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Gene expression systems in Drosophila: a synthesis of time and space
Review
TRENDS in Genetics Vol.20 No.8 August 2004
Gene expression systems in Drosophila: a synthesis of time and space Sean E. McGuire, Gregg Roman and Ronald L. Davis Department of Molecular and Cellular Biology, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA
Until recently, ectopic gene expression in Drosophila was largely accomplished through the use of the heatshock promoter, which provides the experimenter with temporal control over transgene induction, or the GAL4 –UAS system, which provides the experimenter with spatial control over transgene expression. But significant advances have now been made in combining the attributes of temporal and spatial control over gene expression into a single system. In this article, we review the progress on the development and implementation of several gene expression systems that offer control in time and space. These include systems employing the yeast FLP recombinase gene and FRT sites (FLP and/or FRT), tetracycline-responsive transcription factors (Tet-On and Tet-Off), steroid hormone responsive transcription factors (GeneSwitch and ER –GAL4) and temperature-sensitive repressors of the classical GAL4–UAS system (TARGET). ‘Henceforth, space by itself, and time by itself, are doomed to fade away into mere shadows, and only a kind of union of the two will preserve as an independent reality.’ [Herman Minkowski (1906), On relativity and the space-time continuum].
Transgenic techniques in Drosophila melanogaster were developed to enable scientists to drive the expression of an exogenous genetic construct in the living organism and to study its consequence. The types of experiments that can be performed with this general approach range from gene overexpression or misexpression experiments in wild-type organisms, gene rescue experiments in mutant organisms or even gene silencing experiments by expression of interfering products, such as dominant negative proteins or RNA interference (RNAi) constructs. There are, however, limitations with the available gene expression targeting systems that revolve around the degree of control over transgene expression. Conceptually, gene expression systems can be divided into three classes (Table 1). The first class consists of methods that regulate transgene expression in specific temporal windows that are defined by the experimenter. The predominant approach here has been to use a heat-shock promoter to induce gene expression following a simple heat-shock regimen. The second class includes methods that provide for spatially restricted transgene expression, and involve the use of either defined Corresponding author: Ronald L. Davis ([email protected]). Available online 25 June 2004
promoter sequences or enhancer trapping methods to restrict gene expression to the tissues of interest. Clearly, the most useful system for targeted gene expression is the third, which offers the investigator the ability to acutely adjust the level of gene expression within the dimensions of both time and space. Here we review the advances made in the development of gene expression systems with control in both time and space. We further discuss the remarkable opportunities for new types of investigations offered by these advances. Gene expression with control in time Heat-shock promoter One approach to targeted gene expression that enables the experimenter to define the developmental or physiological window in which a transgene is expressed is through the use of a heat-shock promoter [1]. This method provides a high degree of temporal control over the expression of a transgene and also enables the experimenter to modulate the level and persistence of transgene expression by varying the intensity and length of the heat shock [2,3]. However, there are several drawbacks associated with this approach. First, the heat-shock promoters produce a low level of basal transcription that can be significant even under non-heat-shock conditions. Consequently, phenotypic effects can be produced with the expression of certain genes, such as toxin genes, even without an elevation in temperature. Second, the heat shock used to induce the expression of the transgene can itself produce undesired effects on the flies depending on the timing of the heat shock and the inducing temperature [4]. Finally, the main limitation with this approach is that transgene expression is induced in essentially all cells in the organism, which prevents one from delineating the spatial characteristics associated with the phenotypic effects of a transgene. Gene expression with control in space Defined promoters for driving gene expression One method for achieving spatially delineated gene expression is to use defined promoter sequences to drive the expression of a gene of interest. This method has the advantage of enabling the expression of the transgene to be driven with the spatial and temporal characteristics of the cloned promoter. Examples of restricted tissue specific promoters include the glass multimer reporter (GMR) [5],
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Table 1. Gene expression systems in Drosophilaa Gene expression System
Inducer
Basis for operation
Control in time?
Control in space?
Reversible expression?
Heat-shock promoter (hsp)
Heat shock
Heat-inducible promoter
Yes
No
Yes
Defined promoter
–
Defined promoter
–
hsp-FLP –FRT
Heat shock
Yes
GAL4 –UAS-hsp FLP
Heat shock
Yes
Incomplete
No
Tet-ON or Tet-OFF
Doxycycline
Yes
Yes
Yes
Ligand inducible GAL4 chimeras
RU486 DES Estradiol Heat shock
Yeast transcription factor GAL4 and binding sites (UAS) Heat shock inducible yeast flipase recombinase and its genomic target sites (FRT) to excise transcription termination cassette Yeast transcription factor GAL4 and binding sites (UAS) combined with heat shock inducible flipase Doxycycline responsive transcription factor and genomic binding sites (Tet operator) GAL4 DNA binding domain combined with heterologous activation domain and ligand binding domain Heat sensitive repressor of GAL4 (GAL80ts)
Yes, as selected for Yes, as selected for Incomplete
No
GAL4 –UAS
Yes, as selected for No
Yes
Yes
Yes
Yes
Yes
Yes
TARGET a
No No
Abbreviations: FRT, FLP recombinase recognition; UAS, upstream activating sequences.
which drives expression in the eye, and the pan-neuronal embryonic lethal abnormal vision (ELAV) promoter [6]. Examples of ubiquitous promoters include the tubulin and actin5C promoters [7,8]. Overall, this approach has been valuable in the cases where defined promoters have been identified, however, the total number of these has remained limited. GAL4 –UAS system The most widely used system in Drosophila for achieving spatially restricted gene expression is the GAL4 –upstream activating sequences (UAS) system [9] (Figure 1a). In this scheme, a P element carrying the GAL4 transcriptional activator is randomly mobilized throughout the genome, bringing the expression of GAL4 under the control of endogenous tissue-specific enhancers. The target transgene that is cloned downstream of a UAS sequence is then expressed in the same tissue-specific pattern as the GAL4 activator. There are several advantages to this system. First, the transcriptional activator and the target UAS transgene are carried in different parental lines, enabling viability of the parental stocks when toxic transgenes are carried. Second, once generated, a line expressing GAL4 in a given spatial pattern can be crossed to any UAS target line, enabling the GAL4 line to be used as a general resource. Finally, when a given UAS target line is generated, the target gene can be driven anywhere in the fly by crossing to the appropriate GAL4 line. A recent survey of FlyBase (http://flybase.bio.indiana. edu/) revealed 1095 characterized GAL4 lines and 238 lines bearing UAS transgenes. In addition, a new database, GETDB (http://flymap.lab.nig.ac.jp/,dclust/main. html), has been created that profiles the expression patterns and molecular locations of some 4615 GAL4 enhancer trap lines. Furthermore, the Rørth EP collection and the Gene-Search collection have 2300 and www.sciencedirect.com
613 specialized UAS lines, respectively [10,11]. These collections of flies carry special P elements containing the GAL4– UAS promoter facing outward at the end(s) of the P element, so that when the P element is randomly mobilized throughout the genome, it brings the expression of nearby genes under the control of GAL4. These reagents provide a powerful resource for molecular genetic studies in Drosophila, a topic that has been reviewed extensively [2,3,12,13]. One limitation of the GAL4 –UAS system, however, is that it is not generally possible to induce this system. Although the GAL4 transcriptional activator shows a small degree of variation in activity as a function of temperature [9], this variation occurs upon already high levels of basal activity, making the system unsuitable as a gene switch. Additionally, many GAL4 lines demonstrate dynamic expression, with patterns changing throughout development and adulthood. This makes it difficult to define whether rescue experiments using the GAL4– UAS system represent a developmental rescue or an adult rescue of a phenotype. Furthermore, because many, if not most, of the GAL4 lines drive expression early in development, they often can not be used with the UAS transgenes that encode toxic gene products except to induce developmental lethality [14,15]. Gene expression with control in time and space: the synthesis Hsp –FLP–FRT One approach to combine spatial and temporal control of transgene expression used a combination of the heat-shock promoter and the FLP recombinase to excise a transcriptional-termination cassette that is placed between a defined promoter and a transgene. In this approach, the FLP recombinase is cloned downstream of the heat-shock promoter, enabling the experimenter to control the timing of induction of the FLP recombinase, which then excises
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(a) The GAL4–UAS system
GAL4P Enhancer
GAL4
YFG UAS
(b) The GeneSwitch system
(c) The TARGET system
Geneswitch GAL80ts – Hormone
Enhancer
19°C
GAL4P GeneSwitch
UAS
YFG
Enhancer
GAL4 UAS
YFG
Geneswitch + Hormone
30°C GAL4P
Enhancer
GeneSwitch
YFG UAS
Enhancer
GAL4 UAS
YFG
Figure 1. The GAL4 –UAS, GeneSwitch and TARGET systems. (a) The GAL4– UAS System. In the conventional GAL4–UAS system, the yeast transcriptional activator is driven in a specific spatial pattern either by a defined promoter or by an endogenous enhancer. The GAL4 protein, in turn, binds to its cognate UAS binding site and constitutively activates the transcription of your favorite gene (YFG) cloned downstream of the UAS. (b) Ligand-inducible GAL4 chimeras. In these systems, the DNA binding domain of the GAL4 protein is fused either to the p65 activation domain and a mutant progesterone receptor ligand binding domain (GeneSwitch, shown here) or to the estrogen receptor to generate the ligand-inducible chimeric activators. In the absence of hormone, the GeneSwitch is in the ‘off’ state. In the presence of hormone, the GeneSwitch molecule undergoes a conformational change to an active conformation where it can bind to a UAS sequence and activate transcription of YFG. (c) The TARGET System. In this system, the conventional GAL4– UAS system is conditionally regulated by a temperature sensitive allele of GAL80. At 198C, transcription of YFG is repressed, whereas this repression is relieved by a temperature shift to 308C, leading to high levels of expression of YFG in a specific tissue. Both the GeneSwitch and TARGET systems provide spatial and temporal control over gene expression. Modified from Refs [33,34] and reproduced with permission (www.sciencemag.org). Abbreviation: UAS, upstream activating sequences.
the intervening transcription-termination cassette and enables the transgene to be expressed in the tissue defined by the chosen promoter [16]. An alternative approach is to combine the hsp –FLP – FLP recombinase recognition (FRT) method with the GAL4– UAS system. In this approach, a transcriptional-termination cassette is inserted between a defined promoter and GAL4 or between the UAS sequence and the target gene [14,17 –20]. Expression of the transgene is induced in response to the heat-shock induction of FLP expression. These methods differ from the traditional heat-shock promoter system in that the experimenter control of transgene expression is permanently lost after induction. In addition, although the heatshock promoter drives expression ubiquitously throughout the organism, the FLP recombinase produces mosaics of gene expression that vary from animal to animal because the efficiency of recombination is , 100%. These attributes make these systems ideal for mosaic mapping efforts but render them unsuitable for generating populations of flies with the same expression pattern of the transgene. www.sciencedirect.com
Tetracycline-transactivator system Several newer systems have been developed that provide spatial and temporal targeting of gene expression in a manner that is useful for generating homogeneous populations of transgenic animals. One of these is the based on the tetracycline transactivator (tTA), which is regulated by tetracycline (Tet) or its derivatives [21] (Figure 2a). The utility of the Tet system was demonstrated initially by Bujard and colleagues [21]. Similar to the bipartite GAL4– UAS system, the tetracycline transactivator is expressed in a tissue of interest, where it subsequently binds to a tet operator sequence that is placed upstream of a target transgene and activates transcription. In the Tet-Off system, the tTA constitutively activates transcription in the absence of tetracycline, and this activity is silenced in the presence of tetracycline. Bello and colleagues demonstrated the effectiveness of this system in Drosophila by using an eye-specific enhancer to drive the expression of a TetO – LacZ transgene specifically in the eye imaginal disc. LacZ expression was observed as early
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as six hours after the larvae were shifted to food lacking tetracycline. The group also demonstrated the ability to repress the expression of the TetO– LacZ transgene in embryos by feeding the mothers high concentrations of tetracycline. To demonstrate biological function, they used the system to suppress the embryonic and larval lethality that is associated with expression of the Antennapedia homeotic gene under the control of a HoxA7 – tTA construct, and later induced the expression in adults, leading to a bristle phenotype [22]. Stebbins and Yin [23] have subsequently introduced the actin5C promoter to drive the tTA ubiquitously in the fly and incorporated insulating elements into the vectors to reduce position effects on the transgenes.
A second Tet-regulated system (Figure 2b) has been developed in which the presence of doxycycline positively regulates the activity of the tetracycline transactivator (Tet-On) [24]. The Tet-On system was initially reported not to function in Drosophila [22] but Bieshke and colleagues adapted the system for use in the fly [25]. Using the actin5C promoter, transgene induction occurred between eight and 20 hours after feeding, whereas maximal induction of the system required 48 hours of feeding followed by three days of accumulation. Significant levels of b-galactosidase were still observed ten days after removal of the flies from doxycycline. It is not clear at this point whether this represents the stability of the reporter or the persistent activation of the rtTA driver. Long-term
(a) The Tet-Off System
(b) The Tet-On System
– Tetracycline
Enhancer
rtTA
– Tetracycline
tTA
YFG
tTA
rtTA
Enhancer
tet-O
YFG tet-O
tTA rtTA + Tetracycline
Enhancer
+ Tetracycline
tTA
YFG
Enhancer
rtTA
YFG
tet-O
tet-O
(c) The GAL4–UAS-tet system
rtTA
– Tetracycline YFG tet-O GAL4P Enhancer
rtTA
GAL4 UAS
+ Tetracycline rtTA
YFG tet-O TRENDS in Genetics
Figure 2. Schematic diagram of the Tet-Off, Tet-On and GAL4 –UAS– Tet systems. (a) The Tet-Off system. In this system, the tetracycline transactivator protein (tTA) is driven by either a defined promoter or endogenous enhancer in a spatially delineated pattern. In the absence of tetracycline or doxycycline, the transactivator binds to the tet operator and activates transcription of YFG. In the presence of tetracycline or doxycycline, the tTA is maintained in an inactive state, and transcription of your favorite gene (YFG) is silenced. (b) The Tet-On system. In this system, the reverse tetracycline activator (rtTA) is maintained in an inactive state in the absence of tetracycline or doxycycline and transcription of YFG is silenced. In the presence of drug, the rtTA binds to the tet-operator and activates the transcription of YFG. (c) The GAL4 –UAS– Tet system. To capitalize on the expression patterns previously characterized with the conventional GAL4–UAS system, the two tet systems have been combined with the GAL4 –UAS system. The combination of the Tet-On system and the GAL4–UAS system is shown. In this scheme, any GAL4 driver can be used to drive the expression of a UAS –rtTA construct, leading to constitutively high levels of expression of the rtTA in the same cells as the GAL4 driver. The rtTA does not activate the expression of YFG in these cells in the absence of drug. In the presence of the drug, however, high levels of YFG are expressed in these cells, allowing for spatial and temporal control over YFG expression. Abbreviation: UAS, upstream activating sequences. www.sciencedirect.com
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treatment with doxycycline at these levels was also shown to have an adverse impact on the lifespan of the flies. A second generation Tet-On activator, rtTAs-M2, has been generated that yields greater induction capabilities and more stringent regulation [26]. The activator was later modified by removing a putative cryptic splice site, adjusting codon usage and flanking the cassette with insulator sequences, producing an altered rtTA called rtTA-M2-alt TA [27]. With this activator, improved repression of basal transcription, improved kinetics of induction and higher absolute levels of transgene induction, of , 70-fold, have been reported compared with those of the original rtTA transactivator. One drawback with introducing a new system into Drosophila is that the number of useful tissue specific activator lines is small initially. This issue has been addressed by linking both the Tet-Off and Tet-On systems with the GAL4– UAS system [23,27]. In this scheme, a tissue-specific GAL4 line drives the UAS-dependent expression of either tTA or rtTA-M2-alt, which in turn, drives the expression of an additional transgene (Figure 2c). The fold induction observed in these schemes varies greatly depending on the GAL4 driver and target transgenes used but has been reported to be in the range of 30 – 250-fold [23,27]. There are several limitations that are associated with tetracycline systems. The Tet-Off system requires that the animals be exposed to the drug for extended periods. The newer Tet-On system appears to circumvent this issue; however, the full characteristics of this system – including an estimate of fold induction in the presence and absence of doxycycline and the off-rate kinetics – have yet to be described. Furthermore, the ability of the system to be modulated within embryos or pupae, which can not take up the drug by feeding, is unfeasible. Most importantly, although the system is able to capitalize on existing GAL4 drivers to achieve tissue-specific expression of the Tet activators, it currently fails to take advantage of the large numbers of existing UAS reagents, including the Rørth EP collection and the Gene-Search collection [10,11]. In the future, this limitation might be overcome by using tissuespecific rtTA activity to drive the expression of GAL4 and thus make use of these UAS lines. Ligand-inducible GAL4 chimeras A more recent and promising approach for achieving temporal and spatial targeting of gene expression from UAS transgenes is based on hormone inducible GAL4 chimeric proteins (Figure 1b). Both a GAL4– estrogen receptor fusion (GAL4 – ER) and a GAL4– progesterone receptor fusion (GeneSwitch) have recently been demonstrated to function in the fly [15,28,29]. Kinetic analysis of the GAL4 –ER system revealed that b-galactosidase activity reaches detectable levels within 12 hours and maximal levels at 2.5 days after continuous exposure to diethylstilbesterol (DES). Slightly slower induction was observed with b-estradiol. Three days after drug-containing food was withdrawn from the flies the b-galactosidase was undetectable. Han et al. [15] subsequently used the torsolike promoter to drive expression of the GAL4– ER chimera (tslGAL4 – ER) specifically in the border cells of the oocyte. This system was used to drive the expression of the www.sciencedirect.com
I mutant of the A chain of diphtheria toxin (DTI), which is under the control of a UAS sequence. Previous experiments showed that the UAS–DTI transgene, when combined with conventional GAL4 drivers, produces lethality. By contrast, when larvae carrying the tslGAL4 – ER and UAS –DTI transgenes were raised in the absence of DES, normal numbers of progeny were observed, whereas larvae raised in the presence of DES failed to yield any adults. Induction of this system in adult females for threeto-five days with either b-estradiol or DES resulted in the specific elimination of border cells at the anterior edge of the oocyte and posterior follicle cells. Taken together, these experiments demonstrate the utility of the GAL4– ER switch for tissue-specific expression of UAS based transgenes. Another GAL4 chimera was made ligand-inducible by fusing the GAL4 DNA-binding domain to the human progesterone receptor ligand-binding domain (GeneSwitch) [30]. Using the pan-neuronal ELAV enhancer to drive expression of GeneSwitch, Osterwalder and colleagues [28] demonstrated detectable levels of UAS– green fluorescent protein (GFP) expression by western blot as early as five hours after bathing larvae in the anti-progestin, RU486. By 21 hours, the level of GFP protein had become equivalent to that observed in larvae carrying the conventional ELAV– GAL4 driver in combination with the UAS –GFP transgene. Rearing third instar larvae on food containing RU486 resulted in a 51 –60-fold induction of UAS –GFP expression over levels in the absence of RU486. In addition, when mothers were fed RU486, embryos and newly hatched larvae expressed GFP in the nervous system, indicating that RU486 is transferred from mothers to the embryo, where it can activate the GeneSwitch. Driving a UAS– tetanus toxin construct (UAS– TeTxLC) with the conventional ELAV–GAL4 driver prevents embryonic hatching and is uniformly fatal. When the ELAV–GeneSwitch construct was used in combination with the UAS –TeTxLC, however, normal numbers of progeny are recovered in the absence of hormone, indicating little leakiness of the system. No larvae survive to adulthood when they eat food containing as little as 3 mg/ml RU486. GeneSwitch also displays a robust control over the level of gene expression by varying the concentration of RU486 that is fed to larvae [28]. One of the limitations with the ligand inducible GAL4 approach is the lack of multiple tissue-specific expressing driver lines. Therefore, Roman et al. [29] generated enhancer detector constructs containing the RU486 inducible GeneSwitch, and conducted a large-scale screen for expression in the adult head (G. Roman and R. Davis, unpublished). Additional GeneSwitch vector systems have been generated that facilitate rapid cloning of either defined enhancers or promoters upstream of GeneSwitch coding sequences [31]. Kinetic analysis with one of the enhancer detector lines demonstrated that after as little as one hour of RU486 feeding to adult flies, the levels of the reporter b-galactosidase observed at 24 hours were equivalent to those observed in flies that were fed for a full 24 hours. When flies were fed RU486 for 24 hours and then shifted to normal food, b-galactosidase was observed even after six days of withdrawal. It is currently not clear whether this represents the stability of the reporter or the
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persistent activation of the GeneSwitch. The ability to ablate specific tissues with two independent enhancer detector-GeneSwitch lines was demonstrated by driving the expression of a UAS– DTI construct in the fat bodies. Adult flies given chronic administration of RU486 showed ablated fat bodies and 100% lethality after five days of drug treatment. In a test of the GeneSwitch system, Mao et al. [32] fashioned a GeneSwitch driver in which the GeneSwitch cassette was expressed specifically in Drosophila mushroombody neurons using a mushroom-body-specific enhancer. This driver was then used to express a UAS– rutabaga transgene encoding adenylyl cyclase in the memorydefective rutabaga mutant either only during development or only in adult mushroom bodies. This was performed to address the fundamental question of whether the rutabaga gene product is required for the development of the nervous system or for physiological functions in the adult that serve memory formation. Normal rutabaga function restored selectively in the adult mushroom bodies but not during development rescued the memory deficit, thus, answering this question decisively. The TARGET system Given the large number of existing conventional GAL4 and UAS transgenic lines, a powerful addition to the arsenal of ectopic gene expression systems would be a reagent that enabled researchers to capitalize on this universe of well-characterized GAL4 and UAS transgenic lines, while adding the important feature of temporal control of target gene expression. Such a system has recently been developed based on a temperature-sensitive GAL80 protein [33,34]. In yeast, the transcriptional activity of GAL4 is repressed by GAL80 in the absence of galactose. In the presence of galactose, this repression is relieved, enabling GAL4 to activate genes required for galactose metabolism. Recently, a temperature-sensitive variant of GAL80 (GAL80ts) was cloned and used to construct a tubulin promoter-GAL80ts transgene in Drosophila. After introduction into Drosophila, the GAL80ts molecule was shown to regulate GAL4 in a temperature dependent fashion in embryos, larvae, pupae and adults, with optimal repression observed at 198C and derepression beginning at 308C (Figure 1c). This system has been named the TARGET system (temporal and regional gene expression targeting). Kinetic studies of the TARGET system have revealed a slight induction of GFP mRNA as early as 30 min after heat treatment at 328C. Half-maximal levels of RNA were seen at three hours, and by six hours the level of mRNA was roughly equivalent to levels seen in flies carrying the GAL4 driver and the UAS– GFP reporter without the GAL80ts. Estimates of fold induction based on mRNA levels were , 42 – 48-fold. An analysis of the off-rate kinetics showed that mRNA levels returned to half-maximal values by , 15 h, and by 36 h they were at the baseline, uninduced levels. The GAL80ts was shown to repress GAL4 –UAS expression in embryos raised at 198C and shown to enable expression to be induced by shifting the embryos to 308C. Figure 3a illustrates the expression of the reporter UAS – lacZ in the peduncles of the adult www.sciencedirect.com
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mushroom bodies due to the temperature-dependent derepression of GAL4 activity with the TARGET system. Expression is first observed after approximately three hours of incubation at an elevated temperature and accumulates with continued incubation at elevated temperature. The GeneSwitch system exhibits a similar kinetic profile for the induction of UAS– lacZ (Figure 3b). The system has also been used to enable the combination of GAL4 drivers and UAS – toxin constructs that previously could not be combined due to developmental lethality. In particular, the combination of the eye specific GMR– GAL4 driver and a UAS– hid transgene has failed to yield any adult progeny regardless of rearing temperature [14,33]. In the presence of the GAL80ts, however, normal numbers of adults were recovered with eyes that were largely normal in size, shape and pigmentation when the animals were raised at 198C. If the animals were shifted to inducing temperatures at different stages of development, adult flies eclosed with a range of eye phenotypes ranging from the complete lack of any eye structure, eyes that were severely reduced in size, shape and pigmentation, to eyes that were normal in size and shape but lacked pigmentation. The TARGET system has also been used to demonstrate spatial and temporal rescue of the memory defect due to the rutabaga mutation. Mushroom-body-specific GAL4 drivers and a UAS– rutabaga transgene were combined with the GAL80ts to determine whether the rutabaga memory defect was due to a developmental defect in the formation of the mushroom bodies or was due to an acute physiological defect in memory formation. By driving the expression of rutabaga specifically during development, or specifically during adulthood, it was demonstrated that rutabaga expression in the adult mushroom bodies is both necessary and sufficient to rescue the memory defect. Thus, the application of the TARGET system, like the GeneSwitch system, has enabled the dissection of a physiological role for rutabaga in learning and memory from a developmental role in the patterning of the brain. The TARGET system theoretically enables the use of any combination of GAL4 driver and UAS transgene and adds to this the feature of temporal inducibility. In addition to the GAL80ts of the TARGET system described previously, alternative possibilities also exist for temporal regulation of the conventional GAL4– UAS system. One approach has been the use of a light-induced activation of a caged GAL4– VP16 activator [35]. This was used successfully in the embryo to label single cells but its use as a means for generally inducing gene expression in the adult is limited. In addition, one could employ the GAL80 protein in alternative ways. For example, one could place the wild-type GAL80 down stream of the heat-shock promoter to transiently induce GAL80 expression levels and block GAL4 activity. Similarly, one could use specific promoters to drive the expression of wild-type Gal80 in specific cells to constitutively restrict the activity of GAL4 in these cells [36]. Finally, the Tet system could be engineered to regulate temporally the expression of wild-type GAL80 throughout the fly, so as to provide temporal regulation of the conventional GAL4– UAS system.
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(a) 18°C
18°C
32°C
3 hr
6 hr
12 hr
24 hr
24 hr
8 hr
12 hr
24 hr
Uninduced
(b) Feed with RU
Figure 3. Kinetics of reporter expression for TARGET and GeneSwitch. (a) Using the TARGET system, flies carrying the mushroom body GAL4 driver c739, tubP-GAL80ts, and UAS–lacZ were cultured at 188C and subjected to temperature shifts to 328C for various periods of time as indicated. Frontal sections were stained for b-galactosidase (b-gal) activity and photographs taken at the level of the paired ocelli. Arrows indicate the position of one peduncle of the mushroom bodies. Increased levels of b-gal activity occur with increasing periods of incubation at 328C due to the derepression of GAL4. Modified from Ref. [33] and reproduced with permission q2003 AAAS (www.sciencemag.org). (b) Flies carrying a GeneSwitch element driven by a mushroom body enhancer along with UAS– lacZ were fed RU486 for various periods of time as indicated. Frontal sections were stained for b-gal activity and photographs taken at a level near the base of the peduncle. Increased levels of b-gal activity occur with increased time after initiating feeding on RU486. Modified from Ref. [32] and reproduced with permission q2004 National Academy of Sciences, USA. Abbreviation: UAS, upstream activating sequences.
Conclusions and perspectives Significant progress has been made recently towards the development of improved systems for gene expression with control in both time and space. Although the features that constitute an optimal system might vary depending on the biological question of interest, the following features are desirable for many investigations: (i) Rapid on – off kinetics, with immediate initiation of transcription after the induction event and production of the gene product limited only by the rates of transcription and the formation of the mature product. The turn-off step would be limited only by the decay rates of the pre-existing mRNA and the stability of the protein product. (ii) Inert inducer, with no biological affects other than inducing the system. (iii) Control over the level of expression, with optimally a very broad dynamic range to achieve any desired level of gene expression. (iv) Complete off state, with no basal expression in the absence of the inducer. Most of the systems described meet these characteristics to varying degrees. All of the systems demonstrate relatively slow kinetics, with induction taking hours to days, and the abatement taking generally longer. This is inevitable for any system that is based on the induction of transcription because of the lag time for transcription, post-transcriptional processing, translation and posttranslational processing. Similarly, the off-rate kinetics are determined by the subsequent decay of all previously existing components upon withdrawal. For faster kinetics, systems that target the functional protein product will be required [37]. In addition, it is likely that none of the inducing agents for the systems described are completely www.sciencedirect.com
biologically inert. Inducers such as doxycycline have been shown to interfere with normal development. Elevated temperature also can, depending on the level, produce nonspecific effects that might interfere with a given study. All of the systems described above offer some control over the level of gene expression. For instance, providing heat shock at different temperatures, feeding animals different concentrations of RU486 or doxycycline or feeding animals for different times can provide some control over the level of expression. In addition, it is likely that all of the systems have some basal level of expression, although this might not have been detected in previous experiments. Even with the above caveats, the inducible systems described in this review offer new opportunities for the experimental dissection of a wide range of biological questions. They not only enable experimenter control of gene induction or repression in specific tissues but also provide control over the level of gene expression. This provides a broad venue for gene-dose-response experiments. The systems also offer the ability to perform withinorganism controls, by examining the same organism at two different times in the alternative states of gene expression. Gene rescue experiments [32,33] can be performed to determine whether the expression of a gene in a specific tissue at a specific time is sufficient to correct the phenotype of a mutant organism and in the process shed light on the role of that gene. Gene silencing, through expression of RNAi constructs under the control of gene expression systems with control in time and space, provides a new and extremely potent experimental approach to define whether specific genes are necessary for a particular phenotype in the organism.
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In summary, with the development of the systems described in this review, remarkable opportunities now exist to probe the genetics of development, physiology and behavior with both classical and newer genetic tools. Acknowledgements Research in the authors’ laboratories is supported by National Institutes of Health Grant GM63929 and NS19904. Additional support came from the R. P. Doherty-Welch Chair in Science.
References 1 Lis, J.T. et al. (1983) New heat shock puffs and beta-galactosidase activity resulting from transformation of Drosophila with an hsp70-lacZ hybrid gene. Cell 35, 403 – 410 2 Wilder, E. (2000) Ectopic Expression in Drosophila. Methods Mol. Biol. 137, 9 – 14 3 D’Avino, P.P. and Thummel, C.S. (1999) Ectopic expression systems in Drosophila. Methods Enzymol. 306, 129– 142 4 Petersen, N.S. (1990) Effects of heat and chemical stress on development. Adv. Genet. 28, 275 – 296 5 Hay, B.A. et al. (1994) Expression of baculovirus P35 prevents cell death in Drosophila. Development 120, 2121– 2129 6 Yao, K.M. and White, K. (1994) Neural specificity of elav expression: defining a Drosophila promoter for directing expression to the nervous system. J. Neurochem. 63, 41 – 51 7 Bialojan, S. et al. (1984) Characterization and developmental expression of beta tubulin genes in Drosophila melanogaster. EMBO J. 3, 2543 – 2548 8 Fyrberg, E.A. et al. (1983) Transcripts of the six Drosophila actin genes accumulate in a stage- and tissue-specific manner. Cell 33, 115– 123 9 Brand, A.H. and Perrimon, N. (1993) Targeted gene expression as a means of altering cell fates and generating dominant phenotypes. Development 118, 401 – 415 10 Rørth, P.A. (1996) Modular misexpression screen in Drosophila detecting tissue-specific phenotypes. Proc. Natl. Acad. Sci. U. S. A. 93, 12418 – 12422 11 Toba, G. et al. (1999) The gene search system. A method for efficient detection and rapid molecular identification of genes in Drosophila melanogaster. Genetics 151, 725– 737 12 Brand, A.H. et al. (1994) Ectopic expression in Drosophila. Methods Cell Biol. 44, 635 – 654 13 Duffy, J.B. (2002) GAL4 system in Drosophila: a fly geneticist’s Swiss army knife. Genesis 34, 1 – 15 14 Keller, A. et al. (2002) Targeted expression of tetanus neurotoxin interferes with behavioral responses to sensory input in Drosophila. J. Neurobiol. 50, 221 – 233 15 Han, D.D. et al. (2000) Investigating the function of follicular subpopulations during Drosophila oogenesis through hormone-dependent enhancer-targeted cell ablation. Development 127, 573 – 583 16 Struhl, G. and Basler, K. (1993) Organizing activity of wingless protein in Drosophila. Cell 72, 527– 540
17 Nellen, D. et al. (1996) Direct and long-range action of a DPP morphogen gradient. Cell 85, 357– 368 18 Zecca, M. et al. (1996) Direct and long-range action of a wingless morphogen gradient. Cell 87, 833– 844 19 Pignoni, F. and Zipursky, S.L. (1997) Induction of Drosophila eye development by decapentaplegic. Development 124, 271 – 278 20 Ito, K. et al. (1997) The Drosophila mushroom body is a quadruple structure of clonal units each of which contains a virtually identical set of neurones and glial cells. Development 124, 761 – 771 21 Gossen, M. and Bujard, H. (1992) Tight control of gene expression in mammalian cells by tetracycline-responsive promoters. Proc. Natl. Acad. Sci. U. S. A. 89, 5547 – 5551 22 Bello, B. et al. (1998) Spatial and temporal targeting of gene expression in Drosophila by means of a tetracycline-dependent transactivator system. Development 125, 2193 – 2202 23 Stebbins, M.J. and Yin, J.C. (2001) Adaptable doxycycline-regulated gene expression systems for Drosophila. Gene 270, 103 – 111 24 Gossen, M. et al. (1995) Transcriptional activation by tetracyclines in mammalian cells. Science 268, 1766 – 1769 25 Bieschke, E.T. et al. (1998) Doxycycline-induced transgene expression during Drosophila development and aging. Mol. Gen. Genet. 258, 571– 579 26 Urlinger, S. et al. (2000) Exploring the sequence space for tetracyclinedependent transcriptional activators: novel mutations yield expanded range and sensitivity. Proc. Natl. Acad. Sci. U. S. A. 97, 7963 – 7968 27 Stebbins, M.J. et al. (2001) Tetracycline-inducible systems for Drosophila. Proc. Natl. Acad. Sci. U. S. A. 98, 10775 – 10780 28 Osterwalder, T. et al. (2001) A conditional tissue-specific transgene expression system using inducible GAL4. Proc. Natl. Acad. Sci. U. S. A. 98, 12596 – 12601 29 Roman, G. et al. (2001) P[Switch], a system for spatial and temporal control of gene expression in Drosophila melanogaster. Proc. Natl. Acad. Sci. U. S. A. 98, 12602 – 12607 30 Burcin, M.M. et al. (1998) A regulatory system for target gene expression. Front. Biosci. 3, c1 – c7 31 Roman, G. and Davis, R.L. (2002) Conditional expression of UAS-transgenes in the adult eye with a new gene-switch vector system. Genesis 34, 127– 131 32 Mao, Z. et al. (2004) Pharmacogenetic rescue in time and space of the rutabaga memory impairment by using Gene-Switch. Proc. Natl. Acad. Sci. U. S. A. 101, 198 – 203 33 McGuire, S.E. et al. (2003) Spatiotemporal rescue of a memory defect in Drosophila. Science 302, 1765– 1768 34 McGuire, S.E. et al. (2004) Spatiotemporal gene expression targeting with the TARGET and Gene-Switch systems in Drosophila. Sci. STKE 2004, pl4 35 Cambridge, S.B. et al. (1997) Drosophila mitotic domain boundaries as cell fate boundaries. Science 277, 825– 828 36 Kitamoto, T. (2002) Conditional disruption of synaptic transmission induces male-male courtship behavior in Drosophila. Proc. Natl. Acad. Sci. U. S. A. 99, 13232 – 13237 37 Jurgen Dohmen, R. et al. (1994) Heat-inducible degron: A method for constructing temperature-sensitive mutants. Science 263, 1273– 1276
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Gene expression systems in Drosophila: a synthesis of time and space Sean E. McGuire, Gregg Roman and Ronald L. Davis Department of Molecular and Cellular Biology, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA
Until recently, ectopic gene expression in Drosophila was largely accomplished through the use of the heatshock promoter, which provides the experimenter with temporal control over transgene induction, or the GAL4 –UAS system, which provides the experimenter with spatial control over transgene expression. But significant advances have now been made in combining the attributes of temporal and spatial control over gene expression into a single system. In this article, we review the progress on the development and implementation of several gene expression systems that offer control in time and space. These include systems employing the yeast FLP recombinase gene and FRT sites (FLP and/or FRT), tetracycline-responsive transcription factors (Tet-On and Tet-Off), steroid hormone responsive transcription factors (GeneSwitch and ER –GAL4) and temperature-sensitive repressors of the classical GAL4–UAS system (TARGET). ‘Henceforth, space by itself, and time by itself, are doomed to fade away into mere shadows, and only a kind of union of the two will preserve as an independent reality.’ [Herman Minkowski (1906), On relativity and the space-time continuum].
Transgenic techniques in Drosophila melanogaster were developed to enable scientists to drive the expression of an exogenous genetic construct in the living organism and to study its consequence. The types of experiments that can be performed with this general approach range from gene overexpression or misexpression experiments in wild-type organisms, gene rescue experiments in mutant organisms or even gene silencing experiments by expression of interfering products, such as dominant negative proteins or RNA interference (RNAi) constructs. There are, however, limitations with the available gene expression targeting systems that revolve around the degree of control over transgene expression. Conceptually, gene expression systems can be divided into three classes (Table 1). The first class consists of methods that regulate transgene expression in specific temporal windows that are defined by the experimenter. The predominant approach here has been to use a heat-shock promoter to induce gene expression following a simple heat-shock regimen. The second class includes methods that provide for spatially restricted transgene expression, and involve the use of either defined Corresponding author: Ronald L. Davis ([email protected]). Available online 25 June 2004
promoter sequences or enhancer trapping methods to restrict gene expression to the tissues of interest. Clearly, the most useful system for targeted gene expression is the third, which offers the investigator the ability to acutely adjust the level of gene expression within the dimensions of both time and space. Here we review the advances made in the development of gene expression systems with control in both time and space. We further discuss the remarkable opportunities for new types of investigations offered by these advances. Gene expression with control in time Heat-shock promoter One approach to targeted gene expression that enables the experimenter to define the developmental or physiological window in which a transgene is expressed is through the use of a heat-shock promoter [1]. This method provides a high degree of temporal control over the expression of a transgene and also enables the experimenter to modulate the level and persistence of transgene expression by varying the intensity and length of the heat shock [2,3]. However, there are several drawbacks associated with this approach. First, the heat-shock promoters produce a low level of basal transcription that can be significant even under non-heat-shock conditions. Consequently, phenotypic effects can be produced with the expression of certain genes, such as toxin genes, even without an elevation in temperature. Second, the heat shock used to induce the expression of the transgene can itself produce undesired effects on the flies depending on the timing of the heat shock and the inducing temperature [4]. Finally, the main limitation with this approach is that transgene expression is induced in essentially all cells in the organism, which prevents one from delineating the spatial characteristics associated with the phenotypic effects of a transgene. Gene expression with control in space Defined promoters for driving gene expression One method for achieving spatially delineated gene expression is to use defined promoter sequences to drive the expression of a gene of interest. This method has the advantage of enabling the expression of the transgene to be driven with the spatial and temporal characteristics of the cloned promoter. Examples of restricted tissue specific promoters include the glass multimer reporter (GMR) [5],
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Table 1. Gene expression systems in Drosophilaa Gene expression System
Inducer
Basis for operation
Control in time?
Control in space?
Reversible expression?
Heat-shock promoter (hsp)
Heat shock
Heat-inducible promoter
Yes
No
Yes
Defined promoter
–
Defined promoter
–
hsp-FLP –FRT
Heat shock
Yes
GAL4 –UAS-hsp FLP
Heat shock
Yes
Incomplete
No
Tet-ON or Tet-OFF
Doxycycline
Yes
Yes
Yes
Ligand inducible GAL4 chimeras
RU486 DES Estradiol Heat shock
Yeast transcription factor GAL4 and binding sites (UAS) Heat shock inducible yeast flipase recombinase and its genomic target sites (FRT) to excise transcription termination cassette Yeast transcription factor GAL4 and binding sites (UAS) combined with heat shock inducible flipase Doxycycline responsive transcription factor and genomic binding sites (Tet operator) GAL4 DNA binding domain combined with heterologous activation domain and ligand binding domain Heat sensitive repressor of GAL4 (GAL80ts)
Yes, as selected for Yes, as selected for Incomplete
No
GAL4 –UAS
Yes, as selected for No
Yes
Yes
Yes
Yes
Yes
Yes
TARGET a
No No
Abbreviations: FRT, FLP recombinase recognition; UAS, upstream activating sequences.
which drives expression in the eye, and the pan-neuronal embryonic lethal abnormal vision (ELAV) promoter [6]. Examples of ubiquitous promoters include the tubulin and actin5C promoters [7,8]. Overall, this approach has been valuable in the cases where defined promoters have been identified, however, the total number of these has remained limited. GAL4 –UAS system The most widely used system in Drosophila for achieving spatially restricted gene expression is the GAL4 –upstream activating sequences (UAS) system [9] (Figure 1a). In this scheme, a P element carrying the GAL4 transcriptional activator is randomly mobilized throughout the genome, bringing the expression of GAL4 under the control of endogenous tissue-specific enhancers. The target transgene that is cloned downstream of a UAS sequence is then expressed in the same tissue-specific pattern as the GAL4 activator. There are several advantages to this system. First, the transcriptional activator and the target UAS transgene are carried in different parental lines, enabling viability of the parental stocks when toxic transgenes are carried. Second, once generated, a line expressing GAL4 in a given spatial pattern can be crossed to any UAS target line, enabling the GAL4 line to be used as a general resource. Finally, when a given UAS target line is generated, the target gene can be driven anywhere in the fly by crossing to the appropriate GAL4 line. A recent survey of FlyBase (http://flybase.bio.indiana. edu/) revealed 1095 characterized GAL4 lines and 238 lines bearing UAS transgenes. In addition, a new database, GETDB (http://flymap.lab.nig.ac.jp/,dclust/main. html), has been created that profiles the expression patterns and molecular locations of some 4615 GAL4 enhancer trap lines. Furthermore, the Rørth EP collection and the Gene-Search collection have 2300 and www.sciencedirect.com
613 specialized UAS lines, respectively [10,11]. These collections of flies carry special P elements containing the GAL4– UAS promoter facing outward at the end(s) of the P element, so that when the P element is randomly mobilized throughout the genome, it brings the expression of nearby genes under the control of GAL4. These reagents provide a powerful resource for molecular genetic studies in Drosophila, a topic that has been reviewed extensively [2,3,12,13]. One limitation of the GAL4 –UAS system, however, is that it is not generally possible to induce this system. Although the GAL4 transcriptional activator shows a small degree of variation in activity as a function of temperature [9], this variation occurs upon already high levels of basal activity, making the system unsuitable as a gene switch. Additionally, many GAL4 lines demonstrate dynamic expression, with patterns changing throughout development and adulthood. This makes it difficult to define whether rescue experiments using the GAL4– UAS system represent a developmental rescue or an adult rescue of a phenotype. Furthermore, because many, if not most, of the GAL4 lines drive expression early in development, they often can not be used with the UAS transgenes that encode toxic gene products except to induce developmental lethality [14,15]. Gene expression with control in time and space: the synthesis Hsp –FLP–FRT One approach to combine spatial and temporal control of transgene expression used a combination of the heat-shock promoter and the FLP recombinase to excise a transcriptional-termination cassette that is placed between a defined promoter and a transgene. In this approach, the FLP recombinase is cloned downstream of the heat-shock promoter, enabling the experimenter to control the timing of induction of the FLP recombinase, which then excises
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(a) The GAL4–UAS system
GAL4P Enhancer
GAL4
YFG UAS
(b) The GeneSwitch system
(c) The TARGET system
Geneswitch GAL80ts – Hormone
Enhancer
19°C
GAL4P GeneSwitch
UAS
YFG
Enhancer
GAL4 UAS
YFG
Geneswitch + Hormone
30°C GAL4P
Enhancer
GeneSwitch
YFG UAS
Enhancer
GAL4 UAS
YFG
Figure 1. The GAL4 –UAS, GeneSwitch and TARGET systems. (a) The GAL4– UAS System. In the conventional GAL4–UAS system, the yeast transcriptional activator is driven in a specific spatial pattern either by a defined promoter or by an endogenous enhancer. The GAL4 protein, in turn, binds to its cognate UAS binding site and constitutively activates the transcription of your favorite gene (YFG) cloned downstream of the UAS. (b) Ligand-inducible GAL4 chimeras. In these systems, the DNA binding domain of the GAL4 protein is fused either to the p65 activation domain and a mutant progesterone receptor ligand binding domain (GeneSwitch, shown here) or to the estrogen receptor to generate the ligand-inducible chimeric activators. In the absence of hormone, the GeneSwitch is in the ‘off’ state. In the presence of hormone, the GeneSwitch molecule undergoes a conformational change to an active conformation where it can bind to a UAS sequence and activate transcription of YFG. (c) The TARGET System. In this system, the conventional GAL4– UAS system is conditionally regulated by a temperature sensitive allele of GAL80. At 198C, transcription of YFG is repressed, whereas this repression is relieved by a temperature shift to 308C, leading to high levels of expression of YFG in a specific tissue. Both the GeneSwitch and TARGET systems provide spatial and temporal control over gene expression. Modified from Refs [33,34] and reproduced with permission (www.sciencemag.org). Abbreviation: UAS, upstream activating sequences.
the intervening transcription-termination cassette and enables the transgene to be expressed in the tissue defined by the chosen promoter [16]. An alternative approach is to combine the hsp –FLP – FLP recombinase recognition (FRT) method with the GAL4– UAS system. In this approach, a transcriptional-termination cassette is inserted between a defined promoter and GAL4 or between the UAS sequence and the target gene [14,17 –20]. Expression of the transgene is induced in response to the heat-shock induction of FLP expression. These methods differ from the traditional heat-shock promoter system in that the experimenter control of transgene expression is permanently lost after induction. In addition, although the heatshock promoter drives expression ubiquitously throughout the organism, the FLP recombinase produces mosaics of gene expression that vary from animal to animal because the efficiency of recombination is , 100%. These attributes make these systems ideal for mosaic mapping efforts but render them unsuitable for generating populations of flies with the same expression pattern of the transgene. www.sciencedirect.com
Tetracycline-transactivator system Several newer systems have been developed that provide spatial and temporal targeting of gene expression in a manner that is useful for generating homogeneous populations of transgenic animals. One of these is the based on the tetracycline transactivator (tTA), which is regulated by tetracycline (Tet) or its derivatives [21] (Figure 2a). The utility of the Tet system was demonstrated initially by Bujard and colleagues [21]. Similar to the bipartite GAL4– UAS system, the tetracycline transactivator is expressed in a tissue of interest, where it subsequently binds to a tet operator sequence that is placed upstream of a target transgene and activates transcription. In the Tet-Off system, the tTA constitutively activates transcription in the absence of tetracycline, and this activity is silenced in the presence of tetracycline. Bello and colleagues demonstrated the effectiveness of this system in Drosophila by using an eye-specific enhancer to drive the expression of a TetO – LacZ transgene specifically in the eye imaginal disc. LacZ expression was observed as early
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as six hours after the larvae were shifted to food lacking tetracycline. The group also demonstrated the ability to repress the expression of the TetO– LacZ transgene in embryos by feeding the mothers high concentrations of tetracycline. To demonstrate biological function, they used the system to suppress the embryonic and larval lethality that is associated with expression of the Antennapedia homeotic gene under the control of a HoxA7 – tTA construct, and later induced the expression in adults, leading to a bristle phenotype [22]. Stebbins and Yin [23] have subsequently introduced the actin5C promoter to drive the tTA ubiquitously in the fly and incorporated insulating elements into the vectors to reduce position effects on the transgenes.
A second Tet-regulated system (Figure 2b) has been developed in which the presence of doxycycline positively regulates the activity of the tetracycline transactivator (Tet-On) [24]. The Tet-On system was initially reported not to function in Drosophila [22] but Bieshke and colleagues adapted the system for use in the fly [25]. Using the actin5C promoter, transgene induction occurred between eight and 20 hours after feeding, whereas maximal induction of the system required 48 hours of feeding followed by three days of accumulation. Significant levels of b-galactosidase were still observed ten days after removal of the flies from doxycycline. It is not clear at this point whether this represents the stability of the reporter or the persistent activation of the rtTA driver. Long-term
(a) The Tet-Off System
(b) The Tet-On System
– Tetracycline
Enhancer
rtTA
– Tetracycline
tTA
YFG
tTA
rtTA
Enhancer
tet-O
YFG tet-O
tTA rtTA + Tetracycline
Enhancer
+ Tetracycline
tTA
YFG
Enhancer
rtTA
YFG
tet-O
tet-O
(c) The GAL4–UAS-tet system
rtTA
– Tetracycline YFG tet-O GAL4P Enhancer
rtTA
GAL4 UAS
+ Tetracycline rtTA
YFG tet-O TRENDS in Genetics
Figure 2. Schematic diagram of the Tet-Off, Tet-On and GAL4 –UAS– Tet systems. (a) The Tet-Off system. In this system, the tetracycline transactivator protein (tTA) is driven by either a defined promoter or endogenous enhancer in a spatially delineated pattern. In the absence of tetracycline or doxycycline, the transactivator binds to the tet operator and activates transcription of YFG. In the presence of tetracycline or doxycycline, the tTA is maintained in an inactive state, and transcription of your favorite gene (YFG) is silenced. (b) The Tet-On system. In this system, the reverse tetracycline activator (rtTA) is maintained in an inactive state in the absence of tetracycline or doxycycline and transcription of YFG is silenced. In the presence of drug, the rtTA binds to the tet-operator and activates the transcription of YFG. (c) The GAL4 –UAS– Tet system. To capitalize on the expression patterns previously characterized with the conventional GAL4–UAS system, the two tet systems have been combined with the GAL4 –UAS system. The combination of the Tet-On system and the GAL4–UAS system is shown. In this scheme, any GAL4 driver can be used to drive the expression of a UAS –rtTA construct, leading to constitutively high levels of expression of the rtTA in the same cells as the GAL4 driver. The rtTA does not activate the expression of YFG in these cells in the absence of drug. In the presence of the drug, however, high levels of YFG are expressed in these cells, allowing for spatial and temporal control over YFG expression. Abbreviation: UAS, upstream activating sequences. www.sciencedirect.com
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treatment with doxycycline at these levels was also shown to have an adverse impact on the lifespan of the flies. A second generation Tet-On activator, rtTAs-M2, has been generated that yields greater induction capabilities and more stringent regulation [26]. The activator was later modified by removing a putative cryptic splice site, adjusting codon usage and flanking the cassette with insulator sequences, producing an altered rtTA called rtTA-M2-alt TA [27]. With this activator, improved repression of basal transcription, improved kinetics of induction and higher absolute levels of transgene induction, of , 70-fold, have been reported compared with those of the original rtTA transactivator. One drawback with introducing a new system into Drosophila is that the number of useful tissue specific activator lines is small initially. This issue has been addressed by linking both the Tet-Off and Tet-On systems with the GAL4– UAS system [23,27]. In this scheme, a tissue-specific GAL4 line drives the UAS-dependent expression of either tTA or rtTA-M2-alt, which in turn, drives the expression of an additional transgene (Figure 2c). The fold induction observed in these schemes varies greatly depending on the GAL4 driver and target transgenes used but has been reported to be in the range of 30 – 250-fold [23,27]. There are several limitations that are associated with tetracycline systems. The Tet-Off system requires that the animals be exposed to the drug for extended periods. The newer Tet-On system appears to circumvent this issue; however, the full characteristics of this system – including an estimate of fold induction in the presence and absence of doxycycline and the off-rate kinetics – have yet to be described. Furthermore, the ability of the system to be modulated within embryos or pupae, which can not take up the drug by feeding, is unfeasible. Most importantly, although the system is able to capitalize on existing GAL4 drivers to achieve tissue-specific expression of the Tet activators, it currently fails to take advantage of the large numbers of existing UAS reagents, including the Rørth EP collection and the Gene-Search collection [10,11]. In the future, this limitation might be overcome by using tissuespecific rtTA activity to drive the expression of GAL4 and thus make use of these UAS lines. Ligand-inducible GAL4 chimeras A more recent and promising approach for achieving temporal and spatial targeting of gene expression from UAS transgenes is based on hormone inducible GAL4 chimeric proteins (Figure 1b). Both a GAL4– estrogen receptor fusion (GAL4 – ER) and a GAL4– progesterone receptor fusion (GeneSwitch) have recently been demonstrated to function in the fly [15,28,29]. Kinetic analysis of the GAL4 –ER system revealed that b-galactosidase activity reaches detectable levels within 12 hours and maximal levels at 2.5 days after continuous exposure to diethylstilbesterol (DES). Slightly slower induction was observed with b-estradiol. Three days after drug-containing food was withdrawn from the flies the b-galactosidase was undetectable. Han et al. [15] subsequently used the torsolike promoter to drive expression of the GAL4– ER chimera (tslGAL4 – ER) specifically in the border cells of the oocyte. This system was used to drive the expression of the www.sciencedirect.com
I mutant of the A chain of diphtheria toxin (DTI), which is under the control of a UAS sequence. Previous experiments showed that the UAS–DTI transgene, when combined with conventional GAL4 drivers, produces lethality. By contrast, when larvae carrying the tslGAL4 – ER and UAS –DTI transgenes were raised in the absence of DES, normal numbers of progeny were observed, whereas larvae raised in the presence of DES failed to yield any adults. Induction of this system in adult females for threeto-five days with either b-estradiol or DES resulted in the specific elimination of border cells at the anterior edge of the oocyte and posterior follicle cells. Taken together, these experiments demonstrate the utility of the GAL4– ER switch for tissue-specific expression of UAS based transgenes. Another GAL4 chimera was made ligand-inducible by fusing the GAL4 DNA-binding domain to the human progesterone receptor ligand-binding domain (GeneSwitch) [30]. Using the pan-neuronal ELAV enhancer to drive expression of GeneSwitch, Osterwalder and colleagues [28] demonstrated detectable levels of UAS– green fluorescent protein (GFP) expression by western blot as early as five hours after bathing larvae in the anti-progestin, RU486. By 21 hours, the level of GFP protein had become equivalent to that observed in larvae carrying the conventional ELAV– GAL4 driver in combination with the UAS –GFP transgene. Rearing third instar larvae on food containing RU486 resulted in a 51 –60-fold induction of UAS –GFP expression over levels in the absence of RU486. In addition, when mothers were fed RU486, embryos and newly hatched larvae expressed GFP in the nervous system, indicating that RU486 is transferred from mothers to the embryo, where it can activate the GeneSwitch. Driving a UAS– tetanus toxin construct (UAS– TeTxLC) with the conventional ELAV–GAL4 driver prevents embryonic hatching and is uniformly fatal. When the ELAV–GeneSwitch construct was used in combination with the UAS –TeTxLC, however, normal numbers of progeny are recovered in the absence of hormone, indicating little leakiness of the system. No larvae survive to adulthood when they eat food containing as little as 3 mg/ml RU486. GeneSwitch also displays a robust control over the level of gene expression by varying the concentration of RU486 that is fed to larvae [28]. One of the limitations with the ligand inducible GAL4 approach is the lack of multiple tissue-specific expressing driver lines. Therefore, Roman et al. [29] generated enhancer detector constructs containing the RU486 inducible GeneSwitch, and conducted a large-scale screen for expression in the adult head (G. Roman and R. Davis, unpublished). Additional GeneSwitch vector systems have been generated that facilitate rapid cloning of either defined enhancers or promoters upstream of GeneSwitch coding sequences [31]. Kinetic analysis with one of the enhancer detector lines demonstrated that after as little as one hour of RU486 feeding to adult flies, the levels of the reporter b-galactosidase observed at 24 hours were equivalent to those observed in flies that were fed for a full 24 hours. When flies were fed RU486 for 24 hours and then shifted to normal food, b-galactosidase was observed even after six days of withdrawal. It is currently not clear whether this represents the stability of the reporter or the
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persistent activation of the GeneSwitch. The ability to ablate specific tissues with two independent enhancer detector-GeneSwitch lines was demonstrated by driving the expression of a UAS– DTI construct in the fat bodies. Adult flies given chronic administration of RU486 showed ablated fat bodies and 100% lethality after five days of drug treatment. In a test of the GeneSwitch system, Mao et al. [32] fashioned a GeneSwitch driver in which the GeneSwitch cassette was expressed specifically in Drosophila mushroombody neurons using a mushroom-body-specific enhancer. This driver was then used to express a UAS– rutabaga transgene encoding adenylyl cyclase in the memorydefective rutabaga mutant either only during development or only in adult mushroom bodies. This was performed to address the fundamental question of whether the rutabaga gene product is required for the development of the nervous system or for physiological functions in the adult that serve memory formation. Normal rutabaga function restored selectively in the adult mushroom bodies but not during development rescued the memory deficit, thus, answering this question decisively. The TARGET system Given the large number of existing conventional GAL4 and UAS transgenic lines, a powerful addition to the arsenal of ectopic gene expression systems would be a reagent that enabled researchers to capitalize on this universe of well-characterized GAL4 and UAS transgenic lines, while adding the important feature of temporal control of target gene expression. Such a system has recently been developed based on a temperature-sensitive GAL80 protein [33,34]. In yeast, the transcriptional activity of GAL4 is repressed by GAL80 in the absence of galactose. In the presence of galactose, this repression is relieved, enabling GAL4 to activate genes required for galactose metabolism. Recently, a temperature-sensitive variant of GAL80 (GAL80ts) was cloned and used to construct a tubulin promoter-GAL80ts transgene in Drosophila. After introduction into Drosophila, the GAL80ts molecule was shown to regulate GAL4 in a temperature dependent fashion in embryos, larvae, pupae and adults, with optimal repression observed at 198C and derepression beginning at 308C (Figure 1c). This system has been named the TARGET system (temporal and regional gene expression targeting). Kinetic studies of the TARGET system have revealed a slight induction of GFP mRNA as early as 30 min after heat treatment at 328C. Half-maximal levels of RNA were seen at three hours, and by six hours the level of mRNA was roughly equivalent to levels seen in flies carrying the GAL4 driver and the UAS– GFP reporter without the GAL80ts. Estimates of fold induction based on mRNA levels were , 42 – 48-fold. An analysis of the off-rate kinetics showed that mRNA levels returned to half-maximal values by , 15 h, and by 36 h they were at the baseline, uninduced levels. The GAL80ts was shown to repress GAL4 –UAS expression in embryos raised at 198C and shown to enable expression to be induced by shifting the embryos to 308C. Figure 3a illustrates the expression of the reporter UAS – lacZ in the peduncles of the adult www.sciencedirect.com
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mushroom bodies due to the temperature-dependent derepression of GAL4 activity with the TARGET system. Expression is first observed after approximately three hours of incubation at an elevated temperature and accumulates with continued incubation at elevated temperature. The GeneSwitch system exhibits a similar kinetic profile for the induction of UAS– lacZ (Figure 3b). The system has also been used to enable the combination of GAL4 drivers and UAS – toxin constructs that previously could not be combined due to developmental lethality. In particular, the combination of the eye specific GMR– GAL4 driver and a UAS– hid transgene has failed to yield any adult progeny regardless of rearing temperature [14,33]. In the presence of the GAL80ts, however, normal numbers of adults were recovered with eyes that were largely normal in size, shape and pigmentation when the animals were raised at 198C. If the animals were shifted to inducing temperatures at different stages of development, adult flies eclosed with a range of eye phenotypes ranging from the complete lack of any eye structure, eyes that were severely reduced in size, shape and pigmentation, to eyes that were normal in size and shape but lacked pigmentation. The TARGET system has also been used to demonstrate spatial and temporal rescue of the memory defect due to the rutabaga mutation. Mushroom-body-specific GAL4 drivers and a UAS– rutabaga transgene were combined with the GAL80ts to determine whether the rutabaga memory defect was due to a developmental defect in the formation of the mushroom bodies or was due to an acute physiological defect in memory formation. By driving the expression of rutabaga specifically during development, or specifically during adulthood, it was demonstrated that rutabaga expression in the adult mushroom bodies is both necessary and sufficient to rescue the memory defect. Thus, the application of the TARGET system, like the GeneSwitch system, has enabled the dissection of a physiological role for rutabaga in learning and memory from a developmental role in the patterning of the brain. The TARGET system theoretically enables the use of any combination of GAL4 driver and UAS transgene and adds to this the feature of temporal inducibility. In addition to the GAL80ts of the TARGET system described previously, alternative possibilities also exist for temporal regulation of the conventional GAL4– UAS system. One approach has been the use of a light-induced activation of a caged GAL4– VP16 activator [35]. This was used successfully in the embryo to label single cells but its use as a means for generally inducing gene expression in the adult is limited. In addition, one could employ the GAL80 protein in alternative ways. For example, one could place the wild-type GAL80 down stream of the heat-shock promoter to transiently induce GAL80 expression levels and block GAL4 activity. Similarly, one could use specific promoters to drive the expression of wild-type Gal80 in specific cells to constitutively restrict the activity of GAL4 in these cells [36]. Finally, the Tet system could be engineered to regulate temporally the expression of wild-type GAL80 throughout the fly, so as to provide temporal regulation of the conventional GAL4– UAS system.
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(a) 18°C
18°C
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Figure 3. Kinetics of reporter expression for TARGET and GeneSwitch. (a) Using the TARGET system, flies carrying the mushroom body GAL4 driver c739, tubP-GAL80ts, and UAS–lacZ were cultured at 188C and subjected to temperature shifts to 328C for various periods of time as indicated. Frontal sections were stained for b-galactosidase (b-gal) activity and photographs taken at the level of the paired ocelli. Arrows indicate the position of one peduncle of the mushroom bodies. Increased levels of b-gal activity occur with increasing periods of incubation at 328C due to the derepression of GAL4. Modified from Ref. [33] and reproduced with permission q2003 AAAS (www.sciencemag.org). (b) Flies carrying a GeneSwitch element driven by a mushroom body enhancer along with UAS– lacZ were fed RU486 for various periods of time as indicated. Frontal sections were stained for b-gal activity and photographs taken at a level near the base of the peduncle. Increased levels of b-gal activity occur with increased time after initiating feeding on RU486. Modified from Ref. [32] and reproduced with permission q2004 National Academy of Sciences, USA. Abbreviation: UAS, upstream activating sequences.
Conclusions and perspectives Significant progress has been made recently towards the development of improved systems for gene expression with control in both time and space. Although the features that constitute an optimal system might vary depending on the biological question of interest, the following features are desirable for many investigations: (i) Rapid on – off kinetics, with immediate initiation of transcription after the induction event and production of the gene product limited only by the rates of transcription and the formation of the mature product. The turn-off step would be limited only by the decay rates of the pre-existing mRNA and the stability of the protein product. (ii) Inert inducer, with no biological affects other than inducing the system. (iii) Control over the level of expression, with optimally a very broad dynamic range to achieve any desired level of gene expression. (iv) Complete off state, with no basal expression in the absence of the inducer. Most of the systems described meet these characteristics to varying degrees. All of the systems demonstrate relatively slow kinetics, with induction taking hours to days, and the abatement taking generally longer. This is inevitable for any system that is based on the induction of transcription because of the lag time for transcription, post-transcriptional processing, translation and posttranslational processing. Similarly, the off-rate kinetics are determined by the subsequent decay of all previously existing components upon withdrawal. For faster kinetics, systems that target the functional protein product will be required [37]. In addition, it is likely that none of the inducing agents for the systems described are completely www.sciencedirect.com
biologically inert. Inducers such as doxycycline have been shown to interfere with normal development. Elevated temperature also can, depending on the level, produce nonspecific effects that might interfere with a given study. All of the systems described above offer some control over the level of gene expression. For instance, providing heat shock at different temperatures, feeding animals different concentrations of RU486 or doxycycline or feeding animals for different times can provide some control over the level of expression. In addition, it is likely that all of the systems have some basal level of expression, although this might not have been detected in previous experiments. Even with the above caveats, the inducible systems described in this review offer new opportunities for the experimental dissection of a wide range of biological questions. They not only enable experimenter control of gene induction or repression in specific tissues but also provide control over the level of gene expression. This provides a broad venue for gene-dose-response experiments. The systems also offer the ability to perform withinorganism controls, by examining the same organism at two different times in the alternative states of gene expression. Gene rescue experiments [32,33] can be performed to determine whether the expression of a gene in a specific tissue at a specific time is sufficient to correct the phenotype of a mutant organism and in the process shed light on the role of that gene. Gene silencing, through expression of RNAi constructs under the control of gene expression systems with control in time and space, provides a new and extremely potent experimental approach to define whether specific genes are necessary for a particular phenotype in the organism.
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In summary, with the development of the systems described in this review, remarkable opportunities now exist to probe the genetics of development, physiology and behavior with both classical and newer genetic tools. Acknowledgements Research in the authors’ laboratories is supported by National Institutes of Health Grant GM63929 and NS19904. Additional support came from the R. P. Doherty-Welch Chair in Science.
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