InteractiveFly: GeneBrief

white: Biological Overview | References

BIOLOGICAL OVERVIEW

Monoamines such as dopamine, histamine and serotonin (5-HT) are widely distributed throughout the brain of the fly brain, where many of their actions have been investigated. For example, histamine is released from photoreceptor synapses in the lamina neuropile of the visual system. Mutations of the genes white, an important eye pigmentation marker in fly genetics that encodes an ABC transporter, and its binding partner brown, cause neural phenotypes not readily reconciled solely with actions in eye pigmentation. Flies mutant for these genes, and another binding partner, scarlet, have about half the wild-type amount of histamine in the head, as well as reduced 5-HT and dopamine. These differences parallel reductions in immunoreactivity to the corresponding biogenic amines. They also correlate with the amine content of fractions after differential centrifugation of head homogenates. Thus, most of the amine is found in the vesicle-rich fraction of wild-type head homogenates, whereas it is found in the supernatant fractions from white, brown and scarlet flies. White co-expresses in lamina epithelial glia with Ebony, which conjugates histamine to β-alanine. Histamine is then released when the conjugate is hydrolyzed in photoreceptors, by Tan. Mutant white ameliorates the effects of tan on head histamine whereas it exacerbates the effects of ebony. These results are consistent with the proposal that histamine uptake by the epithelial glia may be white dependent. Behavioral abnormalities in white, brown and scarlet mutants could arise because aminergic neurons in the Drosophila brain have reduced amine for release (Borycz, 2008).

Screening pigments such as melanin in vertebrates (e.g., Oyster, 1999) or ommochromes and pteridines in insects (Phillips, 1980) provide obvious means by which eyes screen their photoreceptor neurons from excess light or glare. As a result, corresponding pigmentation mutants, such as albino mammals and white-eyed Drosophila have defective vision, especially at high light intensities. These defects are, however, only partly attributable to an inability to screen stray light. For example, albino mammals of many different species have neurological as well as retinal defects. In Drosophila, although white mutants are positively phototactic their vision is not normal; they lack optomotor responses, for example, and have an abnormal electroretinogram (ERG). In other pigmentation mutants of Drosophila, neurological defects are not restricted to the visual system. Thus, mutants of the kynurenine pathway of tryptophan metabolism, a precursor in the biosynthesis of brown ommochrome pigment, exhibit neural defects. The mutants cardinal (with excess 3-hydroxykynurenine) and cinnabar (with excess kynurenine) exhibit deficits in learning and memory and altered volumetric changes in the mushroom bodies of the brain (Savvateeva, 2000). vermilion mutants (which lacks kynurenines) suffer a gradual decline in learning and memory, and an increase in mushroom body volume. These mutants also have decreased immunoreactivity to the cysteine string protein (CSP), which functions at late stages of Ca²⁺-regulated exocytosis of synaptic vesicles, implicating the action of both genes at synapses. Collectively, these examples indicate the pleiotropic action of pigmentation genes in the nervous system (Borycz, 2008 and references therein).

Pigmentation mutations have readily identifiable phenotypes. white was in fact the first genetic mutant to be isolated in Drosophila (Morgan, 1910), and its obvious eye phenotype leads to its widespread use as a genetic marker. Extreme white alleles and white deficiencies remove both brown and red pigments (Hadorn, 1951). Yet mutants of white, which encodes an ABC transporter, and its binding partner brown (Mount, 1987), have behavioral and other phenotypes not readily reconciled with an action in the eye. For example, volatile general anesthetics reveal behavioral differences attributable to neuronal action (Campbell, 2001), and possibly related, white mutants have reduced performance in spatial learning (Diegelmann, 2006). Preliminary work provides evidence that, in addition to their action in loading pigment granules in the eye, White and its binding partners may be involved in a hitherto unappreciated transport function for biogenic amines (Borycz, 2005a; Borycz, 2008 and references therein).

In order to reveal a neural phenotype for white and its binding partner genes, the neurotransmitter phenotypes of corresponding mutants were examined. This could be examined most readily at one site in the fly's visual system, the first optic neuropile, or lamina, where the synaptic terminals of photoreceptors from the compound eye use histamine as a neurotransmitter, are also large, and vesicle-laden, and contain the highest concentration of the brain's histamine (Borycz, 2005b; Borycz, 2008 and references therein).

ABC transporters are paired heterodimer ATPase transporter proteins with many cellular functions. Specificity of the white gene product is largely determined by its binding partners (Ewart, 1998), Brown and Scarlet each producing different eye pigmentation phenotypes. This study reports that white, brown and scarlet mutants not only lack normal pigment granule contents in their eyes, but also normal biogenic amines in their brains, apparently because their synaptic vesicle contents are altered. Thus all three mutant flies have different neurological phenotypes from the wild-type. Behavioral differences not attributable to eye coloration, but of neural origin, have been reported in these mutants (Campbell, 2001; Diegelmann, 2006). Possibly related to these, misexpression or mislocalization of mini-white, a truncated form of the white gene or wild-type white, generates altered sexual behavior in male flies (Zhang, 1995; Hing, 1996). Although it is not clear what, if any, behavioral features these examples might share, it is possible that all could be regulated by biogenic amines acting as neuromodulators, the release of which is reduced in white (Borycz, 2008).

The amine levels in wild-type fly heads obviously vary, and this may also be true within mutant lines. Thus, the current results for dopamine in the heads of white mutants are 33% higher, and for 5-HT 365% higher than data recently reported (Hardie, 2006). The Hardie paper showed chromatograms of the separation of dopamine and 5-HT from wild-type flies, but not the measured values for each. It was therefore impossible to compare the current wild-type data with theirs. Recently, Sang (2007) reported approximately 300 pg/head for dopamine in a Ddc-GAL4 Drosophila line, which apparently had a w¹¹¹⁸ mutant background, a determination very similar to the current data on this white mutant. By contrast, in another study (Dierick, 2007), basal levels of 5-HT in the head of Canton S average between 60-80 pg/head, 2.5 to 3.3 times less than the current data. These differences could result from genotypic differences in the wild-type, but are more likely the outcome of dietary differences. Thus Drosophila fed with 50 mmol l^-1 5-hydroxytryptophan, the immediate precursor of 5-HT, showed a 15- to 20-fold increase in 5-HT in the head (Dierick, 2007). Close standardization of the medium is thus required when analyzing 5-HT in the head to enable comparisons between different studies. Additional variables include sex and age. Thus, Neckameyer (2000) report more dopamine in males than females, and in younger flies than older. These values refer to whole-body determinations of dopamine, however, not to heads, and although the current samples were from 10 flies, the determinations are reported for a minimum of eight samples taken from flies about 7-days old, so that overall it is presumed that they reflect both sexes and a spectrum of ages (Borycz, 2008).

In brain homogenates it was found that the partition between pellet and supernatant varies both for the particular amine and individual mutant. Intact neurons concentrate neurotransmitter in synaptic vesicles, by a factor of 100 at cholinergic synapses or lower, perhaps 8:1 in Drosophila photoreceptors, which contain most of the photoreceptor histamine. In brain homogenates, however, the equivalent pellet:supernatant ratio for histamine is only about 2.57:1, suggesting that neurotransmitter is lost from synaptic vesicles into the supernatant. This loss could result directly from vesicle damage during homogenization. An alternative, and more likely, explanation is based on the rate of vesicle recycling, calculated for histamine release at R1-R6 (Borycz, 2005b; Stuart, 2007), which suggests that vesicle shedding may still have occurred in homogenates, so as to deplete histamine-containing organelles in the pellet. This rate in vivo is very rapid, sufficient to deplete the terminal by a calculated 11% of its histamine per second, if compensatory histamine recycling were not to occur (Stuart, 2007), and thus to deplete photoreceptor synaptosomes more severely than the synaptosomes of other neurons in the pellet. Release by vesicle shedding within the homogenate would shift histamine from pellet to supernatant, and plausibly follow structural disruption of epithelial glia, sites of ebony action (Stuart, 2007). Supporting this conclusion, the pellet:supernatant ratio is 13.1:1 for dopamine, indicating that retention of vesicular neurotransmitter in the pellet is high and that homogenization per se is non-destructive (Borycz, 2008).

For the mutants, pellet:supernatant ratios are reversed, the wild-type:white mutant ratios for dopamine differing 34-fold, and for 5-HT, 7-fold. These differences suggest that most intravesicular amine found in the wild-type must be absent in the pellet fraction from the mutant. Other amines present in the pellet fractions from other mutants are sufficient to suggest either that only some synaptic vesicles are wholly depleted or that all are only partially depleted (Borycz, 2008).

Each mutation acts specifically on the amine profiles of the brain. Compatible with its suggested role as one half of an ABC-type transporter (Ames, 1986; Mount, 1987), white has the most comprehensive overall action. Differences in the amine phenotype of each mutant are related to those for eye pigment granules. Thus white and brown flies fail to transport guanine (Sullivan, 1979), whereas white and scarlet have reduced uptake of tryptophan and kynurenine (Sullivan, 1975). Transport of both substrates is impaired in white mutants, but there is a broad spectrum of transport substrates, which the current data now suggest may also include biogenic amines. For tryptophan, a precursor of 5-HT, reduced 5-HT was therefore anticipated in white and scarlet mutants, but in fact no significant difference was found from brown mutants. A difference instead was found in head dopamine, between scarlet flies on the one hand, and white and brown flies, on the other. Each mutant has a neurotransmitter phenotype that is proposed to reflect the gene's involvement in amine transport, and the physiology of the corresponding aminergic neurons (Borycz, 2008).

A candidate point of convergence between the amine and pigment phenotypes of white and its binding partners could lie in their respective storage organelles, synaptic vesicles and pigment granules. Pigment granules (Summers, 1982) are ultimately vesicular elaborations of the Golgi apparatus (Shoup, 1966), and synaptic vesicles also arise from the trans-Golgi network. Immunoreactivity to White and Scarlet localizes to the granule membranes (Mackenzie, 2000), and white-dsred tag colocalizes with the endosomal marker Garnet. Synaptic vesicles, which are serviced by AP-3 vesicles (Faúndez, 1998) that transport White (Lloyd, 2002), might therefore be expected to express White. In the lamina, however, White localizes most strongly to epithelial glia, rather than synaptic vesicles (Borycz, 2008).

The same epithelial glia that strongly express both white and ebony (Richardt, 2002), also invaginate R1-R6 terminals at capitate projections, postulated sites for histamine recycling (Fabian-Fine, 2003) that have more multiple heads in mutant white terminals. Brown is a binding partner of White in the eye (Dreesen, 1988), and both brown and white mutants lack White expression in the lamina, as if the two may also be binding partners there. The lack in brown mutants suggests that White protein must first bind to Brown to localize correctly in the lamina. A similar interaction may be necessary to transport or stabilize the Scarlet-White dimer (Mackenzie, 2000). The functional outcome of white in the lamina is unclear, because the mutant differs from wild-type only in being more light-sensitive, reflecting the loss of pigment granules, but possibly also having impaired synaptic transmission (Borycz, 2008 and references therein).

The data identify the interaction between White and Brown best for histamine in the lamina, but white must also function for the other amines, which show similar redistribution between pellet and supernatant fractions, consistent with a shift from organelle-bound storage. It is not clear why the data fail to reveal clear levels of White protein expression elsewhere in the brain. In situ hybridization likewise reveals white in the eye but not the brain, indicating that possible transcription in the brain must be at least an order of magnitude less. However, RT-PCR does reveal reduced but clear expression of white in sine oculis mutants, which lacks compound eyes (Campbell, 2001). Most likely, therefore, transcriptional levels in the brain are too low to detect (Borycz, 2008).

The possibility of a more general effect of white in cells other than the visual system and for other amines than histamine, is hard to address. Glial cells are very slender and enwrapping and lack endosomes that are easily detected, and possible storage sites in alternative neurons that might use other biogenic amines are equally inaccessible. This is why the current study has studied the most accessible neurons in one of the best-characterized neuropiles of the fly's brain, which also has the largest amount of any amine. All other systems pose much less favorable alternatives (Borycz, 2008).

Although the outcome of white's action may lie in a partial loss of intravesicular bioamine, at least in the visual system this action is indirect, and occurs via epithelial glial expression that must affect histamine recycling through the photoreceptor-glial shuttle (Stuart, 2007). Tan mutants accumulate carcinine, which they synthesize but cannot hydrolyze (Borycz, 2002), and so show a large peak of [³H]carcinine, whereas double-mutant white, tan convert less [³H]histamine to [³H]carcinine than do tan single mutants. This decrease is consistent with reduced [³H]histamine uptake by the epithelial glia, and therefore a tentative model is considered in which histamine uptake by the epithelial glia is white dependent. ebony mutants fail to trap [³H]histamine as carcinine, which they cannot synthesize (Borycz, 2002), and thus have no way to retain ingested tritium, thus having less [³H]histamine than wild-type. According to the model for white, double-mutant white; ebony flies would be unable to take up histamine at the epithelial glia, and therefore could not store it at this site. It is therefore proposed that the increased [³H]histamine in white; ebony mutants reflects an uptake outside the visual system. It must be acknowledged that the strength of this interpretation is circumscribed by such alternative expression sites for ebony and tan, by the histaminergic roles of additional lamina glia, and by the possibility that white might also have additional transport functions in epithelial glia. With these qualifications in mind a model is nevertheless predicted in which white acts at the epithelial glia to take up histamine from the synaptic cleft of the photoreceptor (Borycz, 2008).

Given their obvious pigmentation phenotypes, mutants of white and white transgenes have been widely used as genetic markers. One significance of the current findings, therefore, is that many effects attributed to a mutant gene or transgene isolated in a white background may not simply be those of the unknown gene but also of white itself. This is particularly true for many new genes isolated in whole-eye mosaic flies produced by mitotic recombination. The current findings indicate that, as assayed in the synaptic terminals of photoreceptors, white and its binding partner mutants lack normal synaptic vesicle populations and vesicle contents. Although similar changes in the other biogenic amines e have not been localized to neurons, the data reveal parallel deficits in these too. As a result, neurons may have reduced amine for release as either a neurotransmitter or neuromodulator, especially for sustained or high-output levels of transmission, leading to behavioral consequences. The exact behavior will reflect a balance between synthesis, transport and prior release rates of the particular amine. Thus, despite basic similarities, the behavioral phenotypes may vary both in the different mutants and, to some extent, under different physiological conditions (Borycz, 2008).

Influence of the White locus on the courtship behavior of Drosophila males

Since its discovery by Morgan, the Drosophila white gene has become one of the most intensely studied genes and has been widely used as a genetic marker. Earlier reports that over- and misexpression of White protein in Drosophila males leads to male-male courtship implicated white in courtship control. While previous studies suggested that it is the mislocalization of White protein within cells that causes the courtship phenotype, this study has demonstrated that also the lack of extra-retinal White can cause very similar behavioral changes. Moreover, evidence is provided that the lack of White function increases the sexual arousal of males in general, of which the enhanced male-male courtship might be an indirect effect. It was further shown that white mutant flies are not only optomotor blind but also dazzled by the over-flow of light in daylight. Implications of these findings for the proper interpretation of behavioral studies with white mutant flies are discussed (Krstic, 2013).

Since White protein is important for the cellular import of tryptophan, a precursor of serotonin, and serotonin levels are affected in w mutant flies, it is tempting to draw parallels between the current observations and those made in rats, cats, and rabbits where the reduction of serotonin levels increased the sexual arousal of males. These tried to mount not only females but also conspecific males. It is suggested, therefore, that the w mutant phenotypes observed in these assays may at least in part be caused by lower levels of serotonin. In line with this suggestion, recent work showed that w mutant flies display an elevated phototactic personality and individual reaction to light stimuli, while this enhanced reaction is suppressed by w-dependent serotonin. Therefore, it would be interesting to know whether and which behaviors other than courtship are affected by the lack of extra-retinal White and if these could also be induced by pharmacological reduction of serotonin levels (Krstic, 2013).

It has been proposed that increased dopamine levels in Drosophila lead to an increase in general arousal, which results in an elevated behavioral responsiveness and, more specifically, in an enhanced courtship vigor as well as male-male courtship. Surprisingly, however, a similar effect is achieved by a reduction of dopamine levels, which appears to decrease the threshold of arousal, thus rendering flies responsive to a lower intensity of stimuli. Although the effects of lower dopamine levels on courtship behavior still need to be confirmed, results with w¹¹¹⁸ males, whose serotonin as well as dopamine levels are reduced, are consistent with such a bimodal function of dopamine (Krstic, 2013).

The conspicuous mutant phenotype of the w gene was discovered by Morgan in 1910 and was the first reported Drosophila mutant. Ever since, the w gene has been extensively used as a convenient genetic marker. Despite this fact, characterization of the w gene at the molecular level, other than determining its DNA sequence and, derived from it, that of its protein, has been hampered, mainly because the protein is expressed at very low levels. Thus, expression outside the eye, where its expression is obvious from its mutant phenotype, is not well documented. Hence, it is unclear where w is expressed in the central nervous system (CNS) and peripheral nervous system (PNS) of the adult fly. According to FlyBase, w is expressed at low to extremely low levels in the brain and CNS but no evidence for its expression in the PNS is known (Krstic, 2013).

Therefore, based on the behavioral phenotypes of w mutants reported in this study, it is suggested that the lack of White protein in the CNS, other than eye and ocelli, and perhaps in the PNS, increases the overall sexual arousal of males, leading them to indiscriminately court decapitated males and females in the dark, or to chain in daylight when placed in groups of males. This increased sexual arousal of w mutant males may result from an elevated alertness, a reduction of the threshold for stimuli that elicit courtship, or an enhanced sensitivity to pheromone stimuli. While the first two models imply an augmented sensitivity for courtship in the CNS of courting w¹¹¹⁸ males, the third model suggests that these males exhibit an accelerated processing by the CNS of courtship-relevant information and perhaps an increased activity of PNS neurons that receive sensory stimuli for courtship initiation and maintenance. Although in reality a combination of these models may be relevant, further studies are required to discriminate between them. It should be emphasized that the effect of reduced or absent extra-retinal White protein on a male's state of sexual arousal could be indirect. Thus, the absence or reduction of the extra-retinal white function might affect a network of the nervous system that is not involved in courtship control but whose state influences the circuitry regulating courtship behavior (Krstic, 2013).

Contrasting influences of Drosophila white/mini-white on ethanol sensitivity in two different behavioral assays

The fruit fly Drosophila melanogaster has been used extensively to investigate genetic mechanisms of ethanol (EtOH)-related behaviors. Many past studies in flies have manipulated gene expression using transposons carrying the genetic-phenotypic marker mini-white (mini-w), a derivative of the endogenous gene white(w). Whether the mini-w transgenic marker or the endogenous w gene influences behavioral responses to acute EtOH exposure in flies has not been systematically investigated. This study manipulated mini-w and w expression via (1) transposons marked with mini-w, (2) RNAi against mini-w and w, and (3) a null allele of w. EtOH sensitivity and tolerance were assessed using a previously described eRING assay (based on climbing in the presence of EtOH) and an assay based on EtOH-induced sedation. In eRING assays, EtOH-induced impairment of climbing correlated inversely with expression of the mini-w marker from a series of transposon insertions. Additionally, flies harboring a null allele of w or flies with RNAi-mediated knockdown of mini-w were significantly more sensitive to EtOH in eRING assays than controls expressing endogenous w or the mini-w marker. In contrast, EtOH sensitivity and rapid tolerance measured in the EtOH sedation assay were not affected by decreased expression of mini-w or endogenous w in flies. It is concluded that EtOH sensitivity measured in the eRING assay is noticeably influenced by w and mini-w, making eRING problematic for studies on EtOH-related behavior in Drosophila using transgenes marked with mini-w. In contrast, the EtOH sensitivity assay described in this study is a suitable behavioral paradigm for studies on EtOH sensitivity and rapid tolerance in Drosophila including those that use widely available transgenes marked with mini-w (Chan, 2014).

Analysis of a cellular structure observed in the compound eyes of Drosophila white; yata mutants and white mutants

Previous work has identified the Drosophila yata mutant, which showed phenotypes including progressive vacuolization of the white-colored compound eye, progressive shrinkage of the brain and a shortened lifespan. The yata gene was shown to be involved in controlling intracellular trafficking of the APPL protein, which is an orthologue of APP that is a causative molecule of Alzheimer's disease. This study examined the phenotype of the compound eye of the yata mutant using electron microscopy and confocal microscopy. Abnormal cellular structures that seemed to originate from bleb-like structures and contained vesicles and organelles, such as multivesicular bodies and autophagosomes, were observed in aged white; yata mutants and aged white mutants. These structures were not observed in newly eclosed flies, and the presence of the structures was suppressed in flies grown under constant dark conditions after eclosion. The structures were not observed in newly eclosed red-eyed yata mutants or wild-type flies but were observed in very aged red-eyed wild-type flies. Thus, these data suggest that the observed structures are formed as a result of changes associated with exposure to light after eclosion in white mutants, white; yata mutants and aged flies (Arimoto, 2020).

In this study, the phenotypes of the compound eyes of yata mutants were observed in fine detail using electron microscopy. The observations led to the identification of abnormal cellular structures located in the vicinity of rhabdomeres in aged day 15 and day 29 white; yata mutants. In tangential sections, rhabdomeres were connected to the cytoplasm of the cells from which they were derived, and a different side of these rhabdomeres was often also attached to the identified structures. Although the identified structures were surrounded by a plasma membrane, they were often attached to rhabdomeres. The identified structures were usually located near or opposite the central cavity. In horizontal sections, the identified structures were often found in between rod-like rhabdomeres, and were found to be both connected to and separated from rhabdomeres. They were also found lateral to rhabdomeres and embedded in the cytoplasm of photoreceptor neurons, and were also occasionally observed to cause twisting of the rhabdomeres. Because the identified structures seem to adhere to rhabdomeres, there may be adhesive mechanisms between the identified structures and rhabdomeres. In addition, in some electron micrographs, the identified structures were observed to be bleb-like structures that extended from the cytoplasm of photoreceptor neurons from locations near the adherens junction. These observations suggest that the identified structures originate as bleb-like structures from the cytoplasm of photoreceptor neurons and may migrate into the location of rhabdomeres or the cytoplasm of photoreceptor neurons. The identified structures were filled with vesicles, vacuoles and membranous organelles including multivesicular bodies that appeared to be late endosomes, structures with double membranes and electron-dense structures. Analysis using confocal microscopy revealed the accumulation of anti-Atg8a and anti-Rab7 signals near the rhabdomeres in the day 15 white; yata mutants. Anti-Rab7 antibody has been shown to label late endosomes. Anti-Atg8a antibody has previously been shown to specifically detect the Drosophila Atg8a protein in immunostaining of the fat body of third instar larvae. Although previous studies showed that anti-Atg8 antibody can label aggregates of ectopically overexpressed Atg8 protein or Atg8 protein incorporated into protein aggregates in cultured cells, the anti-Atg8a signal observed in the white; yata mutants seems to represent the accumulation of autophagosomes, as it is consistent with the observation that the identified structures contain structures with double membranes, as revealed by electron microscopy. Thus, these data suggest that the identified structures contained autophagosomes and late endosomes. Moreover, electron-dense structures observed in the identified structures appeared to be similar to lysosomes. These findings suggest that the identified structures may contain organelles involved in protein degradation pathways, such as the autophagy-lysosome system (Arimoto, 2020).

In addition to their presence in day 15 and day 29 white; yata mutants, the identified structures were found in day 29 white mutants. However, the identified structures were not found in day 1 white; yata or white mutants. These structures may be similar to 'multivesicular body-like bodies' previously observed in aged day 21 white mutants. These 'multivesicular body-like bodies' were found to occasionally form a peninsula-like structure that extended from the cytoplasm of a photoreceptor neuron near the adherens junction, and this observation is consistent with the bleb-like form of the identified structures that were observed. The identified structures were not found in red-eyed yata mutant and wild-type flies on days 1 and 29. Formation of the identified structures was completely suppressed in day 29 white mutants reared under constant dark conditions after eclosion. These findings suggest that formation of the structure in white mutants is caused by changes in photoreceptor neurons associated with excessive exposure to light after eclosion because pigment granules were absent in the white mutants. In addition, the data showed that the identified structures also formed in very aged day 71 red-eyed wild-type flies. These changes may be a component of the changes associated with ageing in the compound eyes of flies (Arimoto, 2020).

Significantly more identified structures were observed in the day 29 white; yata mutants than in the day 29 white mutants reared under 12-h light/12-h dark conditions. The identified structures were observed in the day 15 white; yata mutants, but not in the day 15 white mutants. Therefore, the yata mutation may enhance formation of the identified structures, although whether the yata mutation enhances formation of the identified structures in flies with a red-eyed background is unclear. The photoreceptor neurons of yata mutants, at least those in the white mutant background, may be more vulnerable to changes associated with exposure to light. In addition, formation of the identified structures was not completely suppressed in day 29 white; yata mutants reared under constant dark conditions after eclosion, although no identified structures were observed in day 29 white mutants reared under constant dark conditions after eclosion. Therefore, the yata mutation may enhance the mechanisms that lead to formation of the identified structures, even in flies reared under constant dark conditions after eclosion. The loss of such yata-dependent mechanisms may cause formation of the identified structures in the background of a null mutation of white or in flies in which pigment granules have been lost. Diverse phenotypes in addition to a white eye colour have been described in white mutants, suggesting a wide range of molecular functions for the white gene. Although why white mutation enhanced formation of the identified structures in white; yata mutants reared under constant dark conditions after eclosion is unclear, this effect may be related to the fact that pigment granules are lysosome-like organelles and that several genes involved in the biogenesis of pigment granules also play a role in lysosome trafficking. Notably, giant multivesicular structures with diameters greater than 3 μm have been observed in the pigment cells of mutants of the deep orange gene, which encodes an endosomal protein required for normal eye colour (Arimoto, 2020).

The data suggest that the identified structures are more frequently observed in ommatidia at the R8 level than in those at the R7 level regardless of genotype or age. These observations suggest that the identified structures form more frequently in a proximal position in the retina than in a distal position in the retina. Although the reason for this location-dependent differential vulnerability is unknown, it may be related to a previous observation that retinal degeneration processes occurred more rapidly in proximal positions remote from the nucleus than in distal positions in rdgA mutants. Therefore, the difference in the frequency of the identified structures between the ommatidia at the R8 and R7 levels may be related to the susceptibility of photoreceptor neurons to degenerative changes (Arimoto, 2020).

Previous studies suggested that yata is involved in regulating the intracellular trafficking of a subset of proteins including APPL and Fasciclin II. However, it has also been suggested that yata does not control the localization of all membrane-bound and secreted proteins transported via vesicular trafficking because the localization of Synaptotagmin was not affected by yata mutation. In addition, yata may also control the trafficking of some unidentified proteins. Therefore, the loss of yata may enhance formation of the identified structures by impairing the localization of APPL, Fasciclin II or other unidentified target proteins or an unidentified molecular function of yata (Arimoto, 2020).

In conclusion, this study identified a cellular structure that contains vesicles, autophagosomes and multivesicular bodies in the compound eyes of aged white; yata mutants, aged white mutants and very aged wild-type flies. The data suggest that these structures are formed as a result of changes associated with exposure to light after eclosion in white mutants, white; yata mutants and aged flies. Further exploration of the mechanisms of formation of the identified structures may clarify their physiological significance (Arimoto, 2020).

White participates in vesicular transepithelial transport of cGMP

Guanosine 3'-5' cyclic monophosphate (cGMP) and adenosine 3'-5' cyclic monophosphate (cAMP) are important regulators of cell and tissue function. However, cGMP and cAMP transport have received relatively limited attention, especially in model organisms where such studies can be conducted in vivo. The Drosophila Malpighian (renal) tubule transports cGMP and cAMP and utilises these as signalling molecules. This study shows via substrate competition and drug inhibition studies that cAMP transport - but not cGMP transport - requires the presence of di- or tri-carboxylates; and that transport of both cyclic nucleotides occurs via ATP binding cassette sub-family G2 (ABCG2), but not via ABC sub-family C (ABCC), transporters. In Drosophila, the white (w) gene is known for the classic eye colour mutation. However, gene expression data show that of all adult tissues, w is most highly expressed in Malpighian tubules. Furthermore, as White is a member of the ABCG2 transporter class, it is a potential candidate for a tubule cGMP transporter. Assay of cGMP transport in w^- (mutant) tubules shows that w is required for cGMP transport but not cAMP transport. Targeted over-expression of w in w^- tubule principal cells significantly increases cGMP transport compared with that in w^- controls. Conversely, treatment of wild-type tubules with cGMP increases w mRNA expression levels, implying that cGMP is a physiologically relevant substrate for White. Immunocytochemical localisation reveals that White is expressed in intracellular vesicles in tubule principal cells, suggesting that White participates in vesicular transepithelial transport of cGMP (Evans, 2008).

The first Drosophila mutation to be identified was white (w), and it was instrumental to Morgan's description of genes and chromosomes (Morgan, 1910). Despite over 3000 publications on w since then, few have investigated the biological function of White protein, tending instead to concentrate on the genetics of w. The most prominent phenotype of w mutants is the pronounced lack of eye colour. However, eye colour phenotypes can also reflect defects in Malpighian tubule structure and function, because several eye pigment precursors [notably the transport of compounds in the xanthommatin biosynthetic pathway are stored and processed in the larval tubule before being released into the pupal haemocoel for uptake by the developing adult eye. The Malpighian tubules of w^- Drosophila are clear or whitish in appearance due to the absence of tryptophan metabolites and pteridines, unlike wild-type tubules, which are yellow in appearance (Evans, 2008 and references therein).

Insect Malpighian tubules are critical for survival and play essential roles in osmoregulation, homeostasis and immune function (Dow, 2005). Excess fluid and solutes are transported and excreted largely across these blind-ended tubules, with selective re-absorption occurring in the rectum. Drosophila tubules provide a unique model for studying transport in a live polarised epithelial tissue, with added benefits of the availability of genetic tools. cGMP and cAMP signalling has been studied in the Drosophila Malpighian tubule for over 10 years, but the transport mechanisms of these cyclic nucleotides have received rather less attention. Nitric oxide (NO)-cGMP signalling was first identified to stimulate fluid transport (Davies, 1995), with more recent work implicating NO signalling in the immune response (McGettigan, 2005). Furthermore, NO-cGMP signalling can be induced by activation of the capa receptor in Dipteran tubules from several species (Pollock, 2004); and so is an important feature of tubule function in insect vectors of disease. Exogenous cAMP also stimulates fluid transport (Davies, 1995; Riegel, 1998), with fluid transport also being stimulated by corticotropin-releasing factor (CRF)-like peptide (Cabrero, 2002) and calcitonin-like peptide (Coast, 2001; Evans, 2008 and references therein).

cGMP and cAMP are transported into the Malpighian tubules (Riegel, 1998); cGMP transport across the tubule (efflux) is modulated by cGMP-dependent phosphodiesterases (cG-PDEs) (Day, 2006). In mammalian systems, cyclic nucleotide transport has been attributed to a number of ATP binding cassette (ABC) transporters and solute carriers (members of the SLC22 family) (Dazert, 2003; Koepsell, 2004; van Aubel, 2002). These transporters have been widely studied in mammals but equivalent transporters have not previously been identified in dipteran insects (Evans, 2008).

White is a member of the ABC transporter subfamily G (Shulenin, 2001), with greatest sequence similarity to human ABCG2. Investigations of the role of White in Malpighian tubules were carried out ~30 years ago, and tentatively identified location of and transport substrates for White. The location of White in the tubules was thought to be either the basolateral membrane (Sullivan, 1980) or the pigment storage vesicle membranes (Sullivan, 1979). Potential substrates for White included tryptophan (Sullivan, 1980), kynurenine (Sullivan, 1975), 3-hydroxykynurenine (Howells, 1977), guanine and riboflavin (Sullivan, 1979; Evans, 2008 and references therein).

This study demonstrates that cGMP transported by the tubule occurs via ABCG2 transporters, and that White is required for cGMP but not cAMP, transport across the Malpighian tubule. Localisation of White to intracellular vesicles in the principal cells in the main, fluid-transporting segment of the tubule, indicates a possible storage-excretion mechanism of cGMP transport via vesicles (Evans, 2008).

Uptake and excretion of cGMP by Drosophila Malpighian tubules has been documented (Riegel, 1998). This study shows that the transport of cGMP is sensitive to the electrical gradient, suggesting that a secondary active transport mechanism is involved. Secreted fluid is capable of stimulating cGMP-dependent protein kinase (cGK) activity, suggesting that as in mammals, cGMP can be transported across the tubule membrane in unaltered form in Drosophila. Although primary active transport could not be shown unambiguously, the ABCG transport ATPase, White, is nonetheless necessary for significant cGMP flux; it must thus, at least, facilitate the diffusion of cGMP. In tubules, White is expressed throughout the cytoplasm in large vesicles of the main segment. These vesicles are probably the pigment vesicles which were suspected to be the location of White in a previous study (Sullivan, 1980). Other sub-families of ABC transporters also show intracellular localisation: ABCC4 transporters localise to the dense granules of platelets, and not at the plasma membrane (Jedlitschky, 2004). Thus, it seems that sub-families of the ABC transporter family, in both vertebrates and invertebrates, can be localised to vesicular structures in the cytoplasm of the cell. The first identified ABCG transporter in Leishmania, LiABCG4, has been shown to localise to the plasma membrane and to post-Golgi secretory vesicles when overexpressed in yeast (Castanys-Munoz, 2007). However, in the parasite, LiABCG4 is mainly localised to the plasma membrane, with some localisation in flagellar pockets; suggesting that the localisation in secretory vesicles in yeast may be due to over-expression of the ABCG4 transporter in the yeast system. Although the vesicular localisation of White in tubule cells may be due to targeted over-expression of w⁺, localisation of White in non-transgenic tubules (Sullivan, 1980) suggests that vesicular localisation for White is not associated with expression artifacts in vivo (Evans, 2008).

The primarily vesicular localisation of White may also indicate trafficking of cGMP across the Malpighian tubules in vesicles, a novel mechanism of transepithelial cGMP transport that would not compromise the integrity of intracellular cGMP signalling pathways. This would explain why fluid transport assays on tubules from w loss-of-function mutants show similar rates of cGMP-induced fluid transport (Davies, 1995; Dow, 1994) to wild-type tubules. The current model is thus that cGMP is transported into the cell by a basolateral plasma membrane cGMP transporter that still remains to be discovered; once in the cell, it can act to stimulate fluid transport. However, White sequesters cGMP into vesicles, contributing [perhaps together with the action of DmPDE6 (Day, 2006) to its clearance from the cell; and these vesicles are excreted from the apical surface of the cell, presumably as part of a general purpose organic solute clearance mechanism. In the absence of White, cGMP is still transported into cells but due to reduced uptake into intracellular vesicles, is transported into the lumen at a much reduced rate. It will be interesting in due course to try to identify the plasma membrane transporter for cGMP. Importantly, this multi-stage transport model explains why it was not possible to demonstrate accumulation of cGMP beyond Nernst-predicted ratios, despite the involvement of a transport ATPase in the process (Evans, 2008).

Although the w gene has almost exclusively been researched in association with its role as an eye colour marker, recent microarray data of adult fly tissues (Chintapalli, 2007) has shown that w is most highly expressed in Malpighian tubule, a tissue enriched for organic anion transporters. Previous studies have shown that White can act as a heteromeric transporter: with Scarlet, it is a tryptophan transporter, responsible for brown eye colour; with Brown, it transports guanine, the precursor of the red pigment in eye (Dreesen, 1988). Mutation of key residues such as glycine 589 in the fifth transmembrane helix of White, significantly reduces guanine transport by White-Brown heterodimers, suggesting the importance of G589 in heterodimerisation and in guanine transport (Mackenzie, 1999). Interestingly, mutation of amino acid 553 in TM5 of ABCG2 (a well-conserved residue corresponding to G589 in White) disrupts function and trafficking of ABCG2, implying conservation of dimerisation function of these residues across evolution (Polgar, 2006; Evans, 2008 and references therein).

Even if White does play a key role in cGMP transport, organic solute transporters are heavily represented in the tubule transcriptome, and so there are other potential candidate transporters. In mammals, transporters of the ABCC class, notably MRP4 (ABCC4) and MRP5 (ABCC5) have been shown to transport cyclic nucleotides out of the cell (Ritter, 2005). Treatment of Drosophila tubules with glibenclamide, a broad-spectrum inhibitor of ABC transporters, results in inhibition of both cAMP and cGMP transport. However, utilising either known inhibitors or competing substrates for ABBC transporters shows that such transporters are not involved in cAMP/cGMP transport by Malpighian tubules. Interestingly, tubules express five of the seven Drosophila homologues of mammalian ABCC transporters (Day, 2006); with one gene, CG9270, being expressed only in tubules of the adult fly. It would be interesting to screen the product of this gene for ABCC transporter function, and to determine its substrate specificity. In any case, the lack of effect of ABCC-specific drugs on the tubule could indicate evolutionary divergence between mammalian and Drosophila ABCC transporters, or may simply reflect inaccessibility of these transporters to specific drugs if the transporters are localised to the apical membrane (Evans, 2008).

This study has shown that cGMP transport by the tubule is specific and possesses distinct properties from cAMP transport. Previous investigations have suggested that there may be some overlapping function of cyclic nucleotide transporters (Riegel, 1998). The different conclusions reached by these different studies may be a reflection of the concentration of each cyclic nucleotide used - the competing cyclic nucleotide was greatly in excess in the Riegel study. This study shows that cAMP transport requires the presence of di- or tri-carboxylates; but that cGMP transport is unaffected by these compounds. cAMP transport probably requires an OAT-like transporter at the basolateral membrane, whereas cGMP is transported via a different mechanism. Thus in the tubules, the mechanisms of transport of cGMP and cAMP are largely independent and specific. This could reflect the importance of these signalling molecules in the tubules. Alternatively, it could reflect the transport mechanisms necessary in a tissue for which there is a requirement for an established potential gradient to enable solute uptake and excretion (Evans, 2008).

Overall, though, the results suggest that the extraordinary abundance of White in the adult Malpighian tubule may reflect a novel role that continues beyond the need to handle visual pigment precursors in the larva and pupa. White is thus a more versatile transport protein that previously suspected (Evans, 2008).

The white gene of encodes a protein with a role in courtship behavior

The white gene has been extensively studied, yet it is still not understood how its ectopic overexpression induces male-male courtship. To investigate the cellular basis of this behavior, the sexual behavior of several classes of mutants was studied. Male-male courtship is seen not only in flies overexpressing the white gene, but also in mutants expected to have mislocalized White protein. This finding confirms that mislocalizing White transporter in the cells in which it is normally expressed will produce male-male courtship behaviors; the courtship behavior is not an indirect consequence of aberrant physiological changes elsewhere in the body. Male-male courtship is also seen in some mutants with altered monoamine metabolism and deficits in learning and memory, but can be distinguished from that produced by White mislocalization by its reduced intensity and locomotor activity. Double mutants overexpressing white and with mutations in genes for serotonergic neurons suggest that male-male courtship produced by mislocalizing White may not be mediated exclusively by serotonergic neurons. Decreased olfactory learning is found in white mutants and in individuals with mutations in the genes for White's binding partners, brown and scarlet. Finally, in cultured Drosophila and mammalian cells, the White transporter is found in the endosomal compartment. The additional genes identified in this study as being involved in male-male courtship increase the repertoire of mutations available to study sexual behavior in Drosophila (Anaka, 2008).

Although white, as well as brown and scarlet, mutants appear to have a deficit in olfactory learning, null and hypomorphic white mutations do not induce any obvious change in male or female sexual behavior. To explain at the cellular level how overexpression of the white eye-color gene in Drosophila induces male-male courtship, the behavior of four classes of mutants was studied: those mutant for the white gene, those mutant for genes encoding the White binding partners, those mutant for genes responsible for correct intracellular localization of White, and those mutant for genes with roles in learning and monoamine neurotransmitter metabolism. The intracellular location of the white-encoded ABC-type transmembrane transporter was also studied (Anaka, 2008).

When the male-male courtship behavior of the hs-mini-white⁺ gene was first noted, it was proposed to result from heat shock-induced ectopic expression of the white gene in cells in which it was not normally expressed (Zhang, 1995). Thus, while interesting, the male-male courtship behavior did not of itself require white to have a neurological role normally. Indeed, male courtship has been shown to depend not only on neural identity and activity, but also on proteins secreted into the hemolymph. However, the white gene is transcribed in the heads of flies lacking eyes and ocelli (Campbell, 2001), so it seems likely that white is transcribed in neural tissue. It has been shown previously (Lloyd, 2002) that male-male courtship is seen in garnet mutants in which the intracellular localization of White is disrupted only in the cells normally producing White. The male-male courtship of garnet mutants is strictly dependent on the presence of the White protein; garnet and white double mutants show no male-male courtship (Lloyd, 2002). This study extends these results to show that male-male courtship is seen in many 'granule-group' mutants, mutants that have defects in the intracellular trafficking of various proteins, including White. The male-male courtship, sexual preference, and unimpaired general locomotion seen in these mutants mimic the behaviors seen in hs-white⁺ males. The seemingly identical male-male courtship behaviors in males overexpressing and mislocalizing the White protein shows that male-male courtship can result from the relocation of the White transmembrane protein within the cells normally producing it, rather than exclusively being an indirect effect of metabolic perturbation of other cells in the body. This evidence, along with the finding of learning defects in white, brown, and scarlet mutants, the reduced performance of white mutants in spatial learning reported by Diegelmann (2006), and finding that white and brown mutants are resistant to the effects of a volatile general anesthetic (Campbell, 2001), together strongly support the assertion that white normally acts in the nervous system (Anaka, 2008).

When a tagged version of the White protein is expressed in cultured cells, it appears to be localized to endosomes. A primarily endosomal location of the native White protein is supported by genetic analysis (Lloyd, 2002) and consistent with immunocytochemical evidence localizing White to endosomal derivatives in pigment cells and similar vesicular structures in Malpighian tubules (Mackenzie, 2000; Evans, 2008). Further, white mutants display distinctive, abnormal, large pigment granules reminiscent of the giant lysosomes found in Chediak-Higashi Syndrome individuals, and at least one of the human homologs of white, ABCG1/ABC8, is also found in a punctate perinuclear distribution consistent with an vesicular location (Lorkowski, 2001), as is another human homolog, ABCC4 (Jedlitschky, 2004). Interestingly, the labeling pattern seen for White is very similar to that shown in cultured cells for one of the splice variants of the Drosophila vesicular monoamine transporter gene (VMATA); the second splice variant, VMATB, by contrast, localizes to the plasma membrane. VMATA is qualified to act as a vesicular transporter for 5-HT in vivo, as well as two other amines, dopamine and octopamine (Greer, 2005), and the similarity in its pattern of expression to that for White suggests that the latter may also be associated with synaptic vesicles or with other endosomal compartments. An endosomal location for the White protein could be expected to alter the morphology, number, or neurotransmitter loading of synaptic vesicles and would thus simplify models proposed to explain the effect of white on behavior. But, this role would need to be demonstrated. Moreover, immunocytochemistry of the native White protein, using an antibody against part of the extracellular loop, between putative transmembrane helices 5 and 6 (Mackenzie, 2000), fails to label the synaptic boutons of the neuromuscular junction. Thus, if White is indeed a synaptic vesicle protein, it is not an abundant one, a finding consistent with its low level of transcription (Campbell, 2001). Previous biochemical analysis of the uptake of small metabolites in white mutants led to the suggestion that White protein functions in the plasma membrane; however, transport of metabolites across an epithelium can be mediated, indirectly, by intracellular transporters (Mackenzie, 2000; Evans, 2008). A plasma-membrane location, however, is compatible with the location of other members of the ABC family and the immunoexpression of native White protein in the glial cells that surround terminals of photoreceptors (Borycz, 2008), whereas the terminals themselves, which use a fourth biogenic amine, histamine, fail to express either isoform of VMAT. These conflicting localization data could be partly reconciled if the white gene product is only transiently associated with the plasma membrane, as is the case for the yeast ABC transporter, STE6 Or if White's cellular location differs for different cells or different neurons (Anaka, 2008).

While the absence or reduction of White protein does have behavioral consequences, it is only its relocation that induces male-male courtship. The different behavioral responses to the absence versus mislocalization of the White protein could arise from multiple causes. There are many transmembrane pumps capable of transporting tryptophan and VMAT has been identified as the major vesicular monoamine transporter (Greer, 2005). If neurotransmitter loading relies on a number of proteins, the absence of White from the synaptic vesicle may have much less effect than its inappropriate relocation to the plasma membrane. Another alternative is that different behavioral effects of the inappropriate placement of White stem from the availability of binding partners in each compartment, since the specificity of the transporter is conferred not by White, but by its binding partner (Ewart, 1998; Schmitz, 2001). Insofar as the White protein can combine with a number of partners to transport a variety of substrates, a change in the intracellular localization of White could perturb the distribution of a broad spectrum of neurotransmitters and other small metabolites. Indeed, Hoyer (2008) show that white-null males are defective in octopamine-associated aggressive behavior. A final possibility is that the male-male courtship behavior is a direct consequence of reduced learning (Anaka, 2008).

Courtship behavior in Drosophila is complex and largely invariant, but it can be modified by learned behaviors. Normally, courtship between mature males is suppressed by a combination of rejection from other males, antiaphrodisiac pheromones, and learning to refrain from male-male courtship after courting immature males, a process termed experience-dependent courtship modulation or courtship conditioning. This male-male courtship may function to transmit the nuances of the courtship song to the young males, but it is without apparent benefit to the older male. As a result, wild-type males rapidly learn to repress this behavior, and the obligate male-female courtship seen in wild-type flies is, in part, a learned behavior (Anaka, 2008 and references therein).

The finding, consistent with that of Nilsson (2000), that flies in which White is mislocalized do not switch their sexual preference, but simply reduce the specificity of targeting their courtship attempts, suggests a learning deficit. Further, vermillion, cinnabar, and raised mutants show both male-male courtship (McRobert, 2003) and a decreased ability to learn (Savvateeva, 2000; McRobert, 2003), although their neurological functions remain undefined. The neurological functions of the proteins encoded by Frequinin 1, Tyramine receptor, rutabaga, ether a go-go, and dunce, which also show male-male courtship, are well defined. Frequinin 1 encodes a synaptic calcium-binding protein essential for signaling and neuromodulation (Cremona, 2001). Unfortunately, effects on learning or memory have not been reported. Tyramine receptor encodes a receptor that signals in response to octopamine and tyramine and, possibly, other amines. Octopamine has a role in learning and memory, as well as aggression. rutabaga (rut), ether a go-go (eag), and dunce (dnc) encode a protein with adenylate cyclase activity, a plasma-membrane voltage-gated potassium channel and 3'5'-cyclic nucleotide phosphodiesterase, respectively. All of these mutants have defects in learning and/or memory, and rutabaga and dunce have been shown to have defects in specific aspects of learned courtship behavior (Anaka, 2008 and references therein).

Thus, the findings that the loss of White protein causes a decreased ability to learn, and that courtship alterations are found in other mutants with decreased capacity for learning, strengthens the link between learning and courtship. This link could be either indirect or direct. Both learning and courtship behavior may be independently influenced by a neurotransmitter, levels of which are influenced by the White ABC transporter, or the male-male courtship seen in flies mislocalizing the White transporter is a consequence of failure to remember previous nonproductive courtship interactions with other males. When the White protein is overproduced from a heat-shock promoter throughout development, wild-type amounts of eye pigment are deposited. This suggests that normal amounts of the White transporter are available in endosomal and related organelles, presumably including synaptic vesicles, so that learning should be normal. Yet, the frequency of male-male courtship in these flies is higher than in those in which the white gene is only transiently overexpressed. This suggests that male-male courtship may not be a direct consequence of impaired learning but, instead, both depend on the proper levels of neurotransmitters and neuromodulators. As such, the identification of numerous additional genes that induce male-male courtship behavior suggests that indiscriminate sexual behavior may be the default state, and further analysis of these genes may help clarify the link between neural function and courtship behavior. An additional implication of these results is that effects due to the white-null background of many transgene strains, with the mini-white gene of the transformation vector providing only partial rescue, or strains marked with visible eye color markers, such as brown, scarlet, or members of the granule group, should be monitored in behavioral assays (Anaka, 2008).

The evidence for a behavioral role for the White protein is clear, and from its biochemical function as a transporter and its intracellular localization to endosomes, a role for White in moderating the loading of synaptic vesicles with neurotransmitter seems plausible. However, the biochemical details of White's action are not yet clear. Numerous studies have linked serotonin to sexual behavior. Feeding male rats or cats p-chlorophenylalanine, an inhibitor of serotonin biosynthesis, and feeding rabbits a diet lacking tryptophan, the amino-acid precursor of serotonin, both induce male homosexual mounting behavior superficially similar to the male-male courtship observed in this study in Drosophila. In Drosophila, serotonin has also been implicated in sexual behavior. Mosaic flies, in which some cells in the brain have been converted from male to female, either by chromosome loss or by selective expression of sex-determination genes, display male-male courtship. Among the key regions of the brain identified in this way was the mushroom body, a region of the brain associated with courtship behavior and containing serotonergic neurons. Genetic evidence suggests that the White-Scarlet heterodimer pumps tryptophan or tryptophan-derived 3-hydroxykynurenine (Ewart, 1998), and in serotonergic neurons, tryptophan is converted into serotonin. This evidence led Zhang and Odenwald (1995) to postulate that overexpression of the white gene alters tryptophan availability to serotonergic neurons But despite some similarities between the neurobiology of male sexual behavior in mammals and Drosophila, the involvement of serotonin in male-male courtship is still ambiguous (Anaka, 2008).

The courtship behavior seen in some of the mutants tested in this work does not necessarily support a role for serotonin as the key neurotransmitter involved in male-male courtship in Drosophila. The Scarlet protein is believed to dimerize with White to form a tryptophan transporter (Mackenzie, 2000). Thus, while a dearth of White-Scarlet dimer could lead to synaptic vesicles deficient in tryptophan and serotonin, male-male courtship occurs only in the absence of Scarlet but not White. This discrepancy could be due to altered localization of White in the absence of its binding partner. Superficially, the male-male courtship, albeit weak, seen in cinnabar and vermillion males, is also consistent with the involvement of serotonin in male-male courtship; both mutants interrupt the tryptophan pathway. Scenarios linking the male-male courtship behavior seen in brown and Punch mutants to a diminution of serotonin levels become more contrived, however. Punch encodes GTP cyclohydrolase 1, and the Brown-White heterodimer is believed to transport GTP (Ewart, 1998). GTP is not a precursor of serotonin, although it is a precursor of tetrahydrobiopterin, a cofactor required for the synthesis of the serotonin as well as dopamine and nitric oxide. If, however, mutations in the brown gene were to cause male-male courtship behavior by decreasing the intravesiclar concentrations of a cofactor for serotonin biosynthesis, serotonin biosynthesis would have to occur in the synaptic vesicle and not in the cytoplasm, as currently thought. Other explanations, which could rationalize how a decrease in the Brown and Scarlet proteins could lower intravesicular serotonin levels, include the possibility that dimerization of Brown and White is necessary for the correct sorting of the White protein or that a decrease in transcription of the brown and scarlet genes induce transcriptional upregulation of the white gene, causing mislocalization of the excess White protein. Although the brown, scarlet, and white genes appear to be concordantly regulated (Rabinow, 1991), neither the amount nor the location of the White protein has been reported in brown and scarlet mutants. Finally, although only one allele was tested in this study, significant levels of male-male courtship were not observed in Ddc mutant males that have greatly decreased amounts of serotonin (Anaka, 2008).

Conclusions on the role of serotonin in mutants previously found to show male-male courtship are also conflicting. In Drosophila, only a limited number of mutations have previously been shown to cause a switch from male-female to male-male courtship behavior. In addition to the male-female mosaics mentioned above, doublesex (dsx) mutants also show male-male courtship. Since doublesex is involved in somatic sex determination, this effect is probably also mediated by alterations of sexually dimorphic neurons. Similarly, the fruitless gene is involved in somatic sex determination, and mutants exhibit distinctive and, for some alleles, exclusive male-male courtship. These mutants show reduced serotonin levels in a subset of abdominal serotonergic neurons; however, the serotonergic neurons of the mushroom body do not express a male-specific form of the Fruitless protein. Further, it is possible to induce male-male courtship behavior by selective disruption of cholinergic peripheral neurons but not neurons in the mushroom body. The dissatisfaction, prospero, quick-to-court, and raised mutants also show male-male courtship; however, the role of these genes, if any, in serotonin metabolism has yet to be elucidated. The dissatisfaction, prospero, and quick-to-court genes are all expressed in neurons, and prospero and dissatisfaction appear to encode transcription factor or related proteins, but it is not known if these gene products are involved in the specification of serotonergic neuron fate. Thus, the relationship between the White transporter protein, serotonin metabolism, and male-male courtship behavior remains suggestive but unclear (Anaka, 2008).

To attempt to clarify the relationship between serotonergic neurons and male-male courtship behavior resulting from White mislocalization, double mutants were made with the hs-mini-white⁺ and three mutations involved in the formation of serotonergic neurons, huckebein, eagle, and fruitless. The finding that males mutant for huckebein or fruitless show male-male courtship superficially supports a role for serotonin in male-male courtship in Drosophila. However, the male-male courtship behavior shown by the huckebein and fruitless mutant flies can be distinguished from that shown by white mislocalization mutants in both the lower frequency of male-male courtship and the reduced locomotor activity. Further, double mutants combining mutant alleles of fruitless, eagle and huckebein, and the hs-mini-white⁺ gene show that the effects of fruitless, eagle, and huckebein are not epistatic, as might be expected if the White transporter protein acted primarily in serotonergic neurons. Although the fact that the alleles used are viable hypomorphs, and so may have only a modest impact on serotonergic neurons, is an important caveat, if mislocalization of the White transporter acts on serotonin metabolism, its effects should be nullified or greatly reduced in mutants with fewer serotonergic neurons. The data did not show this. However, these results differ from those reported by Nilsson (2000), however, who found that double mutants between a mini-white⁺ transgene and the fru^{sa t} allele showed reduced male-female and male-male courtship characteristic of the fru^sat allele alone. Given that the fruitless gene is complex with many sex-specific alternately spliced products, it is possible that this discrepancy reflects the fru allele tested. The fru^sat allele used by Nilsson and fru³ allele used in this work are produced by transposon inserts into different portions of the gene, which are included in different sets of mRNAs. Thus, the data, and conflicting data in the literature, on the role of serotonin in Drosophila courtship behavior, suggest that it is possible that male-male courtship may result from alterations in more than one neuronal pathway or by altering more than one type of neurotransmitter. Because the White protein appears to be a fairly nonspecific transporter, it may affect the transport of different neurotransmitters. Direct examination of the contents of monoamine neurotransmitters in white mutant brains is the subject of a parallel study that has revealed a reduced amine content in the heads of white mutants, as well as a redistribution of the amines in head homogenates, from being concentrated in a synaptosome-rich fraction of the wild type to a supernatant fraction in the mutant (Borycz, 2008). These results are also consistent with the abolition of male aggression (Hoyer, 2008), an octopamine-associated behavior, recently reported in white-null mutants (Anaka, 2008).

There are at least eight human homologs of the Drosophila white gene (Schmitz, 2001). Polymorphisms in one of these, ABCG1, have been associated with mood and panic disorders in males (Nakamura, 1999). Although a direct demonstration of altered white mRNA or protein levels has not been reported, the association of the human homolog of white with panic disorders is reminiscent of the anesthetic resistance reported by Campbell and Nash (2001) and the modest hyperactivity found in the locomotor tests in this study. Further investigation into the role of white/ABCG1 in humans might also reveal a subtle neural function. More recent work has implicated ABCG1 in other conditions, such as cholesterol and phospholipid metabolism problems and as a contributor to the etiology of Tangier disease (Schmitz, 2001). Thus, while the differences between human and Drosophila brains and behaviors are incontestable, the white gene, the oldest and most prosaic mutation in Drosophila, is highly conserved in both structure and function, and as a metabolite transporter with wide specificity, may have multiple biological roles, including a hitherto largely unacknowledged neural role in Drosophila (Anaka, 2008).

Red flag on the white reporter: a versatile insulator abuts the white gene in Drosophila and is omnipresent in mini-white constructs

Much of the research on insulators in Drosophila has been done with transgenic constructs using the white gene (mini-white) as reporter. This study reports that the sequence between the white and CG32795 genes in Drosophila melanogaster contains an insulator of a novel kind. Its functional core is within a 368 bp segment almost contiguous to the white 3'UTR, hence it has been named Wari (white-abutting resident insulator). Though Wari contains no binding sites for known insulator proteins and does not require Su(Hw) or Mod(mdg4) for its activity, it can equally well interact with another copy of Wari and with unrelated Su(Hw)-dependent insulators, gypsy or 1A2. In its natural downstream position, Wari reinforces enhancer blocking by any of the three insulators placed between the enhancer and the promoter; again, Wari-Wari, Wari-gypsy or 1A2-Wari pairing results in mutual neutralization (insulator bypass) when they precede the promoter. The distressing issue is that this element hides in all mini-white constructs employed worldwide to study various insulators and other regulatory elements as well as long-range genomic interactions, and its versatile effects could have seriously influenced the results and conclusions of many works (Chetverina, 2008).

Search PubMed for articles about Drosophila white gene

Ames, G. F. L. (1986). The basis of multidrug resistance in mammalian cells: homology with bacterial transport. Cell 47: 323-324. PubMed ID: 3533273

Anaka, M,, et al. (2008). The white gene of Drosophila melanogaster encodes a protein with a role in courtship behavior. J. Neurogenet. 22(4): 243-76. PubMed ID: 19012054

Arimoto, E., Kawashima, Y., Choi, T., Unagami, M., Akiyama, S., Tomizawa, M., Yano, H., Suzuki, E. and Sone, M. (2020). Analysis of a cellular structure observed in the compound eyes of Drosophila white; yata mutants and white mutants. Biol Open 9(1). PubMed ID: 31862863

Borycz, J., Borycz, J. A., Loubani, M. and Meinertzhagen, I. A. (2002). tan and ebony genes regulate a novel pathway for transmitter metabolism at fly photoreceptor terminals. J. Neurosci. 22,10549-10557. PubMed ID: 18931318

Borycz, J., Borycz, J. A., Kubów A., Lloyd. V. and Meinertzhagen, I. A. (2008). Drosophila ABC transporter mutants white, brown and scarlet have altered contents and distribution of biogenic amines in the brain. J. Exp. Biol. 211(Pt 21): 3454-66. PubMed ID: 18931318

Borycz, J. A., Borycz, J., Kostyleva, R. and Meinertzhagen, I. A. (2005a). Drosophila ABC transporter mutants white, scarlet and brown have an altered head content and distribution of biogenic amines. Abstr. Soc. Neurosci. 31: 30.16

Borycz, J. A., Borycz, J., Kubów, A., Kostyleva, R. and Meinertzhagen, I. A. (2005b). Histamine compartments of the Drosophila brain with an estimate of the quantum content at the photoreceptor synapse. J. Neurophysiol. 93: 1611-1619. PubMed ID: 15738275

Cabrero, P., Radford, J. C., Broderick, K. E., Veenstra, J., Spana, E., Davies, S. and Dow, J. A. T. (2002). The CRF gene of Drosophila melanogaster encodes a diuretic peptide that activates cAMP signalling. J. Exp. Biol. 205: 3799-3807. PubMed ID: 12432004

Campbell, J. L. and Nash, H. A. (2001). Volatile general anesthetics reveal a neurobiological role for the white and brown genes of Drosophila melanogaster. J. Neurobiol. 49: 339-349. PubMed ID: 11745669

Castanys-Munoz, E., Alder-Baerens, N., Pomorski, T., Gamarro, F. and Castanys, S. (2007). A novel ATP-binding cassette transporter from Leishmania is involved in transport of phosphatidylcholine analogues and resistance to alkyl-phospholipids. Mol. Microbiol. 64: 1141-1153. PubMed ID: 17542911

Chan, R. F., Lewellyn, L., DeLoyht, J. M., Sennett, K., Coffman, S., Hewitt, M., Bettinger, J. C., Warrick, J. M. and Grotewiel, M. (2014). Contrasting influences of Drosophila white/mini-white on ethanol sensitivity in two different behavioral assays. Alcohol Clin Exp Res 38: 1582-1593. PubMed ID: 24890118

Chetverina, D., et al. (2008). Red flag on the white reporter: a versatile insulator abuts the white gene in Drosophila and is omnipresent in mini-white constructs. Nucleic Acids Res. 36(3): 929-37. PubMed ID: 18086699

Chintapalli, V. R., Wang, J. and Dow, J. A. (2007). Using FlyAtlas to identify better Drosophila melanogaster models of human disease. Nat. Genet. 39: 715-720. PubMed ID: 17534367

Coast, G. M., Webster, S. G., Schegg, K. M., Tobe, S. S. and Schooley, D. A. (2001). The Drosophila melanogaster homologue of an insect calcitonin-like diuretic peptide stimulates V-ATPase activity in fruit fly Malpighian tubules. J. Exp. Biol. 204: 1795-1804. PubMed ID: 11316500

Cremona, O. and De Camilli, P. (2001). Phosphoinositides in membrane traffic at the synapse. J. Cell Sci. 114(Pt 6): 1041-52. PubMed ID: 11228149

Davies, S. A., Huesmann, G. R., Maddrell, S. H. P., O'Donnell, M. J., Skaer, N. J. V., Dow, J. A. T. and Tublitz, N. J. (1995). CAP2b, a cardioacceleratory peptide, is present in Drosophila and stimulates tubule fluid secretion via cGMP. Am. J. Physiol. 269: R1321-R1326. PubMed ID: 8594932

Day, J. P., Houslay, M. D. and Davies, S. A. (2006). A novel role for a Drosophila homologue of cGMP-specific phosphodiesterase in the active transport of cGMP. Biochem. J. 393: 481-488. PubMed ID: 16232123

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date revised: 12 April 2022

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