BACKGROUND
the red flour beetle tribolium castaneum has become established as an important model system, from a group of insects - the coleoptera - that comprises one fourth of all described animal species  <cit>  including numerous pests .
while the technical amenability of the fruit fly drosophila melanogaster remains unmatched, there are topics which cannot be readily addressed in the fly.
on one hand, evolutionary questions require comparative functional data from several species.
moreover, many processes are derived in drosophila and data from more insect typical taxa is needed.
for instance, segments are specified all at one time in drosophila  instead of sequential formation that is characteristic of most insects ; embryonic legs do not develop in drosophila while insect larvae usually do have functional walking appendages; extraembryonic membranes are highly reduced and the head is involuted during embryogenesis in drosophila, resulting in seemingly headless lavae  <cit> .
on the other hand, certain issues of insect biology cannot be studied in drosophila because it lacks the respective character.
one example are the odoriferous defensive glands that play a crucial role in insect communication and defense but are not found in drosophila.
hence, there is a need for complementary insect model systems for comparative functional work and for studying processes that are difficult or impossible to study in drosophila.
recent development of genetic techniques has rendered tribolium the second best insect model system.
its genome is sequenced  <cit> , germ line transformation in tribolium is as efficient as in drosophila, and several marker and transposon systems for gene transfer are available  <cit> .
based on these systems, an insertional mutagenesis system has been established  <cit>  which has been used to generate a collection of enhancer trap and homozygous lethal lines  <cit> .
most notably, robust rnai techniques have been established.
rnai can be applied by embryo injection but the systemic uptake of dsrna also allows injection of female pupae or adults and analysis of the embryonic phenotype in the offspring.
injection of larvae allows to uncover phenotypes during metamorphosis without interfering with earlier  gene functions  <cit> .
apparently, all tissues can be targeted by rnai  <cit>  and the null mutant phenotype can be phenocopied by rnai in many cases  <cit> .
while knock-down of gene function via rnai is extremely efficient in tribolium, spatio-temporally controlled misexpression of genes has not been possible so far.
binary expression systems have the advantage that any gene can be expressed in tissue-specific patterns and at certain developmental stages - depending on the availability of driver lines  <cit> .
this allows the study of dominant lethal or sterility inducing genetic constructs because the transgene is only activated when the driver and responder activities are combined.
one widely used binary expression system is the gal4/uas system, which consists of a driver construct, where expression of the heterologous transactivator gal <dig> is driven by an inducible or tissue specific enhancer.
in the responder construct, the gene of interest is under the control of the heterologous gal4-controlled upstream activating sequence   <cit> .
for driver and responder, separate transgenic lines are generated and upon crossing these strains, the gene of interest is expressed in the progeny in the pattern defined by the driver.
gal <dig> was identified in the yeast saccharomyces cerevisiae as a regulator of gal <dig>  gal <dig> and other genes induced by galactose  <cit> .
gal <dig> regulates transcription by binding to a  <dig> bp site in the uas  <cit> .
the gal <dig> transactivator consists of two functional domains.
the dna binding domain maps to the first  <dig> amino acids whereas the activation domain maps to two regions, amino acids 148- <dig> and 768- <dig>  in the transactivator version gal4Δ, the activation domain is directly fused to its dna binding domain <cit> .
this results in a smaller protein which has been shown to activate reporter gene expression about twice as effectively as the original gal <dig> in drosophila  <cit> .
in the gal4-vp <dig> version, the activation domain of gal <dig> has been replaced by the highly acidic portion of the herpes simplex virus protein vp <dig> that activates transcription of immediate early viral genes  <cit> .
it was shown that gal4-vp <dig> can efficiently activate transcription in mammalian cells  <cit> .
also in drosophila gal4-vp <dig> is more active than gal <dig>  but it has been shown to be less useful compared to gal4Δ given that many insertions of gal4-vp <dig> constructs appear to be non-functional  <cit> .
after its inception in the mouse  <cit> , the gal4/uas system has been established in drosophila where it has become a standard technique adapted to diverse uses  <cit> .
one of the numerous extensions of this system is its combination with gal <dig>  a protein that binds to the carboxy-terminal amino acids of gal <dig> and inhibits activation of transcription  <cit> .
the gal4/uas binary system has been adapted to silkworm bombyx mori  <cit>  zebrafish  <cit> , xenopus  <cit>  and arabidopsis  <cit> .
with this work we establish the gal4/uas system in tribolium.
we show that both gal4Δ and gal4-vp <dig> transactivate well, with gal4Δ being slightly more efficient.
importantly, we find that the use of tribolium endogenous core promoters is essential for efficient expression of transgenes.
methods
constructs
all transactivator and responder constructs were stably integrated into the genome by transposition using the piggybac vectors pbac, pbac  <cit> , pbac  <cit>  or pxl-bacii, which was generated in a series of minimal piggybac constructs along with pxl-bacii, pxl-bacii, and pxl-bacii by exchanging the ecori-ecorv fragment of pxl-bacii-ecfp  <cit>  with an ecori-nrui fragment carrying the fluorescent marker and the restriction sites asci and fsei  <cit> .
however, the integration efficiency of pxl--bacii derivatives appears to be lower than with the other version containing more piggybac sequence .transactivator plasmids: pbac; pbac  <cit> ; pbac  <cit> ; pbac ; pbac .
responder plasmids: p <cit>  bac  <cit> : pbac; pbac; pxl-bacii; pbac ; heat shock plasmid: pbac  <cit> .
the sequences of those constructs that were functional are available at genbank.
sketches of the constructs are depicted in fig.
<dig>  detailed maps are available from the authors.
dm: drosophila melanogaster, tc: tribolium castaneum; hsp: heat shock promoter; bhsp: basal heat shock promoter/core promoter.
tribolium stocks and germline transformation
tribolium germline transformation was performed according to standard procedure by injecting piggybac constructs  into embryos of the vermillionwhite  strain  <cit> , together with  <dig> ng/μl helper plasmid phspbac  <cit> .
a femto jet  device with pulled and cut borosilicate glass capillaries was used for injections.
injected embryos were kept under humid conditions for two days at 32°c/90°f, afterwards transferred to lower humidity and kept at 32°c/90°f until they hatched.
larvae were collected and transferred to full wheat flour.
adult g <dig> beetles were crossed to vw wild type animals, and transgenic beetles were outcrossed again with vw.
transformation markers and epifluorescence microscopy
as transformation markers, egfp  <cit>  , eyfp  <cit> , ecfp  <cit>  as well as the humanized variant dsred <dig>  <cit>  were used.
the  <dig> × p3-driven expression pattern of the fluorescent markers was detected in the eyes of t. castaneum using a leica mz 16fa fluorescence stereomicroscope with a planachromatic  <dig>  × objective .
filter sets used were specifically designed egfp-lp , ecfp-lp , and dsredwide .
tribolium crosses
to activate the binary expression system, adult beetles selected for the dominant markers of the driver and responder lines, respectively, were crossed together and kept for  <dig> days at 28°c/81°f.
within this period of time, sperm from previous matings will be largely replaced by that of the newly added males.
subsequently, the crosses were transferred to fresh flour for egg collections at 32°c/90°f.
transheterozygous embryos, larvae, pupae or adults were heat shocked and analyzed for tgfp fluorescence.
heat shock conditions
embryonic heat shocks were performed in  <dig>  ml eppendorf tubes in a water bath for  <dig> minutes at 46°c/115°f.
larval, pupal and adult heat shocks were performed in  <dig> ml eppendorf tubes for  <dig> minutes at 46°c/115°f.
for activation of the drosophila constructs in tribolium the animals were incubated 1h at 48°c in an incubator.
detection of tgfp fluorescence in embryos
after the heat shock, eggs were allowed to recover at 32°c , and subsequently aligned on a microscope slide and analyzed for tgfp  fluorescence every hour .
these steps were performed at room temperature.
detection of reporter gene expression by in situ hybridization
for comparison of reporter gene expression, eggs from 0- <dig> hour collections at 25°c/77°f were heat shocked and fixed  <dig> hours later.
whole-mount in-situ hybridizations were performed as described before  <cit>  with probes of similar size and concentration .
staining time was the same for all in-situ hybridizations.
RESULTS
drosophila constructs fail to work consistently in tribolium
tribolium beetles transgenic for fluorescent proteins under the control of the artificial 3xp <dig> enhancer-promoter element exhibit strong fluorescence in larval, pupal and adult eyes as well as parts of the nervous system  <cit> .
similar expression is found in a variety of other arthropods  <cit> .
moreover, several drosophila constructs have been shown to work in other species  <cit> .
hence, our first approach to introduce the gal4/uas system in tribolium was to directly transfer constructs tested in drosophila  <cit>  to tribolium.
we used both the transactivator versions gal4Δ and gal4-vp <dig>  driven by the 3xp <dig> enhancer-promoter  <cit> .
transgenic beetles for each of these constructs were crossed to beetles carrying a responder with lacz under the control of uast  or uasp   <cit> .
due to an endogenous ß-galactosidase-like activity in the beetle eyes  we could not use an enzyme activity assay to detect the lacz-reporter.
by western blot we did not detect ß-galactosidase in transheterozygotes while alpha-tubulin was detected.
the positive control from an extract of d. melanogaster heads expressing lacz via a functional lexagad/ <dig> system  <cit>  was readily detected .
we also tested a responder construct with tc-giant  <cit>  under the control of uast  with a driver consisting of gal <dig> under the control of the drosophila heat-shock promoter .
we expected phenotypes similar to those elicited by heat-shock induced giant misexpression in drosophila namely the loss of at least four segments from first instar cuticles  <cit> .
the analysis of heat shocked offspring for cuticle phenotypes did not reveal significant differences to controls that had not been subjected to heat shock .
in conclusion, our attempts to directly transfer drosophila-based constructs to tribolium failed.
gal4Δ and gal4-vp <dig> activate reporter gene expression via endogenous core promoters
core promoters might influence the efficiency of transcription in a species specific way.
in order to test if the failure in the above experiments was due to the use of drosophila core promoters, we made a set of constructs using tribolium specific core promoters in both driver and responder constructs.
we tested two versions of transactivators, gal4Δ and gal4-vp <dig>  <cit> , both driven by the endogenous heat shock inducible tc-hsp <dig> promoter element  which contains hsf binding sites similar to those found in drosophila heat inducible promoters .
this promoter leads to strong ubiquitous transcription within  <dig> minutes after heat shock in embryos.
however, earliest blastoderm stages are refractory to heat shock induced expression while shortly before the morphological differentiation of extraembryonic from embryonic tissue the response is strong .
the responder, turbogfp  was used as reporter, fused to the sv <dig> early mrna polyadenylation signal, and driven by uas sites placed upstream of a  <dig> bp fragment containing the basal  tc-hsp <dig> promoter .
this core promoter does not drive expression on its own but can do so when combined with enhancer elements .
in order to exclude position effects, we analyzed two independent transgenic lines for each construct, i.e.
four activator lines were each crossed to two responder lines.
self crossed uas responders were included as negative controls.
a  <dig> hour egglay was collected and heat shocked , and then checked for fluorescence every hour.
fastest expression of tgfp in embryos could be observed using the uas-tc-bhsp-tgfp# <dig> line in combination with both gal4Δ driver lines, while the gal4-vp <dig> lines took about one hour longer before tgfp fluorescence could be detected .
the uas-tc-bhsp-tgfp# <dig> line tended to be activated later than the # <dig> line, indicating some position effect.
but also with the # <dig> reporter line, gal4Δ tended to perform better than gal4-vp <dig>  on average, when crossing the two different responder lines to the gal4Δ lines, tgfp fluorescence was visible  <dig>  hours after heat shock, whereas in crosses with the gal4-vp <dig> lines  <dig>  hours were necessary for first detection .
response to gal4Δ is slightly faster than to gal4-vp <dig>  shown is the time when first fluorescence of tgfp was visible after heat shock.
the driver lines tc-hsp-gal4Δ# <dig> and # <dig> as well as tc-hsp-gal4-vp16# <dig> and # <dig> were crossed against the responder lines uas-tc-bhsp-tgfp# <dig> and # <dig>  combinations containing driver lines based on gal4Δ result in earlier responder gene expression  than combinations containing gal4-vp16-based lines .
gal4/uas is widely applicable in different tissues and stages of tribolium
in order to test the potential of the gal4/uas system for stage or tissue specific expression in tribolium, we performed heat shocks in larvae, pupae and adult beetles.
we observed tgfp fluorescence in animals carrying both the gal4Δ and uas constructs  <dig> hours after the heat shock treatment, at all stages tested , whereas immediately after the heat shock no fluorescence was detected .
as negative controls served pupae and adults carrying both constructs which were not exposed to heat shock  and heat-shocked adults carrying either the transactivator  or the responder construct alone .
these controls did not show increased fluorescence compared to wild type .
the same was true for larvae .
this experiment suggests that the gal4Δ system is active in the beetle at all postembryonic stages.
we further analyzed several tissues of adult animals for reporter activity  <dig> h after heat shock, and found the system to be active in all tissues examined including the wings , male and female reproductive organs , as well as the gut .
because a ubiquitous gal <dig> driver is currently not available, we were not able to determine if all cells are responsive to the system.
animals of the same genotype that were not subjected to heat shock did not exhibit fluorescence in any of these tissues .
endogenous promoters are required for efficient function of transgenes in tribolium
these results suggest that the use of endogenous core promoters may be critical for the function of transgenes.
to directly compare the relative efficiencies of different core promoters, we crossed the driver line tc-hsp-gal4Δ# <dig> to different responder lines which were based on non-tribolium core promoters , induced gal4Δ expression by heat shock and detected the transcript of the reporter gene by whole mount in situ hybridization.
to achieve comparable staining levels, all probes were approximately the same size and concentration, and the stainings were developed for the same time.
two independent insertion sites for each responder construct were analyzed to control for integration site effects.
as reference we used the above mentioned reporter lines uas-tc-bhsp-tgfp# <dig> and # <dig> were used .
first, we tested a responder construct containing the drosophila basal hsp <dig> promoter  which has been used successfully in drosophila to drive lacz  <cit> .
we did not detect expression of lacz mrna in the offspring .
next, we tested the scp <dig> core promoter which is an artificial core promoter that contains four core promoter motifs - a tata box from the cytomegalovirus  gene ie <dig>  a initiator  based on adenovirus major late  genes and a drosophila g retrotransposon, a motif ten element  from drosophila tollo and a downstream promoter element  from the drosophila g core promoter - within a single promoter construct.
it directs high levels of transcription by rna polymerase ii in nuclear extracts from drosophila and hela cells, and is more efficient than the cmv or adml core promoters  <cit> .
this construct  contains additional gaga elements to prevent position effects  <cit> .
however, transgenic lines #f <dig> and #m <dig>  carrying the guas-scp1-dsred construct , showed no expression of dsred in embryos when crossed to our driver line and heat shocked .
in order to test whether this failure is tribolium specific, we generated two independent drosophila lines using the same guas-scp1-dsred construct.
these lines were crossed to the dpp-gal <dig>  ap-gal <dig>  da-gal <dig> and ptc-gal <dig> drivers and examined at larval stages.
dsred fluorescence was observed with the dpp-gal <dig> driver, roughly consistent with the dpp pattern in discs, but not with the other drivers.
apparently, the vertebrate core promoter guas-scp <dig> is not working well in insects.
together, these results indicate that neither a core promoter from drosophila nor an artificial promoter optimized for vertebrate cells is efficient in driving expression in tribolium.
assuming that endogenous core promoters are required for efficient transcription, we tested another tribolium core promoter.
the tc-hairy upstream region has been analyzed previously and from these data the putative tc-hairy core promoter was deduced  <cit> .
we added this putative basal tc-hairy promoter  just downstream of the dm-hsp70tata in the uas-dm-hsp-lacz construct and exchanged the reporter lacz with egfp.
hence, this construct  contains both drosophila and tribolium core promoters.
when activated by gal4Δ, strong expression of eyfp could be detected predominantly within the central nervous system in old embryos .
at earlier stages expression was not efficient  and the negative control  did not exhibit detectable expression of eyfp in the absence of gal <dig> transactivator activity .
this surprising expression pattern was identical in two independently generated insertions of the same construct.
we confirmed that tc-hairy is not expressed in the central nervous system in embryos of this developmental stage .
hence, this unexpected restriction to the nervous system is probably not due to the integration site but either due to a hidden specific activity of the tc-hairy core promoter, or due to interactions with cryptic binding sites elsewhere in the construct.
thus, although the tc-hairy core promoter can confer strong expression in tribolium, it may not be able to direct expression in all embryonic tissues.
in a similar way we also tested the efficiency of drosophila promoters in the context of gal <dig> drivers, in combination with the responder uas-tc-bhsp-tgfp # <dig>  dm-hs-gal <dig>  utilizes the upstream region of the drosophila hsp <dig> gene  <cit> .
these heat shock constructs are frequently used in drosophila .
the transactivator line dm-hs-gal <dig> # <dig> induced weak expression of the reporter gene tgfp in single cells of advanced embryos  whereas with the transactivator line dm-hs-gal <dig> # <dig> no tgfp expression was detected .
this indicates that the drosophila heat shock promoter can elicit only weak gal <dig> activity in a subset of tribolium tissues which in addition may depend on the integration site.
to explore this further, we tested the ability of the drosophila hsp <dig> promotor to directly drive egfp in tribolium.
studying four independent transgenic lines based on the construct pbac that showed to be functional in the butterfly bicyclus anynana  <cit>  we found that the results were highly variable in tribolium; two lines were capable of activating expression after prolonged heat shock in pupae , one was only mildly heat-inducible  and one was active independent of heat-shock .
some of these responses varied across developmental stages .
taken together, we find that the drosphila heat shock promotor does show some activity in tribolium but its activity is weak and strongly influenced by position effects.
temperature dependence of the gal4/uas system
in drosophila, it has been shown that gal <dig> activity is enhanced in flies raised at 29°c/84°f compared to lower temperatures  <cit> .
therefore we wanted to test, whether this temperature dependence exists in tribolium as well.
<dig> to  <dig> hours old tribolium embryos derived from heterozygous parents carrying the transactivator tc-hsp-gal4Δ# <dig> and the responder uas-tc-bhsp-tgfp# <dig> were heat shocked, dechorionated, aligned on a microscope slide and kept at different temperatures.
pictures were taken directly after the heat shock and in one hour intervals to check for the onset of tgfp fluorescence.
indeed we found that higher temperatures lead to an earlier onset of fluorescence ranging from  <dig> hours  to 3- <dig> hours  .
however, maximal tgfp activity  appears to be similar at both temperatures.
there is little difference in the kinetics of chromophore maturation at 28°c and 37°c for several gfp variants .
therefore, the earlier onset of tgfp fluorescence at higher temperatures could rather be due to higher gal4Δ activity, due to faster kinetics of the gene regulatory machinery, or both.
tribolium development proceeds twice as fast at 32°c versus 26°c .
in these experiments, maximal tgfp levels did not appear grossly different at these temperatures.
the gal4/uas system is applicable in tribolium embryogenesis
finally, we analyzed, whether the gal4/uas response is sufficiently rapid to analyze gene function during tribolium embryogenesis.
we tested how much time it takes from heat shock to expression of the reporter gene using the best combination of driver and responder lines .
a one hour egglay was collected and embryos were allowed to develop further until the end of germ band elongation.
the embryos were then heat shocked and fixed for in-situ hybridization at different time points after the heat shock.
it was unclear how temperature would affect the system - on the one hand embryonic development is about twice as fast at 32°c compared to 25°c, on the other hand the heat shock may slow down development and recovery might be aided by lower temperature.
hence, treated embryos were kept at 32°c or 25°c after heat shock.
embryos kept at 25°c were fixed immediately, or two, four and six hours after heat shock treatment .
embryos kept at 32°c were fixed directly after heat shock and after two, three and four hours, because faster onset was expected at higher temperatures  .
tgfp expression was detected via in-situ hybridization as an immediate readout of gene expression.
earliest tgfp expression could be observed four hours after heat shock  when incubated at 25°c, whereas embryos kept at 32°c after heat shock showed expression already after two hours .
strong expression was detectable already after three hours at 32°c while six hours were required for a similar expression level at 25°c .
at the stages tested, the morphology of the head and the elongation of appendages provide a good measure for the age of the embryo.
we compared overall morphology, head morphology and the length of the trunk appendages at the time of heat shock  and when the reporter was fully expressed  in at least  <dig> embryos per time point.
we do not find a major difference indicating that the target gene response is fast relative to developmental time.
discussion
we have adopted the gal4/uas system to tribolium and find that gal4Δ  <cit>  is slightly superior to gal4-vp <dig>  <cit> .
this came not unexpected as gal4Δ is a much smaller protein that consists only of the dna binding and activation domains of gal <dig>  as potential toxic effects of gal4-vp <dig> have been observed for drosophila  <cit>  and in other organisms  <cit>  we suggest the use of gal4Δ in tribolium in the future.
the gal4Δ/uas system is active at all stages of tribolium development and activates reporter gene expression in a variety of different tissues indicating broad applicability.
interestingly, the activation of a uas target is relatively fast compared to embryonic development which will allow the use of the system to investigate embryogenesis in addition to postembryonic stages.
in fact, development does not seem to proceed much after heat shock .
this is in line with findings in drosophila where a 15' arrest of development has been observed upon heat shock  <cit> .
like in drosophila, tribolium embryos resume development after some time and do not show elevated lethality or cuticle phenotypes due to the heat shock conditions used .
in contrast, the extremely fast mode of drosophila early development has in many cases hampered the use of the gal4/uas system in the study of some processes.
furthermore, gal <dig> is likely to be fully active at the temperatures commonly used to raise tribolium  because they are similar to the temperature optimum for yeast   <cit> .
in contrast to endogenous core promoters, neither the drosophila heat shock core promoter nor an artificial "super core promoter"  consisting of optimized vertebrate core promoter motifs were effective in driving expression in tribolium.
similar experience has recently been described for the tephritid fruit fly ceratitis capitata  <cit> .
on the other hand, drosophila heat shock constructs have been shown to work in the silkworm bombyx mori  <cit>  and the butterfly bicyclus anynana  <cit> .
moreover, the artificial 3xp <dig> enhancer is driving fluorescent reporters in the tribolium eyes and brain using a drosophila core promoter  <cit> , and the same is true in a wide variety of other organisms  <cit> .
in the light of our results it appears that the 3xp <dig> element is extremely efficient in driving expression and hence is able to override the poor function of the exogenous core promoter.
CONCLUSIONS
the establishment of the gal4/uas system in tribolium allows more profound functional gene analysis by directed expression in this species in the future.
this will further promote tribolium as a model organism where general biological questions can be studied.
to take full benefit of the system, it is essential to generate a collection of driver lines that allow misexpression in different tissues.
to this end it will be expedient to perform random insertion screens with gal4Δ-containing mutator constructs following a procedure similar to the one recently used in the large scale geku insertional mutagenesis screen  <cit> .
one important lesson for future transgenic tools in tribolium and probably also other species is that the use of endogenous promoters is necessary for efficient expression.
even if constructs based on exogenous core promoters show some activity under certain circumstances or in some species, for full functionality, the use of species-specific promoters is essential.
authors' contributions
jbs made the constructs and the experiments using the endogenous tribolium promoters and wrote the paper.
gb designed this part of the study and wrote the paper.
constructs based on drosophila core promoters and their tests in tribolium were done by iv, mw, and gb under the guidance of eaw and mk.
the guas-scp <dig> construct and transgenic lines were contributed by ak and ma.
all authors read and approved of the final version of the paper.
supplementary material
additional file 1
drosophila promoter does not work reliably in tribolium.
a) variants of drosophila-based gal4/uas systems do not show any activity in tribolium castaneum.
uas-dependent lacz driven by a drosophila core promoter  does not lead to detectable protein expression in adult heads  or abdomen , when activated by 3xp3-driven gal4Δ  or gal4-vp <dig> .
negative controls: uast responder alone  and vermilionwhite strain without transgenes .
functionality of the anti-ß-galactosidase antibody was confirmed by an extract of drosophila heads expressing lacz by a functional lexagad/ <dig> system .
additionally, reprobing of the blot with an anti-alpha-tubulin antibody was performed as a loading control.
b) the inducibility of the drosophila heat shock promoter is low in tribolium and its activity subject to position effect.
four independent insertions of a construct with the drosophila heat shock  <dig> promoter driving egfp  <cit>  were tested at the pupal stage .
"+hs" indicates heat shocked animals.
as controls, transheterozygotes without heatshock  and a heatshocked wt control  were included.
line a and e showed some activation at the pupal stage while little activation was observed in lines b and d. moreover, line d showed some constitutive activity.
c) the inducibility of the drosophila heat shock promoter is unreliable and partially stage-dependent in tribolium.
heat shock activation of drosophila heat shock  <dig> promoter driving egfp  <cit>  line a is strong only at the pupal stage.
"+hs" indicates heat shocked animals.
as controls, transheterozygotes without heatshock  and a heatshocked wt control  were included.
click here for file
acknowledgements
we thank xiaohui zeng for help with one construct.
this work was supported by the dfg , by the hfspo , the robert bosch foundation  and the marie curie rtn programme 'zoonet' .

 the red flour beetle tribolium castaneum has become established as an important model system, from a group of insects - the coleoptera - that comprises one fourth of all described animal species including numerous pests. while the technical amenability of the fruit fly drosophila melanogaster remains unmatched, there are topics which cannot be readily addressed in the fly. on one hand, evolutionary questions require comparative functional data from several species. moreover, many processes are derived in drosophila and data from more insect typical taxa is needed. for instance, segments are specified all at one time in drosophila instead of sequential formation that is characteristic of most insects; embryonic legs do not develop in drosophila while insect larvae usually do have functional walking appendages; extraembryonic membranes are highly reduced and the head is involuted during embryogenesis in drosophila, resulting in seemingly headless lavae. on the other hand, certain issues of insect biology cannot be studied in drosophila because it lacks the respective character. one example are the odoriferous defensive glands that play a crucial role in insect communication and defense but are not found in drosophila. hence, there is a need for complementary insect model systems for comparative functional work and for studying processes that are difficult or impossible to study in drosophila. recent development of genetic techniques has rendered tribolium the second best insect model system. its genome is sequenced, germ line transformation in tribolium is as efficient as in drosophila, and several marker and transposon systems for gene transfer are available. based on these systems, an insertional mutagenesis system has been established which has been used to generate a collection of enhancer trap and homozygous lethal lines. most notably, robust rnai techniques have been established. rnai can be applied by embryo injection but the systemic uptake of dsrna also allows injection of female pupae or adults and analysis of the embryonic phenotype in the offspring. injection of larvae allows to uncover phenotypes during metamorphosis without interfering with earlier gene functions. apparently, all tissues can be targeted by rnai and the null mutant phenotype can be phenocopied by rnai in many cases. while knock-down of gene function via rnai is extremely efficient in tribolium, spatio-temporally controlled misexpression of genes has not been possible so far. binary expression systems have the advantage that any gene can be expressed in tissue-specific patterns and at certain developmental stages - depending on the availability of driver lines. this allows the study of dominant lethal or sterility inducing genetic constructs because the transgene is only activated when the driver and responder activities are combined. one widely used binary expression system is the gal4/uas system, which consists of a driver construct, where expression of the heterologous transactivator gal is driven by an inducible or tissue specific enhancer. in the responder construct, the gene of interest is under the control of the heterologous gal4-controlled upstream activating sequence. for driver and responder, separate transgenic lines are generated and upon crossing these strains, the gene of interest is expressed in the progeny in the pattern defined by the driver. gal was identified in the yeast saccharomyces cerevisiae as a regulator of gal gal and other genes induced by galactose. gal regulates transcription by binding to a bp site in the uas. the gal transactivator consists of two functional domains. the dna binding domain maps to the first amino acids whereas the activation domain maps to two regions, amino acids 148- and 768- in the transactivator version gal4Δ, the activation domain is directly fused to its dna binding domain. this results in a smaller protein which has been shown to activate reporter gene expression about twice as effectively as the original gal in drosophila. in the gal4-vp version, the activation domain of gal has been replaced by the highly acidic portion of the herpes simplex virus protein vp that activates transcription of immediate early viral genes. it was shown that gal4-vp can efficiently activate transcription in mammalian cells. also in drosophila gal4-vp is more active than gal but it has been shown to be less useful compared to gal4Δ given that many insertions of gal4-vp constructs appear to be non-functional. after its inception in the mouse, the gal4/uas system has been established in drosophila where it has become a standard technique adapted to diverse uses. one of the numerous extensions of this system is its combination with gal a protein that binds to the carboxy-terminal amino acids of gal and inhibits activation of transcription. the gal4/uas binary system has been adapted to silkworm bombyx mori zebrafish, xenopus and arabidopsis. with this work we establish the gal4/uas system in tribolium. we show that both gal4Δ and gal4-vp transactivate well, with gal4Δ being slightly more efficient. importantly, we find that the use of tribolium endogenous core promoters is essential for efficient expression of transgenes. methods constructs all transactivator and responder constructs were stably integrated into the genome by transposition using the piggybac vectors pbac, pbac, pbac or pxl-bacii, which was generated in a series of minimal piggybac constructs along with pxl-bacii, pxl-bacii, and pxl-bacii by exchanging the ecori-ecorv fragment of pxl-bacii-ecfp with an ecori-nrui fragment carrying the fluorescent marker and the restriction sites asci and fsei. however, the integration efficiency of pxl--bacii derivatives appears to be lower than with the other version containing more piggybac sequence.transactivator plasmids: pbac; pbac; pbac; pbac; pbac. responder plasmids: p bac: pbac; pbac; pxl-bacii; pbac; heat shock plasmid: pbac. the sequences of those constructs that were functional are available at genbank. sketches of the constructs are depicted in fig. detailed maps are available from the authors. dm: drosophila melanogaster, tc: tribolium castaneum; hsp: heat shock promoter; bhsp: basal heat shock promoter/core promoter. tribolium stocks and germline transformation tribolium germline transformation was performed according to standard procedure by injecting piggybac constructs into embryos of the vermillionwhite strain, together with ng/μl helper plasmid phspbac. a femto jet device with pulled and cut borosilicate glass capillaries was used for injections. injected embryos were kept under humid conditions for two days at 32°c/90°f, afterwards transferred to lower humidity and kept at 32°c/90°f until they hatched. larvae were collected and transferred to full wheat flour. adult g beetles were crossed to vw wild type animals, and transgenic beetles were outcrossed again with vw. transformation markers and epifluorescence microscopy as transformation markers, egfp, eyfp, ecfp as well as the humanized variant dsred were used. the × p3-driven expression pattern of the fluorescent markers was detected in the eyes of t. castaneum using a leica mz 16fa fluorescence stereomicroscope with a planachromatic × objective. filter sets used were specifically designed egfp-lp, ecfp-lp, and dsredwide. tribolium crosses to activate the binary expression system, adult beetles selected for the dominant markers of the driver and responder lines, respectively, were crossed together and kept for days at 28°c/81°f. within this period of time, sperm from previous matings will be largely replaced by that of the newly added males. subsequently, the crosses were transferred to fresh flour for egg collections at 32°c/90°f. transheterozygous embryos, larvae, pupae or adults were heat shocked and analyzed for tgfp fluorescence. heat shock conditions embryonic heat shocks were performed in ml eppendorf tubes in a water bath for minutes at 46°c/115°f. larval, pupal and adult heat shocks were performed in ml eppendorf tubes for minutes at 46°c/115°f. for activation of the drosophila constructs in tribolium the animals were incubated 1h at 48°c in an incubator. detection of tgfp fluorescence in embryos after the heat shock, eggs were allowed to recover at 32°c, and subsequently aligned on a microscope slide and analyzed for tgfp fluorescence every hour. these steps were performed at room temperature. detection of reporter gene expression by in situ hybridization for comparison of reporter gene expression, eggs from 0- hour collections at 25°c/77°f were heat shocked and fixed hours later. whole-mount in-situ hybridizations were performed as described before with probes of similar size and concentration. staining time was the same for all in-situ hybridizations.  drosophila constructs fail to work consistently in tribolium tribolium beetles transgenic for fluorescent proteins under the control of the artificial 3xp enhancer-promoter element exhibit strong fluorescence in larval, pupal and adult eyes as well as parts of the nervous system. similar expression is found in a variety of other arthropods. moreover, several drosophila constructs have been shown to work in other species. hence, our first approach to introduce the gal4/uas system in tribolium was to directly transfer constructs tested in drosophila to tribolium. we used both the transactivator versions gal4Δ and gal4-vp driven by the 3xp enhancer-promoter. transgenic beetles for each of these constructs were crossed to beetles carrying a responder with lacz under the control of uast or uasp. due to an endogenous ß-galactosidase-like activity in the beetle eyes we could not use an enzyme activity assay to detect the lacz-reporter. by western blot we did not detect ß-galactosidase in transheterozygotes while alpha-tubulin was detected. the positive control from an extract of d. melanogaster heads expressing lacz via a functional lexagad/ system was readily detected. we also tested a responder construct with tc-giant under the control of uast with a driver consisting of gal under the control of the drosophila heat-shock promoter. we expected phenotypes similar to those elicited by heat-shock induced giant misexpression in drosophila namely the loss of at least four segments from first instar cuticles. the analysis of heat shocked offspring for cuticle phenotypes did not reveal significant differences to controls that had not been subjected to heat shock. in conclusion, our attempts to directly transfer drosophila-based constructs to tribolium failed. gal4Δ and gal4-vp activate reporter gene expression via endogenous core promoters core promoters might influence the efficiency of transcription in a species specific way. in order to test if the failure in the above experiments was due to the use of drosophila core promoters, we made a set of constructs using tribolium specific core promoters in both driver and responder constructs. we tested two versions of transactivators, gal4Δ and gal4-vp, both driven by the endogenous heat shock inducible tc-hsp promoter element which contains hsf binding sites similar to those found in drosophila heat inducible promoters. this promoter leads to strong ubiquitous transcription within minutes after heat shock in embryos. however, earliest blastoderm stages are refractory to heat shock induced expression while shortly before the morphological differentiation of extraembryonic from embryonic tissue the response is strong. the responder, turbogfp was used as reporter, fused to the sv early mrna polyadenylation signal, and driven by uas sites placed upstream of a bp fragment containing the basal tc-hsp promoter. this core promoter does not drive expression on its own but can do so when combined with enhancer elements. in order to exclude position effects, we analyzed two independent transgenic lines for each construct, i.e. four activator lines were each crossed to two responder lines. self crossed uas responders were included as negative controls. a hour egglay was collected and heat shocked, and then checked for fluorescence every hour. fastest expression of tgfp in embryos could be observed using the uas-tc-bhsp-tgfp# line in combination with both gal4Δ driver lines, while the gal4-vp lines took about one hour longer before tgfp fluorescence could be detected. the uas-tc-bhsp-tgfp# line tended to be activated later than the # line, indicating some position effect. but also with the # reporter line, gal4Δ tended to perform better than gal4-vp on average, when crossing the two different responder lines to the gal4Δ lines, tgfp fluorescence was visible hours after heat shock, whereas in crosses with the gal4-vp lines hours were necessary for first detection. response to gal4Δ is slightly faster than to gal4-vp shown is the time when first fluorescence of tgfp was visible after heat shock. the driver lines tc-hsp-gal4Δ# and # as well as tc-hsp-gal4-vp16# and # were crossed against the responder lines uas-tc-bhsp-tgfp# and # combinations containing driver lines based on gal4Δ result in earlier responder gene expression than combinations containing gal4-vp16-based lines. gal4/uas is widely applicable in different tissues and stages of tribolium in order to test the potential of the gal4/uas system for stage or tissue specific expression in tribolium, we performed heat shocks in larvae, pupae and adult beetles. we observed tgfp fluorescence in animals carrying both the gal4Δ and uas constructs hours after the heat shock treatment, at all stages tested, whereas immediately after the heat shock no fluorescence was detected. as negative controls served pupae and adults carrying both constructs which were not exposed to heat shock and heat-shocked adults carrying either the transactivator or the responder construct alone. these controls did not show increased fluorescence compared to wild type. the same was true for larvae. this experiment suggests that the gal4Δ system is active in the beetle at all postembryonic stages. we further analyzed several tissues of adult animals for reporter activity h after heat shock, and found the system to be active in all tissues examined including the wings, male and female reproductive organs, as well as the gut. because a ubiquitous gal driver is currently not available, we were not able to determine if all cells are responsive to the system. animals of the same genotype that were not subjected to heat shock did not exhibit fluorescence in any of these tissues. endogenous promoters are required for efficient function of transgenes in tribolium these results suggest that the use of endogenous core promoters may be critical for the function of transgenes. to directly compare the relative efficiencies of different core promoters, we crossed the driver line tc-hsp-gal4Δ# to different responder lines which were based on non-tribolium core promoters, induced gal4Δ expression by heat shock and detected the transcript of the reporter gene by whole mount in situ hybridization. to achieve comparable staining levels, all probes were approximately the same size and concentration, and the stainings were developed for the same time. two independent insertion sites for each responder construct were analyzed to control for integration site effects. as reference we used the above mentioned reporter lines uas-tc-bhsp-tgfp# and # were used. first, we tested a responder construct containing the drosophila basal hsp promoter which has been used successfully in drosophila to drive lacz. we did not detect expression of lacz mrna in the offspring. next, we tested the scp core promoter which is an artificial core promoter that contains four core promoter motifs - a tata box from the cytomegalovirus gene ie a initiator based on adenovirus major late genes and a drosophila g retrotransposon, a motif ten element from drosophila tollo and a downstream promoter element from the drosophila g core promoter - within a single promoter construct. it directs high levels of transcription by rna polymerase ii in nuclear extracts from drosophila and hela cells, and is more efficient than the cmv or adml core promoters. this construct contains additional gaga elements to prevent position effects. however, transgenic lines #f and #m carrying the guas-scp1-dsred construct, showed no expression of dsred in embryos when crossed to our driver line and heat shocked. in order to test whether this failure is tribolium specific, we generated two independent drosophila lines using the same guas-scp1-dsred construct. these lines were crossed to the dpp-gal ap-gal da-gal and ptc-gal drivers and examined at larval stages. dsred fluorescence was observed with the dpp-gal driver, roughly consistent with the dpp pattern in discs, but not with the other drivers. apparently, the vertebrate core promoter guas-scp is not working well in insects. together, these results indicate that neither a core promoter from drosophila nor an artificial promoter optimized for vertebrate cells is efficient in driving expression in tribolium. assuming that endogenous core promoters are required for efficient transcription, we tested another tribolium core promoter. the tc-hairy upstream region has been analyzed previously and from these data the putative tc-hairy core promoter was deduced. we added this putative basal tc-hairy promoter just downstream of the dm-hsp70tata in the uas-dm-hsp-lacz construct and exchanged the reporter lacz with egfp. hence, this construct contains both drosophila and tribolium core promoters. when activated by gal4Δ, strong expression of eyfp could be detected predominantly within the central nervous system in old embryos. at earlier stages expression was not efficient and the negative control did not exhibit detectable expression of eyfp in the absence of gal transactivator activity. this surprising expression pattern was identical in two independently generated insertions of the same construct. we confirmed that tc-hairy is not expressed in the central nervous system in embryos of this developmental stage. hence, this unexpected restriction to the nervous system is probably not due to the integration site but either due to a hidden specific activity of the tc-hairy core promoter, or due to interactions with cryptic binding sites elsewhere in the construct. thus, although the tc-hairy core promoter can confer strong expression in tribolium, it may not be able to direct expression in all embryonic tissues. in a similar way we also tested the efficiency of drosophila promoters in the context of gal drivers, in combination with the responder uas-tc-bhsp-tgfp # dm-hs-gal utilizes the upstream region of the drosophila hsp gene. these heat shock constructs are frequently used in drosophila. the transactivator line dm-hs-gal # induced weak expression of the reporter gene tgfp in single cells of advanced embryos whereas with the transactivator line dm-hs-gal # no tgfp expression was detected. this indicates that the drosophila heat shock promoter can elicit only weak gal activity in a subset of tribolium tissues which in addition may depend on the integration site. to explore this further, we tested the ability of the drosophila hsp promotor to directly drive egfp in tribolium. studying four independent transgenic lines based on the construct pbac that showed to be functional in the butterfly bicyclus anynana we found that the results were highly variable in tribolium; two lines were capable of activating expression after prolonged heat shock in pupae, one was only mildly heat-inducible and one was active independent of heat-shock. some of these responses varied across developmental stages. taken together, we find that the drosphila heat shock promotor does show some activity in tribolium but its activity is weak and strongly influenced by position effects. temperature dependence of the gal4/uas system in drosophila, it has been shown that gal activity is enhanced in flies raised at 29°c/84°f compared to lower temperatures. therefore we wanted to test, whether this temperature dependence exists in tribolium as well. to hours old tribolium embryos derived from heterozygous parents carrying the transactivator tc-hsp-gal4Δ# and the responder uas-tc-bhsp-tgfp# were heat shocked, dechorionated, aligned on a microscope slide and kept at different temperatures. pictures were taken directly after the heat shock and in one hour intervals to check for the onset of tgfp fluorescence. indeed we found that higher temperatures lead to an earlier onset of fluorescence ranging from hours to 3- hours. however, maximal tgfp activity appears to be similar at both temperatures. there is little difference in the kinetics of chromophore maturation at 28°c and 37°c for several gfp variants. therefore, the earlier onset of tgfp fluorescence at higher temperatures could rather be due to higher gal4Δ activity, due to faster kinetics of the gene regulatory machinery, or both. tribolium development proceeds twice as fast at 32°c versus 26°c. in these experiments, maximal tgfp levels did not appear grossly different at these temperatures. the gal4/uas system is applicable in tribolium embryogenesis finally, we analyzed, whether the gal4/uas response is sufficiently rapid to analyze gene function during tribolium embryogenesis. we tested how much time it takes from heat shock to expression of the reporter gene using the best combination of driver and responder lines. a one hour egglay was collected and embryos were allowed to develop further until the end of germ band elongation. the embryos were then heat shocked and fixed for in-situ hybridization at different time points after the heat shock. it was unclear how temperature would affect the system - on the one hand embryonic development is about twice as fast at 32°c compared to 25°c, on the other hand the heat shock may slow down development and recovery might be aided by lower temperature. hence, treated embryos were kept at 32°c or 25°c after heat shock. embryos kept at 25°c were fixed immediately, or two, four and six hours after heat shock treatment. embryos kept at 32°c were fixed directly after heat shock and after two, three and four hours, because faster onset was expected at higher temperatures. tgfp expression was detected via in-situ hybridization as an immediate readout of gene expression. earliest tgfp expression could be observed four hours after heat shock when incubated at 25°c, whereas embryos kept at 32°c after heat shock showed expression already after two hours. strong expression was detectable already after three hours at 32°c while six hours were required for a similar expression level at 25°c. at the stages tested, the morphology of the head and the elongation of appendages provide a good measure for the age of the embryo. we compared overall morphology, head morphology and the length of the trunk appendages at the time of heat shock and when the reporter was fully expressed in at least embryos per time point. we do not find a major difference indicating that the target gene response is fast relative to developmental time. discussion we have adopted the gal4/uas system to tribolium and find that gal4Δ is slightly superior to gal4-vp. this came not unexpected as gal4Δ is a much smaller protein that consists only of the dna binding and activation domains of gal as potential toxic effects of gal4-vp have been observed for drosophila and in other organisms we suggest the use of gal4Δ in tribolium in the future. the gal4Δ/uas system is active at all stages of tribolium development and activates reporter gene expression in a variety of different tissues indicating broad applicability. interestingly, the activation of a uas target is relatively fast compared to embryonic development which will allow the use of the system to investigate embryogenesis in addition to postembryonic stages. in fact, development does not seem to proceed much after heat shock. this is in line with findings in drosophila where a 15' arrest of development has been observed upon heat shock. like in drosophila, tribolium embryos resume development after some time and do not show elevated lethality or cuticle phenotypes due to the heat shock conditions used. in contrast, the extremely fast mode of drosophila early development has in many cases hampered the use of the gal4/uas system in the study of some processes. furthermore, gal is likely to be fully active at the temperatures commonly used to raise tribolium because they are similar to the temperature optimum for yeast. in contrast to endogenous core promoters, neither the drosophila heat shock core promoter nor an artificial "super core promoter" consisting of optimized vertebrate core promoter motifs were effective in driving expression in tribolium. similar experience has recently been described for the tephritid fruit fly ceratitis capitata. on the other hand, drosophila heat shock constructs have been shown to work in the silkworm bombyx mori and the butterfly bicyclus anynana. moreover, the artificial 3xp enhancer is driving fluorescent reporters in the tribolium eyes and brain using a drosophila core promoter, and the same is true in a wide variety of other organisms. in the light of our results it appears that the 3xp element is extremely efficient in driving expression and hence is able to override the poor function of the exogenous core promoter.  the establishment of the gal4/uas system in tribolium allows more profound functional gene analysis by directed expression in this species in the future. this will further promote tribolium as a model organism where general biological questions can be studied. to take full benefit of the system, it is essential to generate a collection of driver lines that allow misexpression in different tissues. to this end it will be expedient to perform random insertion screens with gal4Δ-containing mutator constructs following a procedure similar to the one recently used in the large scale geku insertional mutagenesis screen. one important lesson for future transgenic tools in tribolium and probably also other species is that the use of endogenous promoters is necessary for efficient expression. even if constructs based on exogenous core promoters show some activity under certain circumstances or in some species, for full functionality, the use of species-specific promoters is essential. authors' contributions jbs made the constructs and the experiments using the endogenous tribolium promoters and wrote the paper. gb designed this part of the study and wrote the paper. constructs based on drosophila core promoters and their tests in tribolium were done by iv, mw, and gb under the guidance of eaw and mk. the guas-scp construct and transgenic lines were contributed by ak and ma. all authors read and approved of the final version of the paper. supplementary material additional file 1 drosophila promoter does not work reliably in tribolium. a) variants of drosophila-based gal4/uas systems do not show any activity in tribolium castaneum. uas-dependent lacz driven by a drosophila core promoter does not lead to detectable protein expression in adult heads or abdomen, when activated by 3xp3-driven gal4Δ or gal4-vp. negative controls: uast responder alone and vermilionwhite strain without transgenes. functionality of the anti-ß-galactosidase antibody was confirmed by an extract of drosophila heads expressing lacz by a functional lexagad/ system. additionally, reprobing of the blot with an anti-alpha-tubulin antibody was performed as a loading control. b) the inducibility of the drosophila heat shock promoter is low in tribolium and its activity subject to position effect. four independent insertions of a construct with the drosophila heat shock promoter driving egfp were tested at the pupal stage. "+hs" indicates heat shocked animals. as controls, transheterozygotes without heatshock and a heatshocked wt control were included. line a and e showed some activation at the pupal stage while little activation was observed in lines b and d. moreover, line d showed some constitutive activity. c) the inducibility of the drosophila heat shock promoter is unreliable and partially stage-dependent in tribolium. heat shock activation of drosophila heat shock promoter driving egfp line a is strong only at the pupal stage. "+hs" indicates heat shocked animals. as controls, transheterozygotes without heatshock and a heatshocked wt control were included. click here for file acknowledgements we thank xiaohui zeng for help with one construct. this work was supported by the dfg, by the hfspo, the robert bosch foundation and the marie curie rtn programme'zoonet'.
both gal4Δ and gal4vp driven by the endogenous heat shock inducible promoter of the tribolium hsp gene are efficient in activating reporter gene expression under the control of the upstream activating sequence. the red flour beetle tribolium castaneum has developed into an insect model system second only to drosophila. here we report the establishment of the gal4/uas binary expression system in tribolium castaneum. the response is quick in relation to the duration of embryonic development in tribolium - hours with segmentation being completed after hours - which makes the study of early embryonic processes possible using this system. by comparing the efficiency of constructs based on tribolium, drosophila, and artificial core promoters, respectively, we find that the use of endogenous core promoters is essential for high-level expression of transgenic constructs. our results support the contention that high-level transgene expression usually requires endogenous regulatory sequences, including endogenous core promoters in tribolium and probably also other model systems. uas driven ubiquitous tgfp fluorescence was observed in embryos within four hours after activation while in-situ hybridization against tgfp revealed expression already after two hours.
 the red flour beetle tribolium castaneum has developed into an insect model system second only to drosophila. moreover, as a coleopteran it represents the most species-rich metazoan taxon which also includes many pest species. the genetic toolbox for tribolium research has expanded in the past years but spatio-temporally controlled misexpression of genes has not been possible so far.  here we report the establishment of the gal4/uas binary expression system in tribolium castaneum. both gal4Δ and gal4vp driven by the endogenous heat shock inducible promoter of the tribolium hsp gene are efficient in activating reporter gene expression under the control of the upstream activating sequence. uas driven ubiquitous tgfp fluorescence was observed in embryos within four hours after activation while in-situ hybridization against tgfp revealed expression already after two hours. the response is quick in relation to the duration of embryonic development in tribolium - hours with segmentation being completed after hours - which makes the study of early embryonic processes possible using this system. by comparing the efficiency of constructs based on tribolium, drosophila, and artificial core promoters, respectively, we find that the use of endogenous core promoters is essential for high-level expression of transgenic constructs.  with the established gal4/uas binary expression system, ectopic misexpression approaches are now feasible in tribolium. our results support the contention that high-level transgene expression usually requires endogenous regulatory sequences, including endogenous core promoters in tribolium and probably also other model systems. 