The Drosophila ebony mutation (Bridges and Morgan, [1923] Publs Carnegie Inst Wash 327:50) reveals a pleiotropic phenotype with cuticular and behavioral defects. To understand Ebony function in the nervous system, particularly in transmission of the visual signal, it is essential to know the cell type and temporal characteristics of its expression throughout development. Therefore, antiserum was raised against an Ebony peptide to detect the protein in whole-mount and slice preparations of Drosophila. Attention was focused on ebony expression in the adult optic neuropiles of the fly. Colocalization of Ebony with neuronal or glial cell markers in frozen sections showed non-neuronal expression of ebony in the lamina and medulla neuropiles. Furthermore, colocalization with glial cell markers demonstrated glial expression of ebony in epithelial glia of the lamina and neuropile glia of the distal medulla. This finding was confirmed for the lamina epithelial glia by electron microscopic examination of immunolabeling by using the diaminobenzidine method. These glia have in common that they match the two sites of histamine release from the compound eye's photoreceptors. Possible ways in which the biochemical activity of Ebony might function with respect to histamine release are considered (Richardt, 2002).
A quantitative reverse transcription polymerase chain reaction (Q-RT-PCR) study was carried using oligonucleotide primers for ebony that flank exons 2 and 3. ebony RNA was found to exhibit diurnal (in LD) and circadian (in DD) oscillations in abundance in adult head tissues, with peak abundance occurring at the beginning of the photoperiod or subjective day. In both conditions, there is an approximate 4-fold difference between the times of peak and trough RNA abundance. Although the abundance of ebony RNA seems to decrease slightly during DD, there is nonetheless still a clear rhythm in abundance with a peak at the beginning of subjective day. ebony RNA cycling is eliminated in tim01 and per01 mutants, in both LD and DD, indicating that it is governed by a PER/TIM-based circadian oscillator (Suh, 2007).
It has been reported that Ebony protein can be detected in the larval nervous system and the adult visual system; in the visual system, Ebony was found to be exclusively localized to neuropile and epithelial glial cells (Richardt, 2002). The spatial localization of Ebony protein within the adult nervous system, including the visual system and protocerebrum, was studied in order to examine the abundance of the protein at different times of day. To determine the locations of Ebony-containing cells throughout the brain, immunostaining was performed using an anti-Ebony antibody and whole mounts of the adult or third-instar larval nervous systems. These studies indicated that the protein localizes to many regions of the brain and ventral nervous system at both developmental stages. Ebony can be detected in larvae within defined cell populations of the brain lobes and ventral nervous system (Richardt, 2002). In adults, Ebony is detected in the optic lobes, protocerebrum, and thoracic ganglia of wild-type animals, whereas Ebony-positive cells are not seen in tissues obtained from an e1 mutant, a known amorph. It is noted that development of the nervous system appears to be grossly normal in the e1 mutant, and the spatial distribution of Tyrosine Hydroxylase, PDF, and TIM proteins are similar in the e1 mutant and the wild-type. Double labeling with anti-Ebony and anti-Repo (Reversed Polarity, a glia-specific antigen) antibodies demonstrated that all Ebony-containing cells of the larval and adult nervous systems are glia. A similar analysis, using anti-Ebony and anti-Elav (a neuronal marker), did not detect any colabeled cells, consistent with the idea that Ebony is exclusively localized to glia. Previous reports demonstrated Ebony localization in glia of the optic lobes (Richardt, 2002), and consistent with these published results, approximately 120 Ebony-containing cells were detected in each adult medulla. Within the adult protocerebrum, approximately 100 Ebony-positive glial cells were detected, and these are located in several different areas of the dorsal and lateral protocerebrum, regions that are known to contain clock neurons. The positions of Ebony glia suggest that most correspond to the neuropile class of glia, as reported by Richardt (2002), but certain Ebony-positive cells are in close proximity to neuronal cell bodies and may represent cell body glia (Suh, 2007).
To determine where in the adult brain Ebony might show circadian changes in abundance, Ebony immunoreactivity was examined in whole mounts of the adult brain at two times of day that correspond to the peak and trough of abundance as determined by protein blotting experiments. Anti-Ebony immunofluorescence signals were higher at the beginning of the day (or subjective day) (ZT2 or CT3.5) than during the late night or subjective night (ZT21 or CT21). Rhythmic changes in Ebony immunofluorescence were observed in all regions of the adult brain, although they were most apparent in the dorsal protocerebrum and the optic medulla. Whereas robust circadian fluctuations in anti-Ebony staining were apparent in wild-type (WT) brains, this was not observed for brains for the tim01 mutant, consistent with a circadian regulation of Ebony protein abundance (Suh, 2007).
How does the molecular clock regulate the rhythmic expression of ebony in glia? To determine the locations of Ebony glia, relative to clock cells, coimmunostaining experiments were performed using anti-Ebony and anti-PER (or TIM) antibodies. This analysis documented colocalization of Ebony and clock proteins in certain glia and a close association of other Ebony glia with PER/TIM-containing neurons or glia. Interestingly, there are Ebony glia adjacent to the small ventral lateral neurons (sLNvs), the dorsal lateral neurons (LNds), and the dorsal neuron 1 (DN1) and dorsal neuron 3 (DN3) groups. In the optic medulla, Ebony and PER (or TIM) colocalize within certain glia—in this region, approximately 60% of the Ebony glia contain PER and TIM. This suggests that ebony expression might be directly regulated by a PER/TIM-based oscillator within certain glial populations (Suh, 2007).
Given that many Ebony glia contain PER and TIM, it was asked whether expression of Ebony in all clock cells (both glia and neurons) would restore normal behavioral rhythmicity in ebony mutants. This is indeed the case; tim-Gal4-driven expression of ebony+ restored normal behavior in e1 homozygotes. It is presumed that the expression of ebony in a TIM-containing subset of the Ebony glia restores rhythmicity, although expression in neurons (using the elav-Gal4 driver) also weakly rescues rhythmicity. Neuronal rescue might be due to production and release of β-alanyl-amine conjugates from BAS-containing neurons (Suh, 2007).
To determine whether Ebony glia might be positioned to regulate pacemaker neuronal function or to be regulated by the pacemaker cells, the anatomical relationship between Ebony- and PDF (pigment-dispersing factor)-containing cells was looked at more closely. PDF neuropeptide is released from the dorsal projections of the small ventral lateral neurons (sLNvs) according to a circadian rhythm, and it is essential for the circadian regulation of locomotor activity. Coimmunostaining with anti-Ebony and anti-PDF antisera demonstrates that Ebony-containing glia are in close proximity to PDF cell bodies and their projections in the adult brain. It would appear that certain processes of the PDF neurons are closely apposed to Ebony-containing glia, suggesting the possibility of neuronal-glial communication (Suh, 2007).
Because Ebony's BAS activity can conjugate β-alanine to biogenic amines, including DA and 5-HT, the distribution of Ebony glia relative to dopaminergic and serotonergic neurons was also examined. Studies in mammals and insects demonstrate roles for biogenic amines in the regulation of locomotor activity; a dramatic example is the hyperlocomotion phenotype of dopamine transporter (DAT) mutants (Giros, 1996; Kume, 2005). Thus, an interesting hypothesis is that the Ebony BAS may terminate biogenic amine transmitter action by sequestering amines in N-β-alanyl-biogenic amine (NBAA) conjugates (Richardt, 2003), and thus play a role in regulating locomotor activity. Coimmunostaining with anti-Ebony and anti-tyrosine hydroxylase (TH) or anti-5-HT antibodies indicated that most or all DA neurons are close to Ebony glia within the larval and adult brains. Indeed, it would appear that these Ebony glia lie in close proximity to projections from DA or 5-HT neurons. This pattern of localization supports the hypothesis that Ebony enzymatic activity, which is restricted to glial cells, may modulate synaptic biogenic amine levels by generating NBAA conjugates. It is an intriguing hypothesis that NBAA release from glia functions as a feedback mechanism to regulate biogenic amine release from aminergic neurons (Suh, 2007).
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).
Two serotonin receptor transcripts, 5-HT2 and 5-HT1A, were found to oscillate with phases of ZT15 and ZT18, respectively. Serotonin is known to be involved in a variety of neuronal processes in animals, including synaptic plasticity, clock entrainment, and mating behavior. The 104 serotonergic neurons in the adult CNS have been mapped, but no studies have been done of either 5-HT receptor localization or receptor mutant phenotypes. Neither of these receptors are orthologs of the mammalian 5-HT receptor implicated in photic clock entrainment; this would be represented by theDrosophila 5-HT7 gene. 5-HT1A belongs in a class of receptors that respond to agonists by decreasing cellular cAMP, while 5-HT2 is homologous to mammalian receptors whose main mode of action involves activation of phospholipase C, a function involved in synaptic plasticity (Claridge-Chang, 2001).
The ebony transcript was found to oscillate, showing a peak of expression around ZT5. ebony oscillation connects with a body of earlier evidence linking ebony to circadian activity rhythms. ebony hypomorphs show severe defects in circadian rhythm including arrhythmicity/aberrant periodicity in the free-running condition, as well as abnormal activity patterns in LD conditions. Ebony is a putative ß-alanyl dopamine synthetase, and hypomorphs show elevated levels of dopamine. Dopamine has been implicated in control of motor behavior, since it induces reflexive locomotion in decapitated flies, and this response is under circadian control. The results suggest that oscillations of ebony contribute to the assembly of rhythmic locomotor behavior. Other evidence points to a role in clock resetting. In addition to impaired entrainment in LD, ebony flies show an abnormal ERG, and ebony is strongly expressed in the lamina and the medulla optic neuropile, a region associated with vision rather than motor control (Claridge-Chang, 2001).
The Drosophila mutant tan (t) shows reciprocal pigmentation defects compared with the ebony (e) mutant. Visual phenotypes, however, are similar in both flies: Electroretinogram (ERG) recordings lack 'on' and 'off' transients, an indication of impaired synaptic transmission to postsynaptic cells L1 and L2. Cloning of tan revealed transcription of the gene in the retina, apparently in photoreceptor cells. Tan was expressed in Escherichia coli and Western blotting and mass spectroscopic analyses was used to confirmed that Tan is expressed as preprotein, followed by proteolytic cleavage into two subunits at a conserved --Gly--Cys-- motif like its fungal ortholog isopenicillin-N N-acyltransferase (IAT). Tan thus belongs to the large family of cysteine peptidases. To discriminate expression of Tan and Ebony in retina and optic neuropils, antisera was raised against specific Tan peptides. Testing for colocalization with GMR-driven n-Syb-GFP labeling revealed that Tan expression is confined to the photoreceptor cells R1-R8. A close proximity of Tan and Ebony expression is evident in lamina cartridges, where three epithelial glia cells envelop the six photoreceptor terminals R1-R6. In the medulla, R7/R8 axonal terminals appeared lined up side by side with glial extensions. This local proximity supports a model for Drosophila visual synaptic transmission in which Tan and Ebony interact biochemically in a putative histamine inactivation and recycling pathway in Drosophila (Wagner, 2007).
It is proposed that Ebony functions within glia to regulate behavioral rhythmicity. To test this hypothesis, the product was selectively expressed in glial cells of ebony mutants and wild-type animals using the Gal4/UAS binary expression system. To determine if increased ebony+ expression perturbed the activity rhythm of wild-type individuals, the glial-specific driver Repo-Gal4 was employed to overexpress the gene in all glia. There were no obvious differences between repo-Gal4;UAS-ebony flies and UAS-ebony control individuals with regard to the robustness of rhythmicity or the percentage of rhythmic flies. It is concluded that Ebony product is not limiting for determination of rhythmicity (Suh, 2007).
Importantly, the glial-specific expression of ebony+ in an ebony null background, using repo-Gal4, completely rescued rhythmicity, demonstrating that expression within glia is sufficient for normal behavior. Furthermore, Ebony BAS activity is required for normal behavior, since the glial expression of an enzymatically dead form of Ebony (a Ser to Ala change in residue 611 of the active site [Richardt, 2003]) did not rescue behavioral rhythms. Interestingly, the behaviorally rescued flies still had a dark body color similar to e1 control flies, directly demonstrating anatomically separable requirements for ebony in pigmentation and behavioral rhythmicity. As expected, actin-Gal4;UAS-ebony+;eAFA/eAFA flies, which express ebony+ ubiquitously, had normal pigmentation and locomotor activity rhythms (Suh, 2007).
Although Ebony protein normally cycles in abundance in wild-type individuals, it was surmised that pan-glial overexpression, using repo-Gal4, might eliminate the protein cycle. To determine if this was the case, Ebony cycling was examined in repo-Gal4;UAS-ebony+ and control flies using immunostaining procedures. Whereas Ebony protein abundance showed circadian oscillations in control flies, the protein seemed to be constitutively high in repo-Gal4;UAS-ebony individuals. Thus, it would appear that a constitutively high level of Ebony in glia can rescue behavioral rhythmicity, even though the protein normally shows circadian changes in abundance in wild-type animals. The possibility cannot be excluded, however, that Ebony cycling persists in a limited glial cell population of repo-Gal4;UAS-ebony+ flies (Suh, 2007).
If activity feeds back to regulate clock function, in some way, then the molecular oscillator might be perturbed by a lack of Ebony product. To ask whether ebony flies have normal oscillator function, per or tim RNA abundance were examined in wild-type and e1 mutant flies. Those studies showed that the temporal pattern of per and tim expression is similar in the two strains; however, tim transcript abundance seemed to be slightly reduced relative to the wild-type in DD conditions. It was noted, however, that the TIM protein abundance rhythm was similar in the wild-type and e1 as assayed by Western analysis. Furthermore, TIM levels cycled normally in both genotypes in several different TIM-containing cell types, indicating normal clock function (Suh, 2007).
As an assay of clock output, rhythms of PDF immunostaining were compared in e1 and the wild-type. In both types of flies, robust rhythms of PDF immunostaining were observed in the dorsal projections of the sLNv neurons, indicative of rhythms of PDF release. It is concluded that molecular clock function and PDF synaptic output are normal in ebony mutants and that Ebony acts downstream of the clock to regulate circadian behavioral rhythms (Suh, 2007).
To test the hypothesis that PDF release controls the ebony transcriptional rhythm, Ebony immunofluorescence was examined at different times of day in wild-type flies and pdf01 mutants, which lack PDF peptide. Ebony protein cycling, as assessed by immunostaining for the protein at the peak and trough of the cycle, was found to be normal in the pdf01 mutant. Furthermore, quantitative RT-PCR measurements using RNA samples from wild-type and pdf01 mutant heads revealed significant cycling of Ebony in both genotypes (during LD and several days of DD) but no differences between the two genotypes. These results indicate that ebony cycling is regulated by a mechanism not involving the neuronal release of PDF (Suh, 2007).
Rhythmicity of the e1 mutant was examined in two other genetic backgrounds to determine if alterations of DA signaling might contribute to the arrhythmicity of ebony mutants. It has been postulated that the Yellow gene product functions in the melanization pathway, and it has been suggested that y mutants might have elevated DA levels (Drapeau, 2003), since the disruption of DOPA-melanin synthesis in yellow mutants is predicted to lead to elevated DA levels, similar to that observed in ebony flies (Hodgetts, 1973). Double mutants carrying e1 and a yellow mutation (y1) exhibited increased arrhythmicity relative to e1 single mutants, suggesting an additive effect of the two mutations. Although this might be due to a further increase in DA levels in the double mutant, the precise biochemical role of Yellow is still quite controversial (Suh, 2007).
In contrast, it is quite likely that the Drosophila dDAT mutant (fumin, fmn), with decreased DAT function (Kume, 2005) has elevated synaptic DA levels. Thus, rhythmicity was examined in double mutants homozygous for both DATfmn and ebony (DATfmn;e1), and those studies revealed an interesting genetic interaction between the two genes. Whereas e1 mutants seem to have normal levels of activity, DATfmn flies are known to exhibit significantly elevated activity levels (Kume, 2005). Not surprisingly, flies carrying both the e1 and DATfmn mutations are arrhythmic, similar to e1, but unexpectedly the double mutant exhibited activity levels more similar to those of e1 flies than to DATfmn individuals; i.e., e1 suppresses the hyperactivity phenotype of DATfmn. Given that fly DAT is localized exclusively to DA neurons (Porzgen, 2001), this result strongly suggests that Ebony enzymatic activity is relevant for dopaminergic neuronal function (Suh, 2007).
In the course of quantitating activity in single and double mutants, activity levels were carefully measured in wild-type flies and ebony mutants at different times of day. Measurements of daily activity levels indicated that e1 flies are slightly less active than wild-type individuals in both LD and DD conditions. Interestingly, however, the reduction in activity selectively occurred during the day portion of the cycle, indicating that the Ebony function is required for high levels of daytime activity. Such a result is consistent with the notion that Ebony (and perhaps N-beta-alanyldopamine) promotes locomotor activity during the daytime (Suh, 2007).
The concentrations of catecholamines were determined in the decuticalarized retinas and brains at different ages in wildtype and ebony Drosophila melanogaster using the HPLC-technique with an electrochemical detector. L-Dopa, dopamine (DA), alpha-methyldopa (alpha-MD) and unidentified compounds X1, X2 and X3 were found in decuticalarized retinas and brains of wildtype and ebony at different ages. Retinas and brains of the mutant ebony have higher concentrations of L-Dopa, DA and alpha-MD than the wildtype. In both wildtype and ebony, the concentrations of X1, X2 and X3 were found to be higher in decuticalarized retinas than in brains. The identity and importance of X1, X2 and X3 are still unknown (Ramadan, 1993).
Body coloration affects how animals interact with the environment. In insects, the rapid evolution of black and brown melanin patterns suggests that these are adaptive traits. The developmental and molecular mechanisms that generate these pigment patterns are largely unknown. This study demonstrates that the regulation and function of the yellow and ebony genes in Drosophila melanogaster play crucial roles in this process. The Yellow protein is required to produce black melanin, and is expressed in a pattern that correlates with the distribution of this pigment. Conversely, Ebony is required to suppress some melanin formation, and is expressed in cells that will produce both melanized and non-melanized cuticle. Ectopic expression of Ebony inhibits melanin formation, but increasing Yellow expression can overcome this effect. In addition, ectopic expression of Yellow is sufficient to induce melanin formation, but only in the absence of Ebony. These results suggest that the patterns and levels of Yellow and Ebony expression together determine the pattern and intensity of melanization. Based on their functions in Drosophila melanogaster, it is proposed that changes in the expression of Yellow and/or Ebony may have evolved with melanin patterns. Consistent with this hypothesis, it was found that Yellow and Ebony are expressed in complementary spatial patterns that correlate with the formation of an evolutionary novel, male-specific pigment pattern in Drosophila biarmipes wings. These findings provide a developmental and genetic framework for understanding the evolution of melanin patterns (Wittkopp, 2002; full text of article).
Similar phenotypic changes have evolved independently in many animal taxa. It is unknown whether independent changes involve the same or different developmental and genetic mechanisms. Myriad pigment patterns in the genus Drosophila offer numerous opportunities to address this question. Previous studies identified regulatory and structural genes involved in the development and diversification of pigmentation in selected species. This study examined Drosophila americana and Drosophila novamexicana, interfertile species that have evolved dramatic pigmentation differences during the few million years since their divergence. Interspecific genetic analysis was used to investigate the contribution of five specific candidate genes and other genomic regions to phenotypic divergence by testing for associations between molecular markers and pigmentation. At least four distinct genomic regions contributed to pigmentation differences, one of which included the ebony gene. Ebony protein was expressed at higher levels in the more yellow D. novamexicana than the heavily melanized D. americana. Because Ebony promotes yellow pigment formation and suppresses melanization, the expression difference and genetic association suggest that evolution at the ebony locus contributed to pigmentation divergence between D. americana and D. novamexicana. Surprisingly, no genetic association with the yellow locus was detected in this study, and Yellow expression was identical in the two species. Evolution at the yellow locus underlies pigmentation divergence among other Drosophila species; thus, similar pigment patterns have evolved through regulatory changes in different genes in different lineages. These findings bear upon understanding classic models of melanism and mimicry (Wittkopp, 2003; full text of article).
Many proteins are used repeatedly in development, but usually the function of the protein is similar in the different contexts. This study reports that the classical Drosophila locus tan encodes a novel enzyme required for two very different cellular functions: hydrolysis of N-β-alanyl dopamine (NBAD) to dopamine during cuticular melanization, and hydrolysis of carcinine to histamine in the metabolism of photoreceptor neurotransmitter. Two tan-like P-element insertions that failed to complement classical tan mutations were isolated. Both are inserted in the 5′ untranslated region of the previously uncharacterized gene CG12120, a putative homolog of fungal isopenicillin-N N-acyltransferase (EC 2.3.1.164). Both P insertions showed abnormally low transcription of the CG12120 mRNA. Ectopic CG12120 expression rescued tan mutant pigmentation phenotypes and caused the production of striking black melanin patterns. Electroretinogram and head histamine assays indicated that CG12120 is required for hydrolysis of carcinine to histamine, which is required for histaminergic neurotransmission. Recombinant CG12120 protein efficiently hydrolyzed both NBAD to dopamine and carcinine to histamine. It is concluded that D. melanogaster CG12120 corresponds to tan. This is likely to be the first molecular genetic characterization of NBAD hydrolase and carcinine hydrolase activity in any organism and is central to the understanding of pigmentation and photoreceptor function (True, 2005; full text of article).
The molecular identification of tan helps clarify a crucial step in dopamine metabolism and melanin biosynthesis in epidermal cells. All developing adult epidermal cells in insects are capable of secreting catecholamine precursors of melanin and sclerotin, and current models propose that the patterns of adult melanin reflect the differential spatial regulation of four parallel branches from the core dopamine pathway catalyzed by tyrosine hydroxylase and dopa decarboxylase. One of the four branches produces dopa melanin, which is under the control of yellow, the exact function of which is unknown, and at least two Yellow-related proteins, Yellow-f and Yellow-f2, which convert dopachrome to 5,6-dihydroxyindole. Dopamine is also secreted and converted into dopamine melanin through an as yet uncharacterized pathway. Areas of the cuticle that are not melanized secrete NBAD, produced by the action of the Ebony protein, resulting in yellow or light tan cuticle, or N-acetyl dopamine, produced by the action of the arylalkylamine N-acyltransferases, which results in transparent cuticle. All of these precursors are extracellularly polymerized and crosslinked to cuticle proteins, probably through the action of a common set of enzymes, including phenol oxidases, the functions of which in the developing cuticle are not well characterized. Tyrosine and catecholamines are also provided to some degree from the hemolymph, and a hemolymph supply of melanin precursors is required for wing pigmentation (True, 2005).
Normal melanization depends in part on Tan function to provide dopamine by hydrolyzing sequestered NBAD. It is currently unclear why this dopamine is produced from NBAD rather than directly from dopa by dopa decarboxylase. One possible explanation for an Ebony-Tan 'shunt' would be if epidermal cells require rapid or precise temporal regulation of dopamine secretion during cuticle development. For example, long-term sequestration of dopamine awaiting this developmental time window could be injurious to the cell. Alternatively, conversion of dopamine to NBAD by Ebony may be a constitutive ancestral state in insects, and conversion of some of this NBAD back to dopamine for melanin production may be a derived condition in some insects. NBAD synthase activity has been demonstrated in lepidopterans, in which NBAD is a precursor to yellow papiliochrome pigment. Isolation and functional characterization of tan and ebony gene homologs from more basal insects will be needed to test these alternative hypotheses (True, 2005).
The production of dopamine melanin depends on Tan function, which in turn depends on Ebony to produce its substrate. As predicted by this relationship, ebony is epistatic to tan. Production of melanin from both dopa and dopamine is an apparent degeneracy that occurs in insects but not vertebrates, which produce melanin primarily from L-dopa. The final dark black color of many insects reflects contributions of both types of melanin, which continuously darken during cuticle maturation and hardening. There is evidence in D. melanogaster that the two melanin pathways are not independent. The presence of Ebony appears to determine whether melanin will be produced, even in the presence of ectopic Yellow, which gains access to the core dopamine pathway upstream at the dopa stage. Only in the absence of Ebony function is ectopic Yellow able to promote ectopic melanin production. This suggests that normally most dopa is converted to dopamine and then to NBAD (or N-acetyl dopamine), but when the dopamine-to-NBAD step is blocked in an ebony mutant more dopa may be available for Yellow-mediated conversion to dopa melanin, possibly because of product inhibition of dopa decarboxylase. Note that back-conversion of dopamine to dopa has not been observed in insects. ebony mutants accumulate excess levels of dopamine, which is shunted to dopamine melanin. This mechanism has long been a candidate for naturally occurring melanism, which is an extremely common type of polymorphism in insects. Thus, ebony itself is a candidate gene for such polymorphisms. However, D. melanogaster ebony mutants do not show the complete dominance typical of naturally occurring melanic alleles in other insects. Another important candidate is tan, which is shown in this study to be mutable, via gain of function, to dominant production of ectopic melanin (True, 2005).
In a broad survey of Drosophila melanogaster population samples, levels of abdominal pigmentation were found to be highly variable and geographically differentiated. A strong positive correlation was found between dark pigmentation and high altitude, suggesting adaptation to specific environments. DNA sequence polymorphism at the candidate gene ebony revealed a clear association with the pigmentation of homozygous third chromosome lines. The darkest lines sequenced had nearly identical haplotypes spanning 14.5 kb upstream of the protein-coding exons of ebony. Thus, natural selection may have elevated the frequency of an allele that confers dark abdominal pigmentation by influencing the regulation of ebony (Pool, 2007).
This study discovered causal genetic variation for the difference in the thoracic trident pigmentation intensity between two wild-derived strains of D. melanogaster. It was the difference in expression level of ebony, which codes for an enzyme in the melanin-synthesis pathway and has pleiotropic effects on vision and behavior (Takahashi, 2007).
The Drosophila photoreceptor cell has long served as a model system for researchers focusing on how animal sensory neurons receive information from their surroundings and translate this information into chemical and electrical messages. Electroretinograph (ERG) analysis of Drosophila mutants has helped to elucidate some of the genes involved in the visual transduction pathway downstream of the photoreceptor cell, and it is now clear that photoreceptor cell signaling is dependent upon the proper release and recycling of the neurotransmitter histamine. While the neurotransmitter transporters responsible for clearing histamine, and its metabolite carcinine, from the synaptic cleft have remained unknown, a strong candidate for a transporter of either substrate is the uncharacterized Inebriated protein. The inebriated gene (ine) encodes a putative neurotransmitter transporter that has been localized to photoreceptor cells in Drosophila and mutations in ine result in an abnormal ERG phenotype in Drosophila. Loss-of-function mutations in ebony, a gene required for the synthesis of carcinine in Drosophila, suppress components of the mutant ine ERG phenotype, while loss-of-function mutations in tan, a gene necessary for the hydrolysis of carcinine in Drosophila, have no effect on the ERG phenotype in ine mutants. By feeding wild-type flies carcinine, components of mutant ine ERGs can be duplicated. Finally, it was demonstrated that treatment with H3 receptor agonists (H3 receptor is a presynaptic G-protein-coupled autoreceptor, a metabotropic histamine receptor, that inhibits histamine release) or inverse agonists rescue several components of the mutant ine ERG phenotype. This sutdy provides pharmacological and genetic epistatic evidence that ine encodes a carcinine neurotransmitter transporter. It is also speculated that the oscillations observed in mutant ine ERG traces are the result of the aberrant activity of a putative H3 receptor (Gavin, 2007).
The findings of this study indicate that the presumed neurotransmitter transporter encoded by the ine gene in Drosophila transports the histamine metabolite carcinine. Using genetic epistasis this study shows that the oscillations observed in mutant ine ERGs require histidine decarboxylase activity and the carcinine-synthesizing enzyme Ebony, but not the carcinine-hydrolyzing enzyme Tan. Treating wild-type flies with carcinine can phenocopy components of the mutant ine ERG phenotype. Finally, by rescuing the ine2-associated phenotype with drugs that target the mammalian H3 receptor, pharmacological evidence is provided for the presence of a yet uncharacterized putative H3 receptor in Drosophila that may be responsible for the ERG oscillations observed in flies carrying mutations in the ine gene (Gavin, 2007).
Previous studies involving intracellular voltage recordings of ine mutants have led to the conclusion that the oscillations observed in ine mutant ERGs are the result of a defect occurring within the photoreceptor cell. These conclusions are supported by expressing ine specifically in photoreceptor cells and demonstrating a rescue of the ine2-associated oscillations. Neurotransmitter transporters are often able to function from either the presynaptic neuron or from neighboring glial cells, as shown at the neuromuscular junction in ine mutants. Glial cell-specific expression of the ine gene in ine2 flies results in a complete rescue of the ine mutant ERG phenotype. It was somewhat unexpected that ine expression in glial cells rescued the ine2 phenotypes, since glial cells have been shown to lack Tan protein and thus would be unable to convert carcinine back to a recycled pool of histamine. However, it is possible that glial cells do express trace amounts of the enzyme Tan to hydrolyze carcinine and generate a renewable source of histamine for photoreceptor cells, and it is also possible that the Inebriated protein is expressed in a non-autonomous manner and can be transported from glial cells to photoreceptors in the fly eye (Gavin, 2007).
The finding that an ERG recording can exhibit oscillations is somewhat surprising. An ERG does not record the electrical response of a single photoreceptor, but rather is a collective measure of the retinal photoresponse. Thus, if the mutant ine-associated ERG defects are indeed localized to the photoreceptor synapse, as the data suggest, then one would expect that different photoreceptors would be excited/inhibited at different timepoints, ultimately resulting in the oscillations simply canceling themselves out. The fact that oscillations are indeed observed, and appear to be due to a defect occurring at the photoreceptor synapse, implies the existence of an uncharacterized and complex synchronization of photoreceptor cell de-/repolarization (Gavin, 2007).
The lack of rescue of ine2-associated oscillations in flies carrying additional mutations in the postsynaptic histamine receptor gene ort, the finding that mutant ine oscillations were detected within single photoreceptor cells, and the observations that the mutant ine phenotype can be rescued when ine is expressed in photoreceptors, all combine to strongly suggest that the oscillation phenotype is likely a result of a defect occurring within the photoreceptor itself. In addition, by crossing ine2 animals with HdcP218 flies, it was demonstrated that the ine2-associated oscillations are dependent upon histamine synthesis. All of these results indicate that histamine is somehow contributing to the aberrant ERG witnessed in ine2 flies, and that histamine appears to be acting on the presynaptic photoreceptor cell to induce this oscillation phenotype. Further epistatic analyses also revealed that Ebony, but not Tan, activity is required for the generation of oscillations in ine2 ERGs. These genetic experiments are consistent with ine encoding either a carcinine importer found in the photoreceptor cell or a carcinine exporter found in glial cells. The homology of Inebriated with other known Na+/Cl- neurotransmitter transporters (which import neurotransmitter into cells) suggests that Inebriated protein is transporting carcinine into the photoreceptor, and not out of glial cells (Gavin, 2007).
While Ebony is known to act on multiple substrates, such as dopamine to generate β-alanyl-dopamine, the requirement of histamine synthesis for the maintenance of ine2-associated oscillations suggests that it is β-alanyl-histamine, or carcinine, that is somehow responsible for the oscillations observed in ine2 ERGs. It should be noted, however, that ebony mutations were not sufficient in rescuing the hyperpolarization response observed in mutant ine ERG traces. The origins of this hyperpolarization response are still unclear and further research will be required to elucidate its exact meaning. In tan mutants, one would predict that there would be a buildup of carcinine. However, this buildup does not give rise to an ERG recording similar to that of ine2. This is due most likely to the presence of functional Inebriated protein in tan mutant flies, which should effectively clear the carcinine from the synaptic cleft for degradation within the photoreceptor cell (Gavin, 2007).
By treating wild-type and ebony11 flies with carcinine and subsequently inducing components of the ine2-ERG phenotype, further evidence is provided suggesting that the sharp depolarization spike, the oscillations, and the hyperpolarization response all seen in ine2-ERGs are due to a buildup of carcinine within the photoreceptor synaptic cleft. While the oscillations observed in carcinine-treated wild-type flies do not mimic exactly the oscillations seen in ine2 ERG recordings, it is presumably difficult to replicate the carcinine and histamine balance occurring in the eyes of ine2 animals. Indeed, treatment of wild-type flies with higher (10%) or lower (1%) concentrations of carcinine were less effective in inducing the oscillations than the described 5% carcinine dose (Gavin, 2007).
It is possible that carcinine is being degraded or modified by the fly before the compound is able to exert its effects at the photoreceptor cell. In order to eliminate the activity of one enzyme known to be involved in carcinine metabolism, tan1 flies were treated with 5% carcinine overnight. Surprisingly, none of the tan1 flies treated with carcinine showed an aberrant ERG phenotype. It was surprising that carcinine treatment had a strong effect in flies of the ebony11, but not the tan1, background. While the results of these tan1 and ebony11 carcinine-treatment experiments are unexpected, one possible explanation may involve the regulation of carcinine clearance/degradation. The tan1 flies presumably suffer from a perpetual excess of carcinine even before exogenous carcinine treatment, and these flies, in order to reduce their sensitivity to this compound, may consequently decrease the levels of a putative carcinine receptor, increase their rate of carcinine degradation, or increase the levels of Inebriated protein for carcinine clearance. However, ebony11 flies are relatively 'naive' to the effects of carcinine, as their ability to synthesize this compound has been greatly diminished, and as a result these animals may have an increased level of the supposed carcinine receptor, a decrease in Inebriated receptor levels or a decrease in carcinine degradation, ultimately making them more sensitive to the effects of carcinine treatment (Gavin, 2007).
It remains to be seen whether or not all of the mutant ine-associated phenotypes, including increased neuronal excitability and increased sensitivity to osmotic stress, are due to the inability of these flies to transport carcinine. It is possible that the Inebriated protein transports other compounds that perhaps share the common feature of β-alanine conjugation. This might help explain why none of the more common neurotransmitters were taken up by ine-transfected Xenopus oocytes. In order to assist in confirming that Inebriated is indeed a carcinine neurotransmitter transporter, in vitro experiments, such as neurotransmitter uptake assays, will need to be performed. In addition, the ability of Inebriated protein to take up other β-alanyl-neurotransmitters/osmolytes also should be examined (Gavin, 2007).
The oscillations present within the photoreceptor response of ine2 ERGs appear as sharp depolarization/repolarization spikes, and this oscillation phenotype is dependent upon both histamine synthesis and Ebony activity, and is sensitive to drugs that target mammalian H3 receptors. It is perplexing that the synthesis of a single metabolite, carcinine, could be responsible for both the depolarization and repolarization spikes observed within ine mutant ERGs. It is speculated that these oscillations are the result of aberrant signaling involving both carcinine and histamine at a putative H3 receptor in Drosophila. H3 receptors are an unusual example of the G-protein coupled receptor family, in that they have partial constitutive activity, resulting in a constant small percentage of stimulated G-proteins that trigger a reduction of histamine synthesis and release as well as a decrease in extracellular calcium inflow. The presence of an H3 receptor agonist, such as histamine, causes an increase in activity of the associated G-protein and therefore a stronger inhibition of both histamine release and calcium inflow. Thus, synaptic histamine serves as a negative regulator for its own release and induces a slight repolarization of a stimulated presynaptic histaminergic neuron by inhibiting presynaptic calcium channels. An H3 receptor inverse agonist is believed to act by blocking the constitutive activity of the H3 receptor, resulting in the liberation from a histamine release checkpoint as well as the release of restrictions on calcium inflow. Recently, it has been shown that carcinine has the ability to act as an inverse agonist of presynaptic H3 receptors in mice. While significant further research is required to confirm this hypothesis, it is surmised that histamine and carcinine are exerting opposing effects on the polarization state of the histaminergic photoreceptor cell by activating or inhibiting presynaptic calcium channels via a putative Drosophila H3 receptor. While a recent search of the Drosophila genome did not uncover any direct homologs to vertebrate metabotropic histamine receptors, the CG7918 gene was listed as a possible candidate for encoding such a receptor, and this gene bears strong homology to genes encoding H3 receptors in mammals. In addition, the ine2-associated oscillations display sensitivity to mammalian H3 receptor agonists and inverse agonists, strengthening the possibility that an H3 receptor does exist in Drosophila. It is still unclear what the origins of the thioperamide-sensitive depolarization spikes are that are observed in ort5 ERGs. The presence of these thioperamide-sensitive spikes in ort5 ERG recordings implies the requirement of some postsynaptic retrograde signal for ERG stability, and this ort-dependent signal may be involved in the sensitization of the putative H3 receptor (Gavin, 2007).
It was unexpected that thioperamide treatment of wild-type flies resulted in the loss of on and off transients within their ERG traces. It is possible that histamine release was so extreme in the presence of the potent thioperamide that histamine levels were nearly depleted in the eye, resulting in the disruption of downstream signaling events. Indeed, treatment of mice with high concentrations of carcinine, which acts as an inverse agonist of H3 receptors similar to thioperamide, was shown to result in significantly lower overall levels of histamine within the brains of treated mice. This model of indirect histamine depletion has also been postulated to occur in ebony mutant flies. The absence of on and off transients in ebony mutant ERG recordings is attributed to the normal release of histamine by photoreceptor cells, but this histamine subsequently lacks the ability to be 'trapped' by β-alanine conjugation, ultimately resulting in histamine diffusing away from the eye. Interestingly, expression of pertussis toxin in photoreceptor and laminar neurons in Drosophila results in a similar loss of on and off transients in ERG traces, and this is believed to be the result of inactivation of an unknown G-protein coupled receptor found in photoreceptor cells that is unlikely to be rhodopsin. It is possible that pertussis toxin was acting within photoreceptor cells upon the putative H3 receptor in this study, resulting in a lack of negative feedback on histamine synthesis/release, eventually causing the exhaustion/depletion of histamine pools. Further research will be required to confirm or dismiss the presence of a histamine/carcinine-sensitive H3 receptor in Drosophila photoreceptor cells (Gavin, 2007).
Search PubMed for articles about Drosophila Ebony
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(24): 10549-57. Medline abstract: 12486147
Claridge-Chang, A., et al. (2001). Circadian regulation of gene expression systems in the Drosophila head. Neuron 32: 657-671. Medline abstract: 11719206
Drapeau, M. D. (2003). A novel hypothesis on the biochemical role of the Drosophila Yellow protein. Biochem. Biophys. Res. Commun. 311: 1-3. Medline abstract: 14575686
Gavin, B. A., Arruda, S. E. and Dolph, P. J. (2007). The role of carcinine in signaling at the Drosophila photoreceptor synapse. PLoS Genet. 3(12): e206. PubMed Citation: 18069895
Giros, B., et al. (1996). Hyperlocomotion and indifference to cocaine and amphetamine in mice lacking the dopamine transporter, Nature 379: 606-612. Medline abstract: 8628395
Hodgetts, R. B. and Konopka, R. J. (1973). Tyrosine and catecholamine metabolism in wild-type Drosophila melanogaster and a mutant, ebony. J. Insect. Physiol. 19(6): 1211-20. Medline abstract: 4196594
Hotta, Y. and Benzer, S. (1969). Abnormal electroretinograms in visual mutants of Drosophila. Nature 222: 354-356. Medline abstract: 5782111
Hovemann, B. T., et al. (1998). The Drosophila ebony gene is closely related to microbial peptide synthetases and shows specific cuticle and nervous system expression. Gene 221(1): 1-9. Medline abstract: 9852943
Kume, K., Kume, S., Park, S. K., Hirsh, J. and Jackson, F. R. (2005). Dopamine is a regulator of arousal in the fruit fly. J. Neurosci. 25(32): 7377-84. Medline abstract: 16093388
Kyriacou, C. P., Burnet, B. and Connolly, K. (1978). The behavioral basis of overdominance in competitive mating success at the ebony locus of Drosophila melanogaster. Anim. Behav. 26: 1195-1206
Newby, L. M. and Jackson, F. R. (1991). Drosophila ebony mutants have altered circadian activity rhythms but normal eclosion rhythms. J. Neurogenet. 7(2-3): 85-101. Medline abstract: 1903161
Perez, M., Schachter, J. and Quesada-Allue, L. A. (2004). Constitutive activity of N-beta-alanyl-catecholamine ligase in insect brain, Neurosci. Lett. 368: 186-191. Medline abstract: 15351446
Pool. J. E. and Aquadro. C. F. (2007). The genetic basis of adaptive pigmentation variation in Drosophila melanogaster. Mol. Ecol. 16(14): 2844-51. Medline abstract: 17614900
Porzgen, S. K., et al. (2001). . The antidepressant-sensitive dopamine transporter in Drosophila melanogaster: A primordial carrier for catecholamines. Mol. Pharmacol. 59: 83-95. Medline abstract: 11125028
Ramadan, H., Alawi, A. A. and Alawi, M. A. (1993). Catecholamines in Drosophila melanogaster (wild type and ebony mutant) decuticalarized retinas and brains. Cell Biol. Int. 17(8):765-71. Medline abstract: 8220304
Richardt, A., et al. (2003). Ebony, a novel nonribosomal peptide synthetase for beta-alanine conjugation with biogenic amines in Drosophila. J. Biol. Chem. 278(42): 41160-6. Medline abstract: 12900414
Richardt, A., Rybak, J., Stortkuhl, K. F., Meinertzhagen, I. A. and Hovemann, B. T. (2002). Ebony protein in the Drosophila nervous system: optic neuropile expression in glial cells. J. Comp. Neurol. 452(1): 93-102. Medline abstract: 12205712
Suh, J. and Jackson, F. R. (2007). Drosophila Ebony activity is required in glia for the circadian regulation of locomotor activity. Neuron 55(3): 435-47. Medline abstract: 17678856
Takahashi, A., Takahashi, K., Ueda, R. and Takano-Shimizu. T. (2007). Natural variation of ebony gene controlling thoracic pigmentation in Drosophila melanogaster. Genetics [Epub ahead of print]. Medline abstract: 17660557
True, J. R., et al. (2005). Drosophila tan encodes a novel hydrolase required in pigmentation and vision. PLoS Genet. 1(5):e63. Medline abstract: 16299587
Ueda, H. R., et al. (2002). Genome-wide transcriptional orchestration of circadian rhythms in Drosophila. J. Biol. Chem. 277: 14048-14052. Medline abstract: 11854264
Wagner, S., et al. (2007). Drosophila photoreceptors express cysteine peptidase tan. J. Comp. Neurol. 500(4): 601-11. Medline abstract: 17154266
Wittkopp, P. J., Williams, B. L., Selegue, J. E. and Carroll, S. B. (2003). Drosophila pigmentation evolution: divergent genotypes underlying convergent phenotypes. Proc. Natl. Acad. Sci. 100(4): 1808-13. Medline abstract: 12574518
Wittkopp, P. J., True, J. R. and Carroll, S. B. (2002). Reciprocal functions of the Drosophila yellow and ebony proteins in the development and evolution of pigment patterns. Development 129(8): 1849-58. Medline abstract: 11934851
date revised: 10 August 2009
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