takeout: Biological Overview | Regulation | Developmental Biology | References
Gene name - takeout
Synonyms - CG11853
Cytological map position - 96C3--5
Function - potential ligand-binding protein
Symbol - to
FlyBase ID: FBgn0039298
Genetic map position -
Classification - related to ligand-binding proteins
Cellular location - secreted
takeout (to) is a Drosophila circadian clock-regulated output gene, a transcriptional target of the central clock. The Takeout amino acid sequence shows similarity to two ligand binding proteins, including juvenile hormone binding protein. Takeout mRNA is expressed in the head and the cardia (proventriculus), crop (foregut), and antennae -- structures related to feeding. to expression is induced by starvation, which is blocked in all arrhythmic central clock mutants, suggesting a direct molecular link between the circadian clock and the feeding/starvation response. A takeout mutant has aberrant locomotor activity and dies rapidly in response to starvation, indicating a link between locomotor activity, survival, and food status. It is proposed that takeout participates in a novel circadian pathway target that conveys temporal and food status information to feeding-relevant metabolisms and activities (Sarov-Blat, 2000).
Although the function of most circadian rhythm output genes is unknown, the Drosophila lark gene encodes an RNA binding protein involved in the regulation of eclosion (McNeil, 1999), and the mammalian transcription factor DBP exhibits remarkable circadian oscillations and has been shown to control the circadian expression of known downstream genes (Lavery, 1999). However, the mechanism(s) by which the central clock conveys temporal information to downstream behavioral and physiological processes is largely unknown. Both direct neural connections and humoral control have been proposed. Potent circadian activities are found in serum, suggesting that cycling humoral molecules constitute the connection between the brain and the peripheral pacemakers in mammals (Sarov-Blat, 2000 and references therein).
There are now several hormones known to be involved in circadian rhythms. Arguably, the best known example is melatonin. Others include vasopressin, leptin, and hypocretin. These hormones are involved in controlling different behavioral and physiological output activities. Vasopressin, an antidiuretic neuropeptide involved in salt and water balance, has been suggested to be directly controlled by the central clock transcription factor heterodimer Clock-BMAL1, the mammalian homolog of Clock-Cycle. A similar picture has recently emerged for Albumin D-binding protein (DBP). a PAR (proline and acidic amino acid-rich) leucine zipper transcription factor that is expressed according to a robust circadian rhythm in the suprachiasmatic nuclei, harboring the circadian master clock, and in most peripheral tissues (Sarov-Blat, 2000 and references therein).
In Drosophila, the humoral control of circadian behavior is indicated by classic transplantation experiments. A first candidate peptide factor has been proposed, and very recent results indicate that this putative hormone (PDF) is necessary for free-running locomotor activity rhythms. Recent evidence suggests that Pdf expression is controlled by the central pacemaker. It would not be surprising if other hormones important for other specific circadian output pathways were found (Sarov-Blat, 2000 and references therein).
The sequence of Takeout is suggestive of a second hormone involved in a circadian output pathway; it resembles a group of lipophilic ligand binding proteins. to was found in a PCR-based cDNA subtraction screen, in which poly(A)+ RNA from heads of cycle null mutant (cyc01) flies was subtracted from wild-type RNA (So, 2000). to gene expression was down-regulated in all of the clock mutants tested. In wild-type flies, TO mRNA exhibits daily cycling expression but with a novel phase, delayed relative to those of the better-characterized clock mRNAs, Period and Timeless. The E-box-containing sequence in the to promoter shows impressive similarities with those of period and timeless. However, it appears that the E box is not involved in the amplitude and phase of the transcriptional cycling of to. The circadian delayed transcriptional phase is therefore most likely the result of indirect regulation through unknown transcription factors (So, 2000).
takeout involvement in feeding was tested by measuring expression levels under starvation conditions. This is because starvation effects are common for genes involved in lipid and glucose metabolism and even in the regulation of appetite. Indeed, TO mRNA levels are increased after 9 to 10 hr of food deprivation, and a 2 hr refeeding reverses the starvation effects. Induction is most striking in the adjacent gastrointestinal tract regions where to expression was not detectable under normal conditions. tim RNA expression in the gut is more widespread than that of to under normal conditions, and to expression appears to expand and coincide with the tim pattern only when the flies are starved. Induction is also observed in the head. The starvation-induced enhanced expression is also detectable at the protein level. The rapid reversal by refeeding is much less obvious at the protein level, most likely because of a delayed effect on protein by mRNA changes. No enhanced expression is observed with heat shock or oxidative stress (Sarov-Blat, 2000).
Interestingly, an increase in to gene expression is most prominent when the flies are deprived of food at the peak of the mRNA cycle, suggesting that functional clock machinery may be required for this starvation-driven increase in expression. Consistent with this notion, induction is not detectable in arrhythmic clock mutants: per01, tim01, and cyc01 flies. The starvation-induced expression of to could therefore be secondary to an effect on a central clock component. To address this possibility, protein levels of Per and Tim were measured under the same starvation protocol. The results show that they are not enhanced, unlike to. Therefore, starvation-induction of to expression is probably not a secondary consequence of general clock gene induction. Rather, it requires and perhaps works through the clock machinery (Sarov-Blat, 2000).
A to deletion mutant was identified during the analysis of to expression levels in various genetic backgrounds. TO mRNA levels are low in the common lab strain ry506. In addition, Takeout protein levels in ry506 are noncycling and reduced 3-fold, when compared to trough levels of a wild-type (wt) strain. Takeout levels in ry506 are also not increased in response to starvation. These effects are not due to the ry506 mutation itself, because other lab strains bearing the same ry mutation showed normal to expression. to maps far from ry: to is located at 96B19-96C6, whereas ry is located at 87D11. The ry506 mutation was generated by gamma-ray mutagenesis, suggesting that a significant DNA lesion might be present at the to locus. To test this directly, a PCR analysis across the to genomic area was performed in the ry506 strain. To facilitate the analysis, the genomic sequence of to was obtained. The PCR results show that the 3' end region of to is missing in ry506 flies. Because PCR using another pair of primers reveals the presence of the normal stop codon, the genomic deletion in this ry506 strain probably removes the polyA cleavage site or a substantial portion of the 3' UTR, resulting in an unstable transcript (Sarov-Blat, 2000).
Because of the to response to starvation, locomotor activity patterns were examined without food. Flies were transferred to locomotor activity monitors under starvation conditions. Over the subsequent two days, average activity events and the percentage of active flies were monitored as a function of time in LD. The latter parameter approximates survival, since the inactive flies are dead or nearly so. Wild-type flies manifest at least three activity peaks, similar to the morning and evening peaks observed under standard LD conditions. The percentage of active flies slowly decreases during the two days without food. By both criteria, ry506 flies become less active more quickly, i.e., exhibit defects in both circadian activity and survival under these conditions. They show only two peaks of activity, the second significantly smaller than the first, and the average death time (50% inactive flies) was 30 hr for wt flies and 20 hr for the ry506 strain. However, the ry506 flies were more active than wt flies during the activity decline that followed the first activity peak (Sarov-Blat, 2000).
To confirm that the differences between ry506 and the wt strain are the result of the to mutation, two transgenes were introduced into a to mutant background: a tim GAL4 gene and a UAS-to gene. This combination leads to potent to expression in all tim-expressing cells. Two different UAS-to transgenes were examined, and in both cases there was a significant increase in the second activity peak, a small third peaklet, and a dramatically improved survival by about 10 hr of starvation. The transgenic rescue strains also show decreased locomotor activity relative to to after the first peak, even when only active flies are analyzed. No differences in survival rate were observed under heat shock and oxidative stress conditions; in these cases, the to mutant is identical to the wild-type strain. The strain differences indicate that to contributes to wild-type activity patterns and survival under starvation conditions (Sarov-Blat, 2000).
Only under starvation conditions were a survival and locomotor activity phenotype of ry506 flies detected, along with convincing to transgenic rescue. The starvation paradigm was inspired by the to expression pattern, which is localized to body tissues that are relevant to food detection and metabolism: the cardia, the crop, and the antennae. mRNA levels are increased by starvation, in the brain as well as these body tissues, and to affects survival and locomotor activity patterns under these conditions. On this basis, it is suggested that the to brain expression is relevant to feeding (Sarov-Blat, 2000).
Before analyzing to body expression and the locomotor activity patterns under starvation conditions, attempts were made to find a to behavioral phenotype under normal conditions. This was also based on a prior report that rosy alleles have a late eclosion phase, although the free-running period of locomotor activity and eclosion is normal. This phenotype was reproducable and a reproducible delayed locomotor activity phase was also found in ry506 flies. But the eclosion profiles and the delayed locomotor activity phase of the ry506 lines are not rescued with a ry+ transgene, and they are also unaffected by the presence or absence of the to deletion (Sarov-Blat, 2000).
to gene expression is not only under starvation control but also under clock gene control. It exhibits daily oscillating expression and is downregulated in all the clock mutants tested. Based on run-on experiments, to cycling is largely transcriptional, as previously described for per and tim. But the TO mRNA cycle peaks several hours after per and tim, and to transcriptional oscillations may not be dependent on a cis-acting E box (Sarov-Blat, 2000).
It is presumed that clock regulation of to expression contributes to metabolic and even behavioral fluctuations that are relevant to food and feeding. This relationship is underscored by the to starvation response and the lack of a response to starvation in the arrhythmic clock mutant backgrounds. The latter observation implies that the upregulation occurs through a clock mechanism or perhaps requires a functional clock. Consistent with this notion, per01 and tim01 flies die even more rapidly than to flies under starvation conditions. As all to positive tissues appear to express per and tim, this relationship between the circadian clock and to expression might be intracellular. It is likely that to transcription rates change in response to starvation. A circadian regulation of important output functions is consistent with the proposed circadian regulation of olfactory organ activity in Drosophila and in other insects, as recently shown by physiological assays. It is also consistent with the regulation of PDF in Drosophila.
Many to expression features recall leptin, NPY, VGF, and hypocretin -- mammalian hormones with proposed behavioral as well as metabolic functions. These shared properties include circadian regulation, a role for peripheral as well as brain tissue, a starvation response, secretion, and a relationship to ligands and receptors (ligand binding proteins). The latter connection comes from sequence analysis, which suggests that the shared property of the to superfamily is ligand binding. Since juvenile hormone and the molecules bound by JP29 are lipophilic, one can speculate that Takeout also has an endogenous lipophilic ligand relevant to feeding. The features displayed by the to superfamily also resemble the well-characterized lipocalin protein family, present mostly in vertebrates. Lipocalins are classified as extracellular transport proteins, typified by the retinol binding protein, RBP. RBP serves to regulate retinol release from the liver and to transport the insoluble retinol to peripheral target tissues. In general, these small, secreted transporters also serve to protect the ligands from degradation or chemical modification in the circulation. Lipocalin family members display low levels of overall sequence conservation, i.e., pairwise sequence identity is often below 20%, the threshold for reliable alignment. However, after the initial identification of this family, a growing number of crystallographic structures have been solved, and they reveal a compelling structural similarity (Sarov-Blat, 2000 and references therein).
The levels of this putative Takeout ligand might be clock-regulated. In this case, Takeout would serve to amplify or modulate the signal strength. Alternatively, the ligand concentration might be temporally constant, and the clock would then create the metabolic rhythm by controlling the rhythmic expression of Takeout -- the ligand binding protein. Takeout might also control the timing or duration of signal activity, resembling a proposed function of some secreted odorant binding proteins. Starvation would then further increase the signal (Sarov-Blat, 2000).
Based on circadian as well as starvation regulation, Takeout may contribute to an anticipation of food availability. Alternatively, it may contribute to a proper response to a change in food status. A specialized version of the second possibility is a response to starvation. This might include behavioral as well as metabolic changes caused by the absence of food. A similar speculation has been made in the case of NPY and mammals. Starvation might elicit an increase in locomotor activity, to stimulate a search for food. A heightened anticipatory event fits with the normal circadian regulation of Takeout expression, which occurs despite the constant presence of food. Alternatively, starvation might cause a shutdown of activity, to conserve energy, for example. Although the Takeout behavioral response to starvation is only now being subjected to experimental analysis, the mutant versus rescued activity patterns suggest that the mutant flies decrease their activity levels less rapidly during the first activity decline after about 10 hr of starvation. This could contribute to the less successful survival of the mutant strain. Alternatively, it could be irrelevant to survival rate and an independent manifestation of Takeout function under starvation conditions. It could also be a more subtle consequence of the Takeout-mediated events that also lead to decreased survival. If the latter is the case, however, it is not simply due to an increased percentage of dead (viz. immobile) flies, because the mutant strain is still hyperactive after 10 hr of starvation when immobile flies are excluded from the data. In any case, the to responses appear specific for starvation, because there is no effect of heat shock or oxidative stress on to gene expression. Moreover, there is no difference in survival between the to and wild-type strains in response to these two other stresses (Sarov-Blat, 2000).
Although a more detailed examination of the behavioral phenotype is required, two molecular issues are now of great interest. It is important to identify the putative Takeout ligand, as well as its putative intracellular receptor. Identification of both of these molecules will provide important tools and should help deepen an understanding of the relationship between food and circadian rhythms (Sarov-Blat, 2000).
Binding of Clock and Cyc to a 21-bp per E-box-containing fragment has been shown using a yeast one-hybrid assay. To determine if Clk and Cyc also bind the takeout (to) 21-bp E-box-containing sequence, similar yeast one-hybrid assays were performed. Binding to the sequence is signaled by activation of the lacZ gene. Both Clk and Cyc are required to bind the wild-type to 21-bp sequence, but they do not bind to the identical sequence containing a mutated E box (CACGTG to CAGCTG (So, 2000).
Previous studies have shown that a 69-bp E-box-containing per upstream sequence fragment is sufficient to drive robust high-amplitude circadian cycling of reporter gene expression in flies. Since the to sequence and the yeast results suggest a similar to E box, a comparable to 80-bp upstream sequence was tested for its effect on in vivo transcription. The sequence was chosen so that the E box sits in the middle of the 80 bp. Because the nuclear run-on data indicate that the to transcription rate is lower than that of per and tim, a trimer of the 80-bp fragment (to80x3-luc) was assayed. At the same time, a mutated E-box reporter construct was also assayed (to80ex3-luc; the same 2-bp transversion mutations used in the yeast one-hybrid assays). As a control, a per 69-bp trimer (per69x3-luc) was examined in parallel (So, 2000).
Flies transformed with to80x3-luc show surprisingly weak cycling of luminescence. Although the cycling was observed in every line, it was not observed in all experiments. Compared to the per69x3-luc control, not only was the cycling amplitude from the to80x3-luc flies much lower, but luciferase expression was much weaker. Therefore, the to E-box-containing 80-bp fragment does not drive robust transcriptional cycling in vivo. Moreover, the mutated version, to80ex3-luc, shows an identical weak cycling pattern, suggesting that this E box is not relevant to the transcriptional pattern. Consistent with this view, overexpression of Clk driven by a heat shock promoter had no detectable effect on to80x3-luc expression, whereas it clearly induces per69x3-luc expression. Note that all of these trimer promoter constructs, wild type and E-box mutated, results in higher luminescence than the monomer constructs, to-luc and per-luc. This is most likely due to cooperative activity of transcription factors that bind to these regulatory elements (So, 2000).
The failure to observe robust expression and transcriptional oscillation with the to 80-bp fragment suggests that the positive yeast two-hybrid result with the to 21-bp fragment is misleading. Therefore larger to fragment was tested in the yeast system. Consistent with the transgenic fly data, binding of Clk and Cyc to the to E box in yeast becomes undetectable when the E-box-containing sequence is extended from 21 bp to 80 bp. A similar negative result is observed with 1.5 kb and 3.0 kb of to upstream sequence. However, binding of Clk and Cyc to the per E box is unaffected by the increase in sequence length from 21 bp to 69 bp. This suggests that there is a major difference between the per and to E-box regions, which explains the different biological activities in flies (So, 2000).
To provide yet another test of the to E box, S2 cells were transferred with a luciferase reporter gene driven by the 3.0-kb to promoter (to-luc). Luminescence was measured in the presence or absence of cotransfected Clk. There is a 3-fold induction of expression of to-luc by the Clk construct, much less than the 60- and 94-fold induction from the per and tim promoter fragments, respectively. Furthermore, an E-box deletion from the 3.0-kb to promoter does not diminish the level of transcriptional activation, unlike the E-box deletions from the per and tim promoters. The results indicate that the to E box may not contribute to clock-regulated to transcription, suggesting that to transcription requires factors other than Clk and Cyc (So, 2000).
To test the in vivo role of the E box in to transcriptional cycling, transgenic flies carrying to-luc were compared with a 21-bp deletion version that removes the E box. Consistent with the S2 cell data, toDeltaE-luc flies also show cycling of luminescence with amplitude and phase comparable to that of to-luc. This indicates that the E box is not required and that additional elements outside this 21-bp region are sufficient for cycling expression (So, 2000).
TO mRNA expression is down-regulated in cyc01 flies and in all other circadian mutants tested. Its level is undetectable in cyc01 and Clkjrk mutants, as measured by RNase protection and Northern blotting. In contrast, there is detectable TO mRNA in all other genotypes tested, though it is substantially lower than that in wild-type flies. Since there is little or no functional CLK-CYC heterodimer in the cyc01 and Clkjrk backgrounds, the simplest way to explain this observation is that to is directly regulated by CLK and CYC. The higher to transcription in per01, tim01, and per01;tim01 double-mutant flies is presumably due to residual functional CLK-CYC heterodimer in these backgrounds. per01 flies reproducibly show a higher level of to expression than tim01, indicating that Per and Tim may differentially regulate to expression. However, the mechanism underlying this difference is still unknown. When mRNA levels at different time points are measured, to does not show a significant cycling pattern in the clock mutants tested (So, 2000).
The to promoter sequence reveals a remarkable sequence identity with the E-box region of the per and tim promoters. In particular, there is a 9-bp sequence identity around this E-box sequence. The other E-box sequences known in circadian genes usually share the 6-bp core sequence or the core sequence with an additional A (CACGTGA), which has been shown to be strongly preferred by the mammalian BMAL1-MOP4 bHLH-PAS transcription factor heterodimer. In fact, the to and per promoters share 13 out of the 18 bp shown to be sufficient to drive transcriptional activation in S2 cells. This is also consistent with the fact that TO mRNA is undetectable in cyc01 and Clkjrk mutants, suggesting that Clk-Cyc regulates to transcription directly. This would be similar to per and tim transcriptional regulation, despite the phase difference (So, 2000).
The Drosophila somatic sex-determination regulatory pathway has been well studied, but little is known about the target genes that it ultimately controls. In a differential screen for sex-specific transcripts expressed in fly heads, a highly male-enriched transcript was identified encoding Takeout, a protein related to a superfamily of factors that bind small lipophilic molecules. Sex-specific takeout transcripts derive from fat body tissue closely associated with the adult brain and are dependent on the sex determination genes doublesex (dsx) and fruitless (fru). The male-specific Doublesex and Fruitless proteins together activate Takeout expression, whereas the female-specific Doublesex protein represses takeout independently of Fru. When cells that normally express takeout are feminized by expression of the Transformer-F protein, male courtship behavior is dramatically reduced, suggesting that male identity in these cells is necessary for behavior. A loss-of-function mutation in the takeout gene reduces male courtship and synergizes with fruitless mutations, suggesting that takeout plays a redundant role with other fru-dependent factors involved in male mating behavior. Comparison of Takeout sequences to the Drosophila genome reveals a family of 20 related secreted factors. Expression analysis of a subset of these genes suggests that the takeout gene family encodes multiple factors with sex-specific functions (Dauwalder, 2002).
To identify genes under the control of the sex-determination regulatory pathway, a PCR-based subtractive hybridization screen was carried out for sex-specific RNAs expressed in adult fly heads. Head RNA of tra-2/tra-2+ phenotypically wild-type XX adult females was subtracted against the head RNA of sibling XX tra-2/tra-2 mutants, and vice versa. The latter flies are transformed into males both somatically and behaviorally. One cDNA clone that hybridized preferentially with sequences from phenotypic males was isolated and studied in more detail. Northern blot hybridizations confirmed that this sequence represents a highly male-specific 1.1-kb mRNA that was expressed primarily in adult heads. Expression of this mRNA was repressed by Tra-2 in females, since XX tra-2 mutants expressed levels similar to wild-type males. The sequence of the clone was later found to be identical to that of takeout, an independently identified gene responsive to circadian rhythms and starvation (Sarov-Blat, 2000). The takeout gene encodes a secreted protein related to circulating carrier proteins of lipophilic factors, such as the juvenile hormone-binding proteins of other insects. Analysis of RNA prepared at different times during the day failed to reveal any significant variation in takeout levels (Dauwalder, 2002).
Takeout expression has been reported in the adult brain as well as in the cardia and other segments of the digestive system, in which it is induced by starvation (Sarov-Blat, 2000). To identify tissues giving rise to male-specific takeout transcripts, RNA in situ hybridizations were performed on parallel serial sections of adult male and female heads. Surprisingly, no takeout RNA was detected within the adult brain using either of two probes from different regions of the takeout transcription unit, even when samples were overstained. Instead, both probes detected high levels of RNA in the fat body that surrounds the brain as well as in a dispersed population of cells in the third antennal segment. Expression was not detected in males homozygous for the to1 mutation. Comparison of simultaneous hybridizations performed on sections from males and females revealed that fat-body expression was male specific, but that expression in the antennae was not. To confirm this, antennae from male and female heads were dissected and low-cycle RT-PCR was carried out. This showed that although overall accumulation of takeout RNA in whole flies is highly male biased, no difference was apparent in male and female antennal RNA levels. It is concluded that antenna-derived takeout messages account for only a small fraction of all takeout RNA, and that sex-specific expression of takeout in adult heads derives primarily from the fat body. Thus, the sex-specific regulation of takeout varies by tissue type (Dauwalder, 2002).
Takeout has been shown to have similarity to six other Drosophila proteins that are also under circadian control (So, 2000; Claridge-Chang, 2001; McDonald, 2001; Lin, 2002). When used to carry out BLAST-P searches of both the translated Drosophila genome sequence and the entire set of predicted Drosophila proteins, Takeout identifies a family of 20 related proteins. The sequences share interspersed regions of conservation that correspond to regions in Takeout that have similarity with circulating juvenile hormone-binding proteins (JHBPs) of other insects. Although the takeout genes are dispersed to several locations in the genome, most are found in clusters of two or three closely linked genes. To determine whether other members of the takeout family are also expressed in a sex-specific manner, the expression of five randomly selected family members (CG1124, CG2016, CG5867, CG7096, and CG11852) was surveyed in adult males and females. Although transcripts from three of these genes accumulated equally in both sexes, RNAs from CG5867 and CG7096 were found to be male enriched in adult heads. These results support the idea that members of the takeout gene family perform sex-specific functions (Dauwalder, 2002).
Because takeout identifies sexually differentiated cells within the adult head, it was asked whether male sexual identity within these cells is important for male-specific courtship behavior. Specific cell types can be sexually transformed from male to female by forcing them to express the female-specific transformer protein (TraF) by use of a tissue-specific promoter. Taking advantage of the yeast GAL4/UAS system, a 1.27-kb takeout promoter fragment was placed upstream of the Gal4 gene and used to drive expression of a UAS-TraF transgene. When expression of five independent takeout-GAL4 insertions was tested by crossing to a strain carrying a UAS-lacZ transgene, activity of the promoter was found consistently to be most prominently distributed in a pattern similar to that found in in situ hybridization experiments. Within adult heads, activity was detected in fat body as well as in a subset of cells within the maxillary palps and antennae. In sections from whole adult fly bodies, a lower level of expression was also detected within the cardia and in fat cells dispersed throughout the abdomen and thorax (Dauwalder, 2002).
Adult males expressing TraF under the direction of takeout-GAL4 transgenes were observed for their ability to court wild-type females in a mating chamber. Results were represented as the courtship index (CI), which is a measure of the time a male spends performing any of the steps of courtship during a fixed observation period. The takeout-GAL4/UAS-traF flies from all three driver lines tested gave markedly reduced courtship indices in relation to controls, reflecting the fact that these males spent much less time courting females. Feminization directed by the takeout promoter severely lowers the probability that a male courts or sustains courtship beyond the initial steps of orienting and following. Although, on occasion, all steps of courtship can be carried out by such males. These results indicate that the takeout gene is active in sexually differentiated cell types that play an important role in promoting male courtship behavior (Dauwalder, 2002).
The requirement for male identity of takeout-expressing cells is further supported by comparison of the results obtained in the different driver lines tested. As an indicator of feminization, levels of endogenous takeout RNA were examined. If takeout-expressing cell types are completely feminized, it would be expected that endogenous RNA expression will be greatly reduced. Feminization driven by nonsex-specific takeout-GAL4 drivers virtually eliminated takeout RNA expression. This argues that these lines are feminized in cell types that normally express takeout. In contrast, feminization by the male-specific takeout-GAL4 driver was incomplete, and endogenous takeout RNA was only slightly reduced in amount. This is presumably due to the anticipated negative feedback regulation that reduces the level of TraF expressed in these flies. Feminization, as measured in this way, was well correlated with the ability of these flies to court females. It is conclude dthat the degree of feminization of takeout-expressing cell types is related to the ability of males to perform courtship behavior (Dauwalder, 2002).
To determine whether takeout function is required for male mating behavior, courtship assays were performed on takeout mutant males. In a previous study (Sarov-Blat, 2000), it was shown that a rearranged mutant allele of takeout (to1) is fortuitously carried in a laboratory strain on the ry506 third chromosome. A ry506 mutant strain carrying an identical rearrangement was obtained, and it was also found not to express takeout RNA. Therefore, this allele is referred to as to1. PCR and Southern blotting experiments on to1 showed that the deletion found previously (Sarov-Blat, 2000) is associated with a chromosome rearrangement breakpoint and is located entirely within a region between 39 nucleotides upstream and 494 nucleotides downstream of the takeout-translation stop codon (Dauwalder, 2002).
When tested for courtship behavior, ry506 to1 mutant males show no reduction in courtship relative to heterozygous siblings. However, takeout mutant flies that are also heterozygous for fru (ry506 fru4 to1/ry506 fru+ to1 and ry506 fru3 to1/ry506 fru+ to1) showed a significant reduction in courtship relative to a variety of control genotypes tested (Dauwalder, 2002).
Although courtship of the above mutant flies is quantitatively reduced, it is not absent. The mutant males are capable of all steps of courtship, but perform them less frequently, and seem to lack motivation to court. Unlike homozygous fru mutants, takeout mutants displayed no male-chaining behavior, suggesting that these males are capable of distinguishing between males and females as potential mates. Taken together, these results confirm that a simultaneous reduction of fruitless and takeout function interferes with male courtship (Dauwalder, 2002).
The original fru3 and fru4 mutant alleles were generated in a ry506 genetic background. Prior to the above studies, it was noticed that the third chromosomes in the fru3 and fru4 strains carry not only these fru and ry mutations, but also the to1 mutant allele. This led to a test of whether previously observed courtship phenotypes associated with these fru alleles might have been enhanced by the presence of the takeout mutation. The courtship of the double mutant flies (to1, fru4) was compared with a recombinant fru4 line (to+, fru4) carrying the allele of takeout from the wild-type strain Canton-S. The presence of the takeout mutation causes a statistically significant reduction in courtship index. However, this effect is small in relation to that of the fru4 mutation alone (Dauwalder, 2002).
How Dsx and Fru affect takeout expression was examined. Since dsx is known to affect sexual differentiation in males and females, it might either activate takeout in males, repress it in females, or both. takeout expression was compared between dsx homozygous mutant animals and their heterozygous siblings. In blot hybridization experiments, XY dsx individuals were found to have takeout RNA levels reduced by 37% relative to XY dsx/+ flies, indicating that the male-specific Dsx-M product functions to activate takeout expression. In chromosomally XX individuals, dsx mutations have an opposite effect. In comparison with XX dsx/+ siblings, XX dsx/dsx animals have levels of takeout mRNA increased by 13-fold, indicating that Dsx-F normally functions to repress takeout expression. Thus, the differential expression in males and females is achieved (at least in part) by dsx-dependent repression in females and activation in males. Curiously, XX dsx/dsx intersexes have more takeout RNA than do XY dsx/dsx intersexes, suggesting that sex-specific factors other than dsx also affect overall takeout expression (Dauwalder, 2002).
The effect of the dominant dsxSWE allele on takeout expression was examined. Due to a deletion in the female-specific exon that results in constitutive male-specific splicing of the dsx pre-mRNA, this allele produces only Dsx-M. XY flies carrying this allele are phenotypically normal males, and do not have reduced takeout expression. However, in XX; dsxSWE/+ animals, the presence of Dsx-M antagonizes Dsx-F function, resulting in intersexual flies that are similar in phenotype to those produced by dsx null mutations. takeout was derepressed to intermediate levels in such intersexes, further supporting the idea that takeout is controlled by dsx (Dauwalder, 2002).
Analysis of fru mutants demonstrated that it affects takeout expression only in males. Repeated Northern analysis of RNA from XY fru adults showed that the levels of takeout RNA present in these individuals was consistently reduced by about 32% relative to fru/+ males. Expression of takeout was not increased by loss of fru function in XX females. This is consistent with the recent finding that functional sex-specific Fru protein is not present in females. Taken together, the above results indicate that both Fru and Dsx function to specify male-specific expression of takeout RNA (Dauwalder, 2002).
Functional analysis of Dsx and Fru has led to the suggestion that they have distinct and complementary roles, with Fru specifying sexual identity of tissues in the CNS that are responsible for courtship behavior, and Dsx specifying sex in other somatic tissues. However, given the observation that dsx mutants also have minor effects on courtship behavior, it is believed that a clear delineation of the roles played by Dsx and Fru will require more information about the specific genes and cell types whose sexual identity these factors specify (Dauwalder, 2002).
This study has provided evidence that the takeout gene is a target of regulation by the somatic sex-determination pathway. Although takeout expression in some tissues is nonsex-specific, the vast majority of takeout RNA derives from fat body within the adult head and is specific to males. Surprisingly, analysis of RNA from mutant flies indicates that sex-specific takeout expression depends on the function of both Dsx and Fru. Although this would seem to contradict the expected restriction of Fru function to the CNS, it is worth noting that the effect of Fru on takeout expression could be mediated indirectly by diffusible factors. In situ hybridization studies localized fru RNA to a variety of specific neurons, but not the fat body cells in which male-specific takeout RNA is most prominently expressed. Interestingly, the only other instance of sexual differentiation outside of the CNS, where sex-specific Fru function is known to be required, is in the formation of the Muscle-of-Lawrence, a male-specific abdominal muscle in which sexual fate is determined through inductive signals that originate from the innervating motor neuron (Dauwalder, 2002).
Both the dsx and fru genes encode alternatively spliced transcripts that encode distinct forms of the Dsx and Fru proteins in males and females. Thus, both genes could potentially play a role in either activating takeout in males or repressing it in females. Full activation of takeout is not achieved in either dsx null or fru hypomorphic mutant XY individuals and, instead, takeout RNA is present at levels intermediate between those found in males and females. In chromosomal females, only Dsx is required for repression of takeout. The fact that fru mutants do not affect takeout expression is consistent with experiments suggesting that the female-specific form of fru mRNA is not translated into a functional protein. Moreover, all sex-specific Fru functions so far identified have been found in males. Therefore, although a sex-specific Fru mRNA is produced in females that potentially encodes a protein, there is currently no evidence that it functions to regulate sexual differentiation (Dauwalder, 2002).
The fact that dsx is capable of both activating and repressing takeout expression reflects the dsx gene's unusual ability to perform opposite functions in males and females by producing distinct proteins in the two sexes through alternative pre-mRNA splicing. The male-specific (Dsx-M) and the female-specific (Dsx-F) proteins share a common DM domain, which is required for DNA binding. The two proteins differ at their C termini, a region promoting dimerization in both forms. Three potential Dsx-binding sites are located within 1 kb upstream of the takeout translation initiation codon, but further studies will be required to determine whether Dsx proteins associate directly with the takeout promoter. Taken together, the results presented here suggest that Dsx-F and Dsx-M can each either activate or repress the activity of downstream genes. Presumably, the effect Dsx has on any particular gene is also determined by other regulators interacting with the gene's promoter (Dauwalder, 2002).
The male-specific expression of its RNA in tissues closely associated with the adult brain suggested that secreted Takeout protein might affect male-specific behaviors that occur during courtship and mating. After outcrossing it from its original genetic background, it was found that a takeout mutation reduces the ability of males to court and mate with wild-type females. Moreover, a significant synergistic effect on male courtship behavior was observed when to1 was combined with either of two strong hypomorphic mutations in the fru gene to produce flies simultaneously reduced in both takeout and fruitless function. In fru heterozygotes, which have normal male courtship, reduction in takeout function caused a significant reduction in courtship index. Likewise, the effects of fru homozygous mutants on courtship were enhanced by reduction in takeout function. The fact that takeout only affects courtship in certain genetic backgrounds and the observed interaction of takeout and fru mutations, suggests the possibility that the takeout protein acts redundantly on courtship with genes under the control of fru. Redundantly functioning genes might include those from the takeout family itself, some of which were found in this study to also be male-specifically expressed in adult heads. Consistent with this idea, the feminization of takeout-expressing cells in XY individuals using a takeout promoter driven Tra-F cDNA results in a reduction of courtship behavior of males toward females. This effect on behavior clearly exceeds that observed in takeout mutants, suggesting that other sex-specific factors involved in behavior are affected in the feminized tissues (Dauwalder, 2002).
Studies on Drosophila sexual mosaics have identified a region in the posterior brain as the primary tissue in which male differentiation is required for courtship behavior. However, these studies did not exclude the possibility that the male identity of other tissues are also necessary, and subsequent studies support this idea. The effects on behavior observed when Tra-F is driven by a takeout promoter fragment suggest that tissues outside of the adult brain affect behavior. The takeout-GAL4 transgene used to drive Tra-F was unable to produce detectable activity in the CNS. Since these males were strongly reduced in courtship behavior, it seems likely that male differentiation is necessary in a takeout-expressing tissue outside of the CNS. It is worth noting here, however, that the possibility cannot be excluded that an undetectable level of TraF expression under control of the GAL4 driver in the CNS or in other cell types contributes to the observed effects (Dauwalder, 2002).
Cells within the maxillary palp and third antennal segments in which the takeout promoter is active, could potentially mediate the perception of female pheromones. However, the requirement for these tissues in the courtship of females by males is unclear. The ablation of a large proportion of chemosensory sensillae in the antennae does not result in impaired courtship, and other studies indicate that chemosensory organs on the proximal legs mediate the pheromonal response that stimulates courtship. Feminization of the antennal lobes, brain structures to which the antennal neurons project, leads to nondiscriminatory courtship of males toward both males and females, but does not lower courtship toward females. Current evidence for the involvement of maxillary palps in pheromone perception indicates that they mediate inhibitory rather than stimulatory effects on courtship (Dauwalder, 2002).
The only Takeout-expressing tissue in which there is clear evidence for sexual differentiation is the fat body surrounding the male brain, suggesting that it is responsible for the observed effect on courtship. How might fat body affect courtship behavior? These cells are an important source of products secreted into the hemolymph, which circulate throughout the adult body. The juvenile hormone-binding proteins from other insects, to which Takeout is related, are synthesized in the fat body and secreted into the hemolymph, where they are thought to carry juvenile hormone or other small lipophilic ligands to target cells (Dauwalder, 2002).
takeout has been shown to become induced in starving flies and to prolong their survival. This raises the intriguing issue of how the starvation response might be related to mating behavior. One possibility is that takeout is involved in a mechanism governing how males expend their energy when faced with nutrient deprivation. It is easy to imagine that pathways exist for managing the choice between foraging and courtship behavior that are critical for the male's survival and reproductive success. The observation that, in addition to sex and nutrition, takeout also responds to circadian rhythms suggests that it integrates a variety of signals that affect the adult male's behavior (Dauwalder, 2002).
Takeout protein levels oscillate during the circadian cycle. Takeout levels exhibit daily oscillations and peak at around ZT21 to ZT2 (9 hours after lights out until 2 hours after lights on), a 3 to 4 hr delay from the mRNA peak. The effects of central clock mutations on Takeout are similar to the effects on TO RNA: in all four arrhythmic clock mutations, the protein does not cycle. Among the different clock mutations, the highest levels are in per01 and tim01and lowest in cyc01 and Clkjrk. The weak signal in cyc01 and Clkjrk may be due to a basal level of noncycling Takeout expression. Alternatively, the residual signal may reflect a cross-reacting protein (Sarov-Blat. 2000).
TO body mRNA levels might be low because of a very restricted tissue distribution. Indeed, in situ hybridization reveals that TO mRNA is localized to just two areas of the alimentary canal: an inner part of the cardia and the crop. The cardia is a highly folded epithelial structure at the anterior end (gut ectoderm) of the stomach, in the thorax. Its function has not been well defined in fruit flies. The crop is a saccular anterior midgut structure for storing and passing liquid food to the stomach. There was also intense staining of TO mRNA in the antennae, which is an olfactory organ in insects. Therefore, TO mRNA is present in structures related to feeding and smelling, in addition to being in the brain (Sarov-Blat. 2000).
In wild-type flies, TO mRNA levels exhibit a daily fluctuation in both cycling LD and constant dark conditions. The cycling in free-running conditions indicates that this property is a function of the endogenous clock rather than light driven. The amplitude (peak-to-trough ratio) of cycling is about 5, significantly smaller than those of per (~10) and tim (>10) mRNA expression. Interestingly, from several Northern blot analyses and RNase protection assays, the mRNA levels peak at about ZT17 to ZT20, a 2- to 5-h phase delay with respect to the per and tim cycling profiles (So, 2000).
Since to expression is down-regulated in the clock mutants, it was of interest to learn if the regulation is directly via Clk and Cyc. To determine the localization of to expression, mRNA in situ hybridization to fly head sections was performed. For comparison, clock-expressing cells in the brain were also visualized by assaying tim expression as well as Clk and cyc expression. The in situ results show very similar expression patterns at each gene's high time points. All four genes are expressed throughout the brain cortex, especially in the region between the optic lobe and the central complex, where the lateral neurons are located. They are also expressed in photoreceptor cells, although the to photoreceptor signal is generally lower than that in the brain cortex. However, to is not observed in other tim-expressing cells such as the glial cells in the optic lobe, the central body in the central complex, and the proboscis. In general, the tim expression pattern is very similar to that of Clk and cyc. In bodies, the to expression pattern is a subset of the tim expression pattern. The data here suggest that to is expressed in a significant subset of clock-expressing cells in the head. However, due to the low resolution of mRNA in situ hybridization, it is possibile that to is expressed in cells adjacent to clock cells (So, 2000).
Mechanisms composing Drosophila's clock are conserved within the animal kingdom. To learn how such clocks influence behavioral and physiological rhythms, the complement of circadian transcripts in adult Drosophila heads was determined. High-density oligonucleotide arrays were used to collect data in the form of three 12-point time course experiments spanning a total of 6 days. Analyses of 24 hr Fourier components of the expression patterns revealed significant oscillations for ~400 transcripts. Based on secondary filters and experimental verifications, a subset of 158 genes showed particularly robust cycling and many oscillatory phases. Circadian expression is associated with genes involved in diverse biological processes, including learning and memory/synapse function, vision, olfaction, locomotion, detoxification, and areas of metabolism. Data collected from three different clock mutants (per0, tim01, and ClkJrk), are consistent with both known and novel regulatory mechanisms controlling circadian transcription (Claridge-Chang, 2001).
A genome-wide expression analysis was performed aimed at identifying all transcripts from the fruit fly head that exhibit circadian oscillations in their expression. By taking time points every 4 hr, a data set was obtained that has a high enough sampling rate to reliably extract 24 hr Fourier components. Time course experiments spanning a day of entrainment followed by a day of free-running were performed to take advantage of both the self-sustaining property of circadian patterns and the improved amplitude and synchrony of circadian patterns found during entrainment. 36 RNA isolates from wild-type adult fruit fly heads, representing three 2 day time courses, were analyzed on high-density oligonucleotide arrays. Each array contained 14,010 probe sets (each composed of 14 pairs of oligonucleotide features) including ~13,600 genes annotated from complete sequence determination of the Drosophila genome. To identify different regulatory patterns underlying circadian transcript oscillations, four-point time course data was colleced from three strains of mutant flies with defects in clock genes (per0, tim01, and ClkJrk) during a single day of entrainment. Because all previously known clock-controlled genes cease to oscillate in these mutants but exhibit changes in their average absolute expression levels, the analysis of the mutant data was focused on changes in absolute expression levels rather than on evaluations of periodicity (Claridge-Chang, 2001).
To organize the 158 statistically significant circadian transcripts in a way that was informed by the data, hierarchical clustering was performed. Both the log ratio wild-type data (normalized per experiment) and the log ratios for each of the three clock mutants (normalized to the entire data set) were included to achieve clusters that have both a more or less uniform phase and a uniform pattern of responses to defects in the circadian clock. One of the most interesting clusters generated by this organization is the per cluster. This cluster contains genes that have an expression peak around ZT16 and a tendency to be reduced in expression in the ClkJrk mutant. Strikingly, all genes previously known to show this pattern of oscillation (per, tim, vri) are found in this cluster (Claridge-Chang, 2001).
The genes in a second cluster (Clock cluster are primarily grouped together based on their peak phase (average phase ZT2). By virtue of the mutant expression data, several subclusters within this phase group can be identified. The known circadian genes Clock and takeout (to) are part of this cluster. Clk is found in a clustered pair with the leucyl aminopeptidase gene CG9285. In terms of chromosomal organization, to, CG11891, and CG10513 map closely together on chromosome 3R. Two additional circadian genes in this chromosomal region (CG11852, CG10553). Interestingly, the Clk cluster contains three pairs of homologous genes with very similar expression patterns: the UDP-glycosyl transferase genes Ugt35a and Ugt35b, the enteropeptidase genes CG9645 and CG9649, and the long-chain fatty acid transporter genes CG6178 and CG11407. In the first two cases, the homologous genes are also directly adjacent to each other on the chromosome. An overview of the map positions of all circadian genes in this study is available as supplemental information online (http://www.neuron.org/cgi/content/full/32/4/657/DC1). Apart from Ugt35a and Ugt35b, several other genes with a predicted function in detoxification are members of the Clk cluster (CG17524, CG8993, CG3174, Cyp6a21). It may also be noteworthy that the genes for three oxidoreductases found in this group [Photoreceptor dehydrogenase (Pdh), CG15093, CG12116] have almost identical phases (ZT3) (Claridge-Chang, 2001).
A major challenge in translating the positive effects of dietary restriction (DR) for the improvement of human health is the development of therapeutic mimics. One approach to finding DR mimics is based upon identification of the proximal effectors of DR life span extension. Whole genome profiling of DR in Drosophila shows a large number of changes in gene expression, making it difficult to establish which changes are involved in life span determination as opposed to other unrelated physiological changes. Comparative whole genome expression profiling was used to discover genes whose change in expression is shared between DR and two molecular genetic life span extending interventions related to DR, increased dSir2 and decreased Dmp53 activity. Twenty-one genes were found to be shared among the three related life span extending interventions. One of these genes, takeout, thought to be involved in circadian rhythms, feeding behavior and juvenile hormone binding is also increased in four other life span extending conditions: Rpd3, Indy, chico and methuselah. takeout is shown to be involved in longevity determination by specifically increasing adult takeout expression and extending life span. These studies demonstrate the power of comparative whole genome transcriptional profiling for identifying specific downstream elements of the DR life span extending pathway (Bauer, 2010).
The set of common DR induced genes found represents genes important in life span extension as well as genes associated with other nutrient induced physiological functions not directly related to life span, such as decreased fertility. A comparative approach can be used to enrich for genes more specifically related to life span extension by examining life span extending interventions related to DR that do not have some of the same untoward effects as DR. Expression of dSir2 and DN-Dmp53 are two life span extending interventions that are part of the DR life span extending pathway in flies, but do not have decreased fertility. The whole genome expression profiles of flies on a DR diet and long-lived dSir2 expressing flies on a normal diet show a substantial overlap in changes in gene expression, supporting the observations linking dSir2 and DR. As predicted, while DR has many GO categories associated with downregulation of fertility, fewer are seen with dSir2 long-lived flies and none in DN-Dmp53 expressing long-lived flies (Bauer, 2010).
Comparisons of whole genome profiles of flies on DR, expressing dSir2 and expressing DN-Dmp53 revealed a small set of 21 commonly genes predicted to be enriched for genes involved in longevity regulation. takeout was selected to be further examined based upon takeout's known role in regulating feeding behavior and the starvation response as well as its presence in a set of upregulated genes from transcriptional profiles of another life span extending mutant in the fly, Indy. Examination of takeout mRNA levels showed that in addition to takeout being upregulated in DR from three different fly backgrounds it is also upregulated in four additional separate life span extending mutants chico,Rpd3, methuselah and Indy. Confirmation of takeout's role in longevity determination was demonstrated by overexpression in the fat body or nervous system of adult flies and extending life span (Bauer, 2010).
The level of expression of takeout in the overexpression studies is similar to the induction seen with DR, however lifespan extension by takeout over-expression is less than what is observed with DR. This effect may be due to the w1118 background used in these particular experiments, which is known to have a reduced DR response compared to other backgrounds. Alternatively, takeout may be only one of several genes in the DR life span extending pathway that can positively influence lifespan. Other genes, including the additional 19 upregulated genes identified through comparative transcriptional profiling may increase lifespan incrementally, adding up to the lifespan extension total seen in DR or through other genetic interventions (Bauer, 2010).
The mechanism by which increased to expression leads to life span extension is not known. Interestingly, takeout is regulated in a circadian fashion. Increasingly, the link between the circadian system, food intake and aging has been observed. The finding that expression of takeout from any of three different tissues (adult neurons, pericerebral fat body, abdominal fat body) extends life span suggests that the life span related functions of takeout could be due to its hypothesized function as a secreted Juvenile Hormone (JH) binding protein. Although it is not known if the JH binding domain of takeout is functional, reduction of JH levels have been linked to increased longevity in grasshoppers. takeout may bind JH in the hemolymph, thereby reducing JH bioavailability. It has been speculated that the insect ecdysone-JH system may be the functional equivalent of the mammalian thyroid hormone-prolactin axis, which controls important aspects of mammalian basal metabolism. Therefore, proteins such as takeout may be important mediators, linking a nutrient sensing network (DR, dSir2, insulin/insulin-like signaling) with an effector network (JH signaling), which in turn controls behavioral and physiological adaptation pathways (Bauer, 2010).
The current data suggest that multi-factorial gene expression profiling can be successfully used to enrich for genes directly involved in the regulation of longevity, filtering out the noise of other physiological processes. Further refinement of this unbiased approach will be invaluable for discovering factors and signaling pathways involved in aging and lifespan regulation by a variety of modalities and for the identification of targets for specific therapeutic interventions (Bauer, 2010).
Search PubMed for articles about Drosophila takeout
Bauer, J., et al. (2010). Comparative transcriptional profiling identifies takeout as a gene that regulates life span. Aging 2(5): 298-310. PubMed Citation: 20519778
Claridge-Chang, A., et al. (2001). Circadian regulation of gene expression systems in the Drosophila head. Neuron 32: 657-671. 11719206
Dauwalder, B., et al. (2002). The Drosophila takeout gene is regulated by the somatic sex-determination pathway and affects male courtship behavior. Genes Dev. 16: 2879-2892. 12435630
Du, G., Ng, C.S. and Prestwich, G.D. (1994). Odorant binding by a pheromone binding protein: active site mapping by photoaffinity labeling. Biochemistry 16: 4812-4819. 8161541
Lavery, D., et al. (1999). Circadian expression of the steroid 15 alpha-hydroxylase (Cyp2a4) and coumarin 7-hydroxylase (Cyp2a5) genes in mouse liver is regulated by the PAR leucine zipper transcription factor DBP. Mol. Cell. Biol. 19: 6488-6499. 10490589
Lin, Y., et al. (2002). Influence of the period-dependent circadian clock on diurnal, circadian, and aperiodic gene expression in Drosophila melanogaster. Proc. Natl. Acad. Sci. 99: 9562-9567. 12089325
Lorenz, L. J., Hall, J. C. and Rosbash, M. (1989). Expression of a Drosophila mRNA is under circadian clock control during pupation. Development 107: 869-880. 2517256
McDonald, M. J. and Rosbash, M. (2001). Microarray analysis and organization of circadian gene expression in Drosophila. Cell 107: 567-578. 11733057
McNeil, G. P., Zhang, X., Genova, G., and Jackson, F. R. (1999). A molecular rhythm mediating circadian clock output in Drosophila. Neuron 20: 297-303. 9491990
Sarov-Blat, L., So, W. V., Liu, L. and Rosbash, M. (2000). The Drosophila takeout gene is a novel molecular link between circadian rhythms and feeding behavior. Cell 101: 647-656. 10892651
So, W. V., et al. (2000). takeout, a novel Drosophila gene under circadian clock transcriptional regulation. Mol. and Cell. Biol. 20: 6935-6944. 10958689
Touhara, K., et al. (1993). Ligand binding by recombinant insect juvenile hormone binding protein. Biochemistry 32: 2068-2075. 8448166
Wojtasek, H., and Prestwich, G. D. (1995). Key disulfide bonds in an insect hormone binding protein: cDNA cloning of the juvenile hormone binding protein of Heliothis virescens and ligand binding by native and mutant forms. Biochemistry 34: 5234-5241. 7711043
date revised:20 April 2012
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