BACKGROUND
the most widely used strategies to investigate the function of genes in medaka  are the analyses of mutants, miss-expression of wild type genes or their variants by mrna injection and gene specific translational inhibition by morpholino injections  <cit> .
however, the phenotype of a given mutation mainly reflects the first temporal function of the affected gene in embryonic development, obscuring possible later functions.
similarly, mrna and morpholinos exert their functions immediately following injection, providing information only on the early role of the gene of interest.
a detailed analysis of gene function in a given process can thus be a difficult task.
the gal4/uas system provides an alternative and more specific strategy to analyze specific functions of a gene  <cit> .
the direct application of the drosophila gal4-uas approach, by the generation of transgenic lines, has been established successfully in zebrafish  <cit> .
however, the generation of different transgenic activator and effector lines may be a time- and space-consuming task, and expression levels in these transgenic lines are weak, probably due to a limited transactivation potential of gal <dig> in fish.
gal4/vp <dig>  a fusion of the yeast gal <dig> dna-binding domain with the strong vp <dig> transactivation domain of the herpes simplex virus  <cit>  can be used to enhance transactivation efficacy.
yet, strong transcriptional activators can cause unspecific promoter squelching  <cit>  resulting in retardation of embryogenesis  <cit> .
nonetheless, the gal4/vp16-uas system has been used in zebrafish in transient approaches resulting in mosaic, but easily detectable transgene expression  <cit> .
we have applied the gal4/vp16-uas system for transient transactivation in a heat-shock inducible transgenic gal4/vp <dig> activator line.
generation of transgenic medaka lines, which allow the induction of the gal4/vp <dig> activator to 'physiological'  levels was achieved by using a 5' truncated version of the zebrafish heat-shock promoter hsp <dig>  <cit> .
using a heat-shock promoter to drive expression of the gal4/vp <dig> activator allows tight temporal control of activator and effector  gene expression.
to trace transgene expression in cells of living embryos we have used the cyan fluorescent and yellow fluorescent proteins .
combination with the meganuclease  transgenesis system  <cit>  and the direct-inverted repeats  of the sleeping beauty  transposon system  <cit>  yielded high numbers of transgene expressing cells.
thus, in contrast to the entirely mosaic nature of a transient approach reported thus far, the combined use of a transgenic activator line with systems enhancing even dna distribution or early integration allows uniform expression of injected effector genes upon induction by heat-shock treatment without an immediate need to generate transgenic uas lines.
RESULTS
generation of a heat-shock inducible transgenic gal4/vp <dig> activator line
dna injection leading to mosaic expression in g <dig> allows in vivo tracing of transgene-expressing cells and observation of effects exerted by the transgene through application of fluorescent markers  <cit> .
however, elucidation of biological questions sometimes requires ubiquitous expression of transgenes in a temporally controlled manner.
while the mn protocol strongly reduces mosaicism, it does so only in a fraction of injected embryos .
this can be improved by the use of transgenic animals providing inducible and sufficient expression in all cells.
the idea is to combine stable heat-shock inducible expression of the gal4/vp <dig> activator with transient expression of effector genes upon microinjection.
the effector constructs are uniformly distributed in the entire embryo due to the presence of the sb direct-inverted repeats  <cit> .
we have designed two activator/reporter vectors containing gal4/vp <dig> under control of a  <dig>  kb fragment of the zebrafish  hsp <dig> promoter  or a  <dig>  kb 5' truncated fragment of zfhsp <dig>  respectively .
both vectors contain cfp downstream of several uas elements as an internal reporter.
the ir/drs of sb and two i-scei meganuclease sites flank this entire expression cassette .
the internal reporter provides a direct read-out for activator expression.
a third vector , containing yfp downstream of several uas elements and flanked by ir/drs, was designed as an independent reporter .
it has been shown that the gal4/vp <dig> activator can interfere with general transcription by titrating the basal transcription machinery  <cit> .
we observed developmental retardation and malformation in all embryos injected with gal4/vp <dig> driven by ubiquitous promoters.
similarly, co-injection of high concentrations of gal4/vp <dig> mrna  with pcg <dig> wcs always resulted in developmental malformations .
however, dna co-injections did not affect embryonic development in transient experiments when the hsp <dig> promoter was used to control gal4/vp <dig> expression .
moreover, co-injections of low concentrations of gal4/vp <dig> mrna  with the activator/reporter construct pcg <dig> wcs also showed no effects on embryogenesis , suggesting that the toxicity of the activator depends on the expression level.
the truncated version of the zfhsp <dig> promoter fragment used in the activator/reporter construct pcg <dig> wcs showed a moderate activation upon heat-shock treatment.
this allowed adjusting the induction levels by varying the heat-shock duration.
a transgenic medaka line was established  in which expression levels directly correlated with the heat-shock duration.
extended heat-shocks resulted in very high expression levels, but also caused retardation phenotypes due to the strong transactivation potential of the gal4/vp <dig> fusion protein.
comparable phenotypes were not observed in heat-shock treated wild type embryos.
depending on the developmental stage at the time of induction, the duration of heat-shock treatment was adjusted to induce gal4/vp <dig> and reporter expression without interfering with embryonic development.
induction periods ranged from one minute of heat-shock at 37°c at early stages  to  <dig> minutes at later stages .
on top of uniform cfp expression in the entire embryo and yolk upon heat-shock, some regions of the embryo showed additional responsiveness of the reporter .
microinjection experiments and rt-pcr revealed that reporter gene  expression in transgenic fish is mediated by gal4/vp <dig>  offspring of pcg <dig> -wcs/t transgenic fish was injected with gal4/vp <dig> mrna  at the one-cell stage without heat-shock treatment.
injected embryos exhibited uniform expression of cfp shortly after the onset of zygotic transcription at the mid-blastula transition  <cit> , indicating that cfp expression was induced in response to gal4/vp <dig> .
we applied rt-pcr for the dose/response analysis of activator and reporter mrna .
transcripts of gal4/vp <dig> were detectable already  <dig> minutes after a heat-shock of  <dig> seconds at 37°c.
following a steady increase until about three hours after induction, gal4/vp <dig> messages were degraded between five and ten hours to undetectable levels after twenty hours.
cfp mrna was first detected after two hours and transcript levels were still increasing after  <dig> h. this indicates that the transcription of the reporter cfp is controlled by gal4/vp <dig> protein and that active gal4/vp <dig> is still present when the amount of its transcripts already dropped below detectable levels .
activation of an independent reporter upon injection into the transgenic activator line pcg <dig> wcs/t
we tested the gal4/vp <dig> activator line pcg <dig> wcs/t as a tool to induce expression of an independent reporter upon injection of plasmid dna .
transgenic embryos were injected with different concentrations of the reporter pcg <dig> y .
injected embryos were subjected to heat-shock treatment at different developmental stages for various periods of time, kept at 28°c thereafter and monitored for activator and effector expression during the following days.
due to the sb ir/drs flanking the expression cassette, the independent reporter was distributed equally in the entire embryo resulting in ubiquitous expression of yfp, entirely co-localizing with the internal reporter .
additional mosaic clones of cells expressing yfp at higher levels presumably reflect higher plasmid concentrations in these cells .
however, yfp expression levels appeared relatively independent from the dna concentration, but were directly correlated to the expression levels of the activator or internal reporter, respectively.
CONCLUSIONS
here we show that the gal4/vp16-uas transactivation system can be efficiently used in medaka.
by using fluorescent proteins as internal or independent reporter, cells co-expressing the activator and the gene of interest can be visualized directly.
transparency of these fish embryos allows the evaluation of the cellular fate and response to ectopically expressed genes by time-lapse analyses.
the combination with inducible promoters permits temporal control of effector gene expression and enables the modulation of the response intensity by adjusting the duration of the heat-shock treatment.
this inducible system can be used in transient experiments to study the behavior of transgene expressing cells in an otherwise wild type environment.
the combination with the mn and sb system offers to tailor a range of different levels of mosaicism .
a transgenic gal4/vp <dig> activator line was generated, which provides a powerful tool to induce activator and effector gene expression in a ubiquitous manner at a given time-point .
when used in microinjection approaches of reporter vectors containing ir/drs, our transgenic activator line allows ubiquitous and uniform expression of the reporter gene without the need to generate transgenic effector  lines .
in addition to temporal control mediated by the heat-shock promoter, induction using a focused laser-beam  <cit>  could provide precise spatial control of the effector gene expression.

 the most widely used strategies to investigate the function of genes in medaka are the analyses of mutants, miss-expression of wild type genes or their variants by mrna injection and gene specific translational inhibition by morpholino injections. however, the phenotype of a given mutation mainly reflects the first temporal function of the affected gene in embryonic development, obscuring possible later functions. similarly, mrna and morpholinos exert their functions immediately following injection, providing information only on the early role of the gene of interest. a detailed analysis of gene function in a given process can thus be a difficult task. the gal4/uas system provides an alternative and more specific strategy to analyze specific functions of a gene. the direct application of the drosophila gal4-uas approach, by the generation of transgenic lines, has been established successfully in zebrafish. however, the generation of different transgenic activator and effector lines may be a time- and space-consuming task, and expression levels in these transgenic lines are weak, probably due to a limited transactivation potential of gal in fish. gal4/vp a fusion of the yeast gal dna-binding domain with the strong vp transactivation domain of the herpes simplex virus can be used to enhance transactivation efficacy. yet, strong transcriptional activators can cause unspecific promoter squelching resulting in retardation of embryogenesis. nonetheless, the gal4/vp16-uas system has been used in zebrafish in transient approaches resulting in mosaic, but easily detectable transgene expression. we have applied the gal4/vp16-uas system for transient transactivation in a heat-shock inducible transgenic gal4/vp activator line. generation of transgenic medaka lines, which allow the induction of the gal4/vp activator to'physiological' levels was achieved by using a 5' truncated version of the zebrafish heat-shock promoter hsp. using a heat-shock promoter to drive expression of the gal4/vp activator allows tight temporal control of activator and effector gene expression. to trace transgene expression in cells of living embryos we have used the cyan fluorescent and yellow fluorescent proteins. combination with the meganuclease transgenesis system and the direct-inverted repeats of the sleeping beauty transposon system yielded high numbers of transgene expressing cells. thus, in contrast to the entirely mosaic nature of a transient approach reported thus far, the combined use of a transgenic activator line with systems enhancing even dna distribution or early integration allows uniform expression of injected effector genes upon induction by heat-shock treatment without an immediate need to generate transgenic uas lines.  generation of a heat-shock inducible transgenic gal4/vp activator line dna injection leading to mosaic expression in g allows in vivo tracing of transgene-expressing cells and observation of effects exerted by the transgene through application of fluorescent markers. however, elucidation of biological questions sometimes requires ubiquitous expression of transgenes in a temporally controlled manner. while the mn protocol strongly reduces mosaicism, it does so only in a fraction of injected embryos. this can be improved by the use of transgenic animals providing inducible and sufficient expression in all cells. the idea is to combine stable heat-shock inducible expression of the gal4/vp activator with transient expression of effector genes upon microinjection. the effector constructs are uniformly distributed in the entire embryo due to the presence of the sb direct-inverted repeats. we have designed two activator/reporter vectors containing gal4/vp under control of a kb fragment of the zebrafish hsp promoter or a kb 5' truncated fragment of zfhsp respectively. both vectors contain cfp downstream of several uas elements as an internal reporter. the ir/drs of sb and two i-scei meganuclease sites flank this entire expression cassette. the internal reporter provides a direct read-out for activator expression. a third vector, containing yfp downstream of several uas elements and flanked by ir/drs, was designed as an independent reporter. it has been shown that the gal4/vp activator can interfere with general transcription by titrating the basal transcription machinery. we observed developmental retardation and malformation in all embryos injected with gal4/vp driven by ubiquitous promoters. similarly, co-injection of high concentrations of gal4/vp mrna with pcg wcs always resulted in developmental malformations. however, dna co-injections did not affect embryonic development in transient experiments when the hsp promoter was used to control gal4/vp expression. moreover, co-injections of low concentrations of gal4/vp mrna with the activator/reporter construct pcg wcs also showed no effects on embryogenesis, suggesting that the toxicity of the activator depends on the expression level. the truncated version of the zfhsp promoter fragment used in the activator/reporter construct pcg wcs showed a moderate activation upon heat-shock treatment. this allowed adjusting the induction levels by varying the heat-shock duration. a transgenic medaka line was established in which expression levels directly correlated with the heat-shock duration. extended heat-shocks resulted in very high expression levels, but also caused retardation phenotypes due to the strong transactivation potential of the gal4/vp fusion protein. comparable phenotypes were not observed in heat-shock treated wild type embryos. depending on the developmental stage at the time of induction, the duration of heat-shock treatment was adjusted to induce gal4/vp and reporter expression without interfering with embryonic development. induction periods ranged from one minute of heat-shock at 37°c at early stages to minutes at later stages. on top of uniform cfp expression in the entire embryo and yolk upon heat-shock, some regions of the embryo showed additional responsiveness of the reporter. microinjection experiments and rt-pcr revealed that reporter gene expression in transgenic fish is mediated by gal4/vp offspring of pcg -wcs/t transgenic fish was injected with gal4/vp mrna at the one-cell stage without heat-shock treatment. injected embryos exhibited uniform expression of cfp shortly after the onset of zygotic transcription at the mid-blastula transition, indicating that cfp expression was induced in response to gal4/vp. we applied rt-pcr for the dose/response analysis of activator and reporter mrna. transcripts of gal4/vp were detectable already minutes after a heat-shock of seconds at 37°c. following a steady increase until about three hours after induction, gal4/vp messages were degraded between five and ten hours to undetectable levels after twenty hours. cfp mrna was first detected after two hours and transcript levels were still increasing after h. this indicates that the transcription of the reporter cfp is controlled by gal4/vp protein and that active gal4/vp is still present when the amount of its transcripts already dropped below detectable levels. activation of an independent reporter upon injection into the transgenic activator line pcg wcs/t we tested the gal4/vp activator line pcg wcs/t as a tool to induce expression of an independent reporter upon injection of plasmid dna. transgenic embryos were injected with different concentrations of the reporter pcg y. injected embryos were subjected to heat-shock treatment at different developmental stages for various periods of time, kept at 28°c thereafter and monitored for activator and effector expression during the following days. due to the sb ir/drs flanking the expression cassette, the independent reporter was distributed equally in the entire embryo resulting in ubiquitous expression of yfp, entirely co-localizing with the internal reporter. additional mosaic clones of cells expressing yfp at higher levels presumably reflect higher plasmid concentrations in these cells. however, yfp expression levels appeared relatively independent from the dna concentration, but were directly correlated to the expression levels of the activator or internal reporter, respectively.  here we show that the gal4/vp16-uas transactivation system can be efficiently used in medaka. by using fluorescent proteins as internal or independent reporter, cells co-expressing the activator and the gene of interest can be visualized directly. transparency of these fish embryos allows the evaluation of the cellular fate and response to ectopically expressed genes by time-lapse analyses. the combination with inducible promoters permits temporal control of effector gene expression and enables the modulation of the response intensity by adjusting the duration of the heat-shock treatment. this inducible system can be used in transient experiments to study the behavior of transgene expressing cells in an otherwise wild type environment. the combination with the mn and sb system offers to tailor a range of different levels of mosaicism. a transgenic gal4/vp activator line was generated, which provides a powerful tool to induce activator and effector gene expression in a ubiquitous manner at a given time-point. when used in microinjection approaches of reporter vectors containing ir/drs, our transgenic activator line allows ubiquitous and uniform expression of the reporter gene without the need to generate transgenic effector lines. in addition to temporal control mediated by the heat-shock promoter, induction using a focused laser-beam could provide precise spatial control of the effector gene expression. 
genetic interference by dna, mrna or morpholino injection is a widely used approach to study gene function in developmental biology. however, the lack of temporal control over the activity of interfering molecules often hampers investigation of gene function required during later stages of embryogenesis. the combinatorial properties of this expression system provide a powerful tool for the analysis of gene function during embryonic and larval development in fish by ectopic expression of gene products. we have generated a transgenic gal4/vp activator line that is heat-shock inducible, thereby providing a tool to drive the expression of specific effector genes via gal4/vp merging the gal4/vp16-uas system with the i-scei meganuclease and the sleeping beauty transposon system allows inducible gene expression in an entirely uniform manner without the need to generate transgenic effector lines. to elucidate the roles of genes during embryogenesis a precise temporal control of transgene expression levels in the developing organism is on demand.
 genetic interference by dna, mrna or morpholino injection is a widely used approach to study gene function in developmental biology. however, the lack of temporal control over the activity of interfering molecules often hampers investigation of gene function required during later stages of embryogenesis. to elucidate the roles of genes during embryogenesis a precise temporal control of transgene expression levels in the developing organism is on demand.  we have generated a transgenic gal4/vp activator line that is heat-shock inducible, thereby providing a tool to drive the expression of specific effector genes via gal4/vp merging the gal4/vp16-uas system with the i-scei meganuclease and the sleeping beauty transposon system allows inducible gene expression in an entirely uniform manner without the need to generate transgenic effector lines. combination of this system with fluorescent protein reporters furthermore facilitates the direct visualization of transgene expressing cells in live embryos.  the combinatorial properties of this expression system provide a powerful tool for the analysis of gene function during embryonic and larval development in fish by ectopic expression of gene products. 