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,1
* European Molecular Biology Laboratory, Heidelberg D-69117, Germany
Institute of Molecular Biology and Biotechnology, FORTH, GR71110 Heraklion, Crete, Greece
1 Corresponding author: Institute of Molecular Biology and Biotechnology, FORTH, Vassilika Vouton, P.O. Box 1527, GR 711 10 Heraklion, Crete, Greece.
E-mail: loukeris{at}imbb.forth.gr
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ABSTRACT |
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An important reverse genetic approach is to regulate discrete temporal and spatial expression of transgenes. Two methods are generally used for this purpose. One directs temporal expression by DNA elements such as the heat-shock protein 70 promoter, which achieves high levels of inducibility following temperature elevation (LIS et al. 1983). Limitations of this method include "leakiness" of the promoter, difficulty in interpreting phenotypes due to the pleiotropic effects of heat-shock regimes, and the ubiquitous spatial expression of the transgene (KREBS and FEDER 1997). The second approach utilizes binary systems such as the GAL4-UAS system, used extensively in Drosophila (BRAND and PERRIMON 1993), and the widely used "tet" conditional expression system (GOSSEN and BUJARD 1992, 2002). The latter is based on transcriptional activators (TA) responsive to tetracycline or analogs such as doxycycline (dox). These are relatively benign drugs that readily cross lipid bilayers, thus penetrating most compartments of the body (BERENS and HILLEN 2003). The tet system requires two sets of transgenic lines: driver lines expressing TAs under control of tissue and/or temporally specific promoters and responder lines carrying a cDNA of interest under the transcriptional control of upstream tet operator-derived response elements (TetO) to which these TAs can bind. Thus, in progeny of crosses between driver and responder lines, the cDNA of interest is under dual regulation: by the specific promoter that controls TA production and by the presence/absence of dox that regulates TA activity. A major advantage is that two alternative TAs exist that are affected by dox in the opposite manner. The original transactivator (GOSSEN and BUJARD 1992) is inactivated by dox, preventing transcription ("Tet-Off" system), whereas the reverse transactivator (URLINGER et al. 2000) is activated by dox, leading to rtTA binding to TetO and thereby permitting transcription ("Tet-On" system).
We have initially chosen to control production of tet-dependent transactivators using a promoter sequence from the A. gambiae SRPN10 locus (DANIELLI et al. 2003). Previous analysis established SRPN10 as a potentially important innate immunity locus, which is activated by parasite invasion of the midgut epithelium and is also expressed in a subset of hemocytes and the pericardial cells (DANIELLI et al. 2003). Transcript and protein analysis also suggested that SRPN10 is synthesized in larval and pupal stages. Attempts to block parasite transmission by the expression of Plasmodium inhibitors in transgenic mosquitoes would be greatly facilitated by systems that ensure conditional regulation of the transgene in mosquito tissues encountered by the parasite and that respond to invasion.
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MATERIALS AND METHODS |
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RESULTS |
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Pericardial cell and hemocyte-specific expression modulated by dox and blood feeding:
Using appropriate fluoresence filter sets, larvae and pupae that carried driver or responder transposons were readily identified by their green or red fluorescence, respectively; carriers of both markers in the progeny of crosses between driver and responder lines were easily distinguished (Figure 1C). The identification of Actin5C-eGFP- and Actin5C-dsRed-positive adults was reliable, but required practice to distinguish the GFP signal, which is substantially blocked by the adult cuticle; the dsRed signal is obvious at all postembryonic stages. Double carriers showed tissue-specific ß-galactosidase expression in both larval and adult mosquitoes, primarily in pericardial cells and a set of cells (typically 5–10 µm in diameter) predominantly found attached to the trachea, but also observed in tissues, including ovaries, fat body, and flight muscle, as well as in isolated hemolymph (which we believe are hemocytes, as discussed below, and refer to them as such herein). Furthermore, this expression was appropriately modulated by the presence or absence of dox (Figures 2–4). This section describes this predominant ß-galactosidase expression pattern and its modulation, while a detailed comparison of dox-regulated gene expression, the complex pattern of expression in the midgut, and a low level of background staining in the responder lines are considered later.
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A majority of individuals from crosses with the tTA2 line showed a greater intensity of staining than those derived from the tTA1 line, but significantly there was the same tissue distribution of expression (not shown). In addition, all individuals from a line made homozygous for both tTA1 and TetOPLacZ6 insertions also showed the same tissue distribution and greater intensity of staining compared to individuals heterozygous for both insertions (not shown).
The pattern of rtTA-driven gene expression was assessed in a similar manner in the progeny of crosses with TetOPLacZ responder lines. Initial tests in adults determined that addition of dox (100 µg/ml–10 mg/ml) to the sucrose food source for 24 hr induced ß-gal activity in the pericardial cells and hemocytes (data not shown). Again, blood feeding substantially increased the level of ß-gal staining in both cell types (Figure 2I vs. 2L; Figure 3D). Strong induction of expression was also detected in the larval progeny of these crosses after addition of dox at 10 ng/ml or higher into the larval breeding water (Figure 4B). No significant difference in the dox-dependent expression patterns was observed in crosses between all the different rtTA lines and two responder lines selected for assay.
Background expression:
In the absence of transactivators, only low background levels of X-gal staining were observed in certain tissues of certain responder lines, but not in wild-type controls. Three responder lines showed ß-gal staining in the hindgut epithelium and in stripes along the hypodermis in larval stages, but not in the adult. In all but one responder line a small number of sporadic larval and adult midgut cells also stained, as did the imaginal rings (adult precursors) of the posterior (indicated in Figure 5, B and C) and anterior midgut valves (CLEMENTS 1992) in late fourth instar larvae in the absence of transactivators. One TetOPLacZ line failed to show any transactivator-dependent or background staining. Two lines (TetOPLacZ 5 and 6) characterized by the presence or absence of hindgut and hypodermal background were kept for future analysis.
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Following dox treatment of rtTA/tetOPLacZ larvae, newly emerged adults showed ß-gal staining in association with the meconium formed by the remnants of the larval midgut (Figure 5J; CLEMENTS 1992), but virtually none in the new midgut epithelium. In aged adults, specific ß-gal activity was undetectable without dox treatment (Figure 6A, a) but was strongly induced following exposure to dox (Figure 6A, b), with kinetics similar to those of pericardial cell staining (Figure 6B).
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Dox regulation of tTA and rtTA:
Since the SRPN10 promoter is active in both larvae and adults we were able to compare the ability of the two tet systems to switch transactivation at different developmental stages. For analysis of dox-regulated expression in larvae, we characterized the effects of various dox concentrations in the breeding water. Continuous exposure to 1 µg/ml dox caused reduced growth rate and increased failure to eclose, whereas no detrimental effect was observed with exposure to <100 ng/ml (data not shown). In tTA/tetOPLacZ fourth instar larvae kept under continuous dox treatment, pericardial cell staining indicated a concentration-dependent inhibition of tTA transactivation by dox (Figure 4A). The inhibition was detectable at 0.1 ng/ml and complete at 1 ng/ml. Removal of dox at the second instar stage led to expression by the fourth instar, indicating significant clearance of dox within 4 days.
To regulate expression in adults, dox was added to the sucrose pad used for feeding. Initial experiments indicated that adults could tolerate up to 1 mg/ml continuous exposure without obvious detrimental effects. Above this concentration, increased death rate was observed (not shown). Most experiments with adults were thus performed using a concentration of 100 µg/ml. Exposure of tTA-bearing adults to dox at this concentration for 48 hr prior to blood feeding did not eliminate but reduced substantially (>12-fold) the percentage of stained hemocytes (Figure 3D) and the intensity of staining in pericardial cells relative to nontreated controls (Figure 2A vs. 2B). The intensity of pericardial cell staining in sugar-fed and blood-fed tTA/tetOPLacZ mosquitoes was further reduced by continuous exposure to dox (100 µg) over a further 3-day period compared to the respective non-dox-treated controls, but was not completely eliminated in either group (not shown).
The switching of gene expression by rtTA, as monitored by ß-gal staining, is clearer. In the absence of dox, no rtTA-dependent reporter gene expression could be detected in either larvae or adults, yet Tet-On switching was effective after dox application at both stages (Figures 2–4). Interestingly, as shown in Figure 4, higher concentrations of dox in second instar larvae were required to activate the rtTA driver (10 ng/ml) than to inhibit tTA (1 ng/ml). We also determined that removal of dox from the water during the second instar of rtTA/tetOPLacZ larvae significantly reduced the staining intensity observed at fourth instar, as compared to those larvae under continuous exposure (Figure 4B).
The kinetics of appearance of ß-gal activity in adult rtTA/tetOPLacZ female mosquitoes was followed after supplementing the sucrose used for ad libidum feeding of the adults with dox solution. Detectable ß-gal activity in the pericardial cells and midgut was absent at 2 hr but observed after 7 hr of exposure to dox, while at later times (24 and 48 hr) substantially higher accumulation of activity was observed in both tissues (Figure 6B).
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSLITERATURE CITED |
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A possible but unlikely explanation for the lack of active tTA expression in the midgut might be position effects on all tTA insertions. A more reasonable explanation, however, is the difference in the composition of the alternative transactivators. At the outset of this work we used the original tTA, which functions efficiently in Drosophila (BELLO et al. 1998) and carries the original, unmodified bacterial sequence and complete VP16 activation sequence (GOSSEN and BUJARD 1992). The rtTA, introduced later in the work, was synthetically "restructured" according to human codon usage and carries three copies of the short activation domain of VP16 (URLINGER et al. 2000). This transactivator is significantly more stable than previous versions (URLINGER et al. 2000) and less prone to transcription factor "squelching" and toxicity, which occur after Gal4 or VP16 overexpression in some organisms or tissues (GILL and PTASHNE 1988; DUFFY 2002). These modifications may make the rtTA specifically more active than tTA in the mosquito midgut. If so, we would expect pronounced midgut expression by the use of the next generation tTA (URLINGER et al. 2000), which has been similarly modified.
In the progeny of crosses between driver and responder lines, small cells (typically 5–10 µm) expressing LacZ, under dox regulation, have been found attached to tissues including trachea, fat body, ovaries, and epidermis, as well as circulating in the hemolymph. In this report we have constantly referred to these cells as hemocytes. Cells of the same morphology and distribution have also been previously reported as hemocytes in mosquitoes (CLEMENTS 1992; HILLYER and CHRISTENSEN 2002) and are described as belonging to the plasmatocyte class of hemocytes, thought to be professional phagocytes in Drosophila (MEISTER and LAGUEUX 2003). Cells of this description in the mosquito have been shown to express proteins homologous to those found in hemocytes of other insects or associated with immune responses (MULLER et al. 1999; DANIELLI et al. 2000, 2003; LEVASHINA et al. 2001; BLANDIN et al. 2004; OSTA et al. 2004). Significantly, SRPN10 gene products are included among these proteins (DANIELLI et al. 2003).
An intriguing finding has been the increase in the percentage of ß-gal-expressing hemocytes observed following blood feeding, which is enhanced at 21° in the tTA lines that have been most extensively examined. This change is not affected by a parasite burden and appears blood-meal specific as the percentages are not significantly different between sugar-fed mosquitoes at alternative temperatures. Blood-meal ingestion by female mosquitoes triggers a series of physiological responses among which the best studied are probably digestion and vitellogenesis. We have previously reported a complex organization of the SRPN10 promoter, according to Genomatix Matinspector analysis (DANIELLI et al. 2003), which indicated the presence of multiple putative binding sites for transcription factors controlling morphogenesis and other processes. The promoter fragment used in this study carries putative binding sites for Dorsal, activator protein 1 (AP1), c-REL, CCAAT enhancer binding protein, ADF-1, and GATA factors. The latter participate in numerous morphogenetic and physiological events, including hematopoiesis (MEISTER and LAGUEUX 2003), through synergistic interactions with other transcriptional activators (MERIKA and ORKIN 1995; BRODU et al. 1999; WATANABE et al. 2000). In Aedes aegypti it has been shown that the concentration of GATA activity in the fat body increases dramatically following an infective blood meal, contributing substantially to the extremely high levels of vitellogenin expression (KOKOZA et al. 2001). Therefore, we cannot exclude the possibility that the blood meal triggers similar regulatory circuits in the hemocytes and that the higher fraction of hemocytes expressing LacZ following a blood meal correlates with increased GATA activity. However, there is no further evidence to substantiate these suggestions. It is also not clear why the increase in the percentage of LacZ-expressing hemocytes should be greater after blood feeding and maintenance at the lower temperature, yet it is reasonable to speculate that physiological and molecular responses affecting hemocyte gene expression during metabolism of the blood meal may also be induced by changes in environmental temperature.
In adult midgut cells invaded by P. berghei, transcriptional induction from the SRPN10 locus correlates with morphological manifestations of apoptotic cell death, suggesting a possible implication of SRPN10 in this process (DANIELLI et al. 2003). In midguts isolated from infected A. stephensi, these invaded and apoptotic cells are easily identified by their characteristic rounded shape and are found bulging away from the midgut epithelium. In dox-treated, P. berghei-infected rtTA/tetOPLacZ mosquitoes, clearly higher reporter gene activity was detected in these cells compared to the rest of the midgut epithelium, indicating that the promoter fragment used reflects, in addition, this aspect of SRPN10 regulation.
Strong reporter expression in late fourth instar midguts is also consistent with the hypothesis that the SRPN10 promoter is regulated in cells fated to undergo cell death. In mosquitoes the larval midgut epithelium is destroyed during morphogenesis and replaced by adult tissue during pupation (CLEMENTS 1992). Cytologically distinct alterations first occur during the prepupal stage in the gastric cecum before spreading to the posterior and then the anterior midgut (CLEMENTS 1992). In Drosophila, it is known that the extensive midgut remodeling begins with programmed cell death triggered by the ecdysone pulse that marks the onset of larval-to-pupal metamorphosis; cell death is also first apparent in the ceca 
4 hr before pupal formation and then spreads to the entire epithelium at pupation (JIANG et al. 1997). The ß-gal staining patterns that we observe correlate well with the onset of morphogenesis of the mosquito midgut and suggest a general role for SRPN10 in programmed cell death.
Transactivation by both Tet-On and Tet-Off systems can be switched by dox supplements in a broad developmental range from second instar larvae to adult stages. The very low effective concentrations used to switch expression suggest that dox penetration in larvae is highly efficient. Dox intake through feeding is also efficient in the adult, where addition of dox to the sucrose food source is sufficient to control pericardial cell, hemocyte, and midgut expression. Higher dox concentrations are needed to "switch on" rtTA than to "switch off" tTA-dependent transcription of the same transgene. This is consistent with the in vitro binding characteristics of the two activators with dox and the empirical results of their use in mammalian cells (GOSSEN and BUJARD 2002). The regulation imparted by rtTA is "tighter" operationally (cf. Figures 2 and 3D) in that ß-galactosidase activity is undetectable in the absence of dox, but is rapidly induced in its presence. However, it is not known to what extent the observed difference between the systems may simply reflect the slow turnover of ß-galactosidase, since in testing the tTA system the kinetics of ß-gal degradation will be a major factor in determining the efficiency of observed regulation. The 1- to 2-day half-life of ß-gal shown in mammals (SMITH et al. 1995), if applicable to mosquitoes, would make the total elimination of staining difficult to achieve in the time course of these experiments. Later time points (not shown), up to 7 days after continuous dox treatment, show a continuing loss of ß-gal activity relative to nontreated controls. Despite this continued inhibition, we cannot be sure that repression is complete in the adult stage. Nevertheless, it must be borne in mind that during larval stages, where dox can be given before LacZ expression is evident, it is possible to eliminate ß-galactosidase activity throughout mosquito development. In future applications of the systems, specific characteristics such as protein half-life and transcriptional strength of the requisite driver construct will be important parameters that may need to be assessed for each gene under study. At this stage we can say that both systems are suitable for use in the mosquito since dox concentrations needed for regulation are below levels that affect growth and eclosion.
Background expression:
The limited background LacZ expression in hindgut, hypodermis, imaginal rings, and sporadic midgut cells in responder lines in the absence of transactivators indicates that there is some "leakage" from the TetOPLacZ expression cassette. Three of six responder lines with independent insertions all show similar background expression patterns, suggesting that sequences within the expression cassette can promote expression in certain contexts. Similarly, specific transient fluorescence has been detected in the hindgut following injection into wild-type embryos of a plasmid carrying yellow FP under regulation by the TetOP promoter (our unpublished results). Moreover, hypodermis and hindgut-specific expression has also been observed in stable transgenic A. gambiae lines carrying alternative promoter-GFP fusions (J. R. CLAYTON and G. J. LYCETT, unpublished results). The present report is the first to examine mosquito promoter function by specific localization of reporter gene activity in mosquitoes and further work is required to determine whether the background patterns are common.
The hybrid promoter used in the present study and in Drosophila (BELLO et al. 1998) consists of a heptamer of tetO-binding sites fused to the P transposase minimal promoter. In pilot experiments on transfected A. gambiae cell lines, this promoter showed lower background activity than a TetO-heat-shock protein 70 promoter alternative (data not shown) and was thus the choice for this study. However, analysis of a broad range of alternative minimal promoter sequences is often necessary to achieve low background and specific expression in certain cell types (HOFFMANN et al. 1997; LEUCHTENBERGER et al. 2001). Clearly, this may be of critical importance in certain applications of the system to mosquitoes. It must be emphasized, however, that despite a low level of background expression being observed, we have succeeded in identifying those transgenic responder lines that give minimal background yet retain high levels of responsiveness to dox.
Utility of the tet systems in Anopheles:
Undoubtedly, numerous genes must play key roles in mosquito development, innate immunity, and parasite transmission. This supposition has recently been experimentally supported by RNAi silencing approaches in the mosquito (BLANDIN et al. 2002, 2004; OSTA et al. 2004). Rigorous characterization of the function of these genes will depend on conditional expression systems such as those demonstrated in this article. The major advantages of conditional binary systems include the ability to regulate gene expression by an exogenous molecule such as doxycycline (and so monitor phenotypic changes within the same genotypic background), the ability to target gene expression to specific tissues or stages, and their logistic efficiency (a matter of importance because of the labor intensity of Anopheline strain maintenance). Rather than generating multiple "unitary" lines anew, it would be possible with the tet systems to generate a collection of reusable drivers, each specifying a precisely defined expression pattern. The phenotypes caused by expression or silencing a gene of interest in the available driver patterns could then be assessed by crossing these with a newly generated line bearing the appropriate responder construct.
Apart from their importance for the functional analysis of genes, the tet systems may represent enormously valuable enhancements of the sterile insect techniques envisioned for mosquito control (THOMAS et al. 2000; BENEDICT and ROBINSON 2003) or even localized species eradication (KRAFSUR et al. 1986). Such methods involve mass release of males (either sterile or carrying female-specific dominant lethal genes) to reduce the effective breeding population. The logistics of generating males for release would be greatly facilitated by a genetic sexing method, in which the breeding stocks needed would be routinely maintained and then switched to strictly male-only mass production by addition or removal of a simple compound to food, such as doxycycline. The efficacy of this approach has been demonstrated by utilizing the tTA system in Drosophila (THOMAS et al. 2000; HORN and WIMMER 2003) and the present report makes its adoption feasible in Anopheles.
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ACKNOWLEDGEMENTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSLITERATURE CITED |
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LITERATURE CITED |
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