes express in the brain without a Gal4 driver via endogenous regulatory elements. A surprising finding of our newly constructed UAS‐DTH1m is that it expresses DTH at an undetectable level when regulated by dopaminergic GAL4 drivers even when fully rescuing DA, indicating that DTH immunostaining is not necessarily a valid marker for DA expression. This finding necessitated optimizing DA immunohistochemistry, showing details of DA innervation to the mushroom body and the central complex. When DA rescue is limited to specific DA neurons, DA does not diffuse beyond the DTH‐expressing terminals, such that DA signaling can be limited to very specific brain regions.     

Domain specific genetic mosaic system in the Drosophila eye

Genetic mosaic approach is commonly used in the Drosophila eye by completely abolishing or misexpressing a gene within a subset of cells to unravel its role during development. Classical genetic mosaic approach involves random clone generation in all developing fields. Consequently, a large sample size needs to be screened to generate and analyze clones in specific domains of the developing eye. To address domain specific functions of genes during axial patterning, we have developed a system for generating mosaic clones by combining Gal4/UAS and flippase (FLP)/FRT system which will allow generation of loss‐of‐function as well as gain‐of‐function clones on the dorsal and ventral eye margins. We used the bifid‐Gal4 driver to drive expression of UAS‐FLP. This reagent can have multiple applications in (i) studying spatio‐temporal function of a gene during dorso‐ventral (DV) axis specification in the eye, (ii) analyzing genetic epistasis of genes involved in DV patterning, and (iii) conducting genome wide screens in a domain specific manner. genesis 51:68–74, 2013. © 2012 Wiley Periodicals, Inc.     

Mapping and Application of Enhancer-trap Flippase Expression in Larval and Adult Drosophila CNS

The Gal4/ UAS binary method is powerful for gene and neural circuitry manipulation in Drosophila. For most neurobiological studies, however, Gal4 expression is rarely tissue-specific enough to allow for precise correlation of the circuit with behavioral readouts. To overcome this major hurdle, we recently developed the FINGR method to achieve a more restrictive Gal4 expression in the tissue of interest. The FINGR method has three components: 1) the traditional Gal4/UAS system; 2) a set of FLP/FRT-mediated Gal80 converting tools; and 3) enhancer-trap FLP (ET-FLP). Gal4 is used to define the primary neural circuitry of interest. Paring the Gal4 with a UAS-effector, such as UAS-MJD78Q or UAS-Shi^ts, regulates the neuronal activity, which is in turn manifested by alterations in the fly behavior. With an additional UAS-reporter such as UAS-GFP, the neural circuit involved in the specific behavior can be simultaneously mapped for morphological analysis. For Gal4 lines with broad expression, Gal4 expression can be restricted by using two complementary Gal80-converting tools: tub^P>Gal80> (''flip out'') and tub^P>stop>Gal80 (''flip in''). Finally, investigators can turn Gal80 on or off, respectively, with the help of tissue-specific ET-FLP. In the flip-in mode, Gal80 will repress Gal4 expression wherever Gal4 and ET-FLP intersect. In the flip-out mode, Gal80 will relieve Gal4 repression in cells in which Gal4 and FLP overlap. Both approaches enable the restriction of the number of cells in the Gal4-defined circuitry, but in an inverse pattern. The FINGR method is compatible with the vast collection of Gal4 lines in the fly community and highly versatile for traditional clonal analysis and for neural circuit mapping. In this protocol, we demonstrate the mapping of FLP expression patterns in select ET-FLPx2 lines and the effectiveness of the FINGR method in photoreceptor cells. The principle of the FINGR method should also be applicable to other genetic model organisms in which Gal4/UAS, Gal80, and FLP/FRT are used.     

Anchor negatively regulates BMP signalling to control Drosophila wing development

G protein-coupled receptors play particularly important roles in many organisms. The novel Drosophila gene anchor is an orthologue of vertebrate GPR155. However, the roles of anchor in molecular functions and biological processes, especially in wing development, remain unknown. Knockdown of anchor resulted in an increased wing size and additional and thickened veins. These abnormal wing phenotypes were similar to those observed in BMP signalling gain-of-function experiments. We observed that the BMP signalling indicator p-Mad was significantly increased in wing discs in which anchor RNAi was induced in larvae and accumulated abnormally in intervein regions in pupae. Furthermore, the expression of target genes of the BMP signalling pathway was examined using a lacZ reporter, and the results indicated that omb and sal were substantially increased in anchor-knockdown wing discs. An investigation of genetic interactions between Anchor and the BMP signalling pathway revealed that the thickened and ectopic vein tissues were rescued by knocking down BMP levels. These results suggested that Anchor functions to negatively regulate BMP signalling during wing development and vein formation.