InteractiveFly: GeneBrief

ebony: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References


Gene name - ebony

Synonyms -

Cytological map position- 93C7-93D1

Function - enzyme

Keywords - pigmentation, behavioral rhythmicity, photoperiod response, glia, photoreceptor activity, biogenic amine metabolism

Symbol - e

FlyBase ID: FBgn0000527

Genetic map position - 3R: 17,055,552..17,062,900 [-]

Classification - β-alanyl-dopamine synthase

Cellular location - cytoplasmic



NCBI links: Precomputed BLAST | EntrezGene

Recent literature
Johnson, W. C., Ordway, A. J., Watada, M., Pruitt, J. N., Williams, T. M. and Rebeiz, M. (2015). Genetic changes to a transcriptional silencer element confers phenotypic diversity within and between Drosophila species. PLoS Genet 11: e1005279. PubMed ID: 26115430
Summary:
The modification of transcriptional regulation has become increasingly appreciated as a major contributor to morphological evolution. However, the role of negative-acting control elements (e.g. silencers) in generating morphological diversity has been generally overlooked relative to positive-acting "enhancer" elements. The highly variable body coloration patterns among Drosophilid insects represents a powerful model system in which the molecular alterations that underlie phenotypic diversity can be defined. In a survey of pigment phenotypes among geographically disparate Japanese populations of Drosophila auraria, a remarkable degree of variation was discovered in male-specific abdominal coloration. In testing the expression patterns of the major pigment-producing enzymes, phenotypes were found that were uniquely correlated with differences in the expression of ebony, a gene required for yellow-colored cuticle. Assays of ebony's transcriptional control region indicated that a lightly pigmented strain harbored cis-regulatory mutations that caused correlated changes in its expression. Through a series of chimeric reporter constructs between light and dark strain

Miyagi, R., Akiyama, N., Osada, N. and Takahashi, A. (2015). Complex patterns of cis-regulatory polymorphisms in ebony underlie standing pigmentation variation in Drosophila melanogaster. Mol Ecol [Epub ahead of print]. PubMed ID: 26503353
Summary:
Pigmentation traits in adult Drosophila were used to investigate how phenotypic variations of continuous ecological traits can be maintained in a natural population. First, pigmentation variation in the adult female was measured at seven different body positions in 20 strains from the Drosophila Genetic Reference Panel (DGRP) originating from a natural population in North Carolina. Next, allele-specific expression levels of four genes were quantified by amplicon sequencing. Among those genes, ebony was significantly associated with pigmentation intensity of the thoracic segment. Detailed sequence analysis of the gene regulatory regions of this gene indicated that many different functional cis-regulatory alleles are segregating in the population and that variations outside the core enhancer element could potentially play important roles in the regulation of gene expression. In contrast, sequence analysis in the core cis-regulatory region of tan indicated that SNPs within the region are significantly associated with allele-specific expression level of this gene. Collectively, the data suggest that the underlying genetic differences in the cis-regulatory regions that control intraspecific pigmentation variation can be more complex than those of interspecific pigmentation trait differences, where causal genetic changes are typically confined to modular enhancer elements.

Dembeck, L. M., Huang, W., Carbone, M. A. and Mackay, T. F. (2015). Genetic basis of natural variation in body pigmentation in Drosophila melanogaster. Fly (Austin): [Epub ahead of print]. PubMed ID: 26554300

Body pigmentation in insects and other organisms is typically variable within and between species and is often associated with fitness. Regulatory variants with large effects at bab1, t and e affect variation in abdominal pigmentation in several populations of Drosophila melanogaster. A genome wide association (GWA) analysis of variation in abdominal pigmentation was performed using the inbred, sequenced lines of the Drosophila Genetic Reference Panel (DGRP). The large effects of regulatory variants were confirmed in bab1, t and e. These analyses were, however, imperfect proxies for the effects of segregating variants. This study describes the results of an extreme quantitative trait locus (xQTL) GWA analysis of female body pigmentation in an outbred population derived from light and dark DGRP lines. The effects on pigmentation of 28 genes implicated by the DGRP GWA study were replicated, including bab1, t and e and seven genes previously validated by RNAi and/or mutant analyses. Many additional loci were identified. The genetic architecture of Drosophila pigmentation is complex, with a few major genes and many other loci with smaller effects.


BIOLOGICAL OVERVIEW

ebony encodes an β-alanyl-dopamine synthase regulating β-alanyl conjugation of dopamine and histamine, thus 'trapping' these biogenic amines preventing their further function (see Borycz, 2002 and Richardt, 2003). This enzymatic activity regulates fly pigmentation, photoreceptor activity and behavioral rhythmicity. It has been suggested that glia may be required for normal circadian behavior, but glial factors required for rhythmicity have not been identified in any system. This study shows that a circadian rhythm in Drosophila Ebony (N-β-alanyl-biogenic amine synthetase) abundance can be visualized in adult glia and that glial expression of Ebony rescues the altered circadian behavior of ebony mutants. Molecular oscillator function and clock neuron output are normal in ebony mutants, verifying a role for Ebony downstream of the clock. Surprisingly, the ebony oscillation persists in flies lacking PDF neuropeptide, indicating it is regulated by an autonomous glial oscillator or another neuronal factor. The proximity of Ebony-containing glia to aminergic neurons and genetic interaction results suggest a function in dopaminergic signaling. A model for ebony function is presented wherein Ebony glia participate in the clock control of dopaminergic function and the orchestration of circadian activity rhythms (Suh, 2007).

Because life has evolved in the presence of daily geophysical cycles, most organisms have acquired the ability to adapt the timing of physiological processes to external cycles using an intrinsic time-keeping device called a circadian clock. Both forward genetic and molecular screens in Drosophila and other organisms have identified genes encoding integral components of the circadian oscillator. In the fruit fly, the core oscillator mechanism governing behavioral rhythmicity is comprised of two interconnected molecular loops that result in circadian changes in PER and TIM clock protein abundance and the cyclical feedback repression of clock gene transcription. In addition to the core transcriptional loops, posttranscriptional factors have been identified that are required for the modulation of clock protein stability, activity, or nuclear entry. Although there has been significant progress in delineating clock mechanisms, less is known about the molecular and cellular output pathways that control organismal physiology and behavior (Suh, 2007).

Two behaviors are widely employed to assay circadian rhythmicity in Drosophila: eclosion (the emergence of the adult from the pupal case) and adult locomotor activity. Mutation of a clock element affects rhythms in both eclosion and activity, since the same or a molecularly similar clock regulates both behaviors. In contrast, several mutations have been reported to affect only one of these two behaviors; i.e., to have rhythm-specific effects on circadian behavior. These findings indicate that genetically separable output pathways mediate the circadian control of the two different processes. Mutations in ebony, for example, selectively perturb the locomotor activity rhythm, causing arrhythmicity, but have no effect on the adult eclosion rhythm (Newby, 1991). Such a rhythm-specific effect suggests that Ebony acts downstream of the clock mechanism to orchestrate the circadian control of locomotor activity (Suh, 2007).

Multiple microarray-based studies have identified Drosophila transcripts exhibiting rhythmic daily changes in abundance. These studies verified cycling for all of the known clock genes and, importantly, identified hundreds of other genes that show robust circadian changes in abundance within head tissues. Of note, ebony RNA was shown to exhibit robust circadian cycling in two independent studies (Claridge-Chang, 2001; Ueda, 2002). These results are consistent with the behavioral studies discussed above, which suggest that Ebony protein functions in a clock output pathway (Suh, 2007).

The most obvious phenotype of ebony mutants is defective sclerotization and cuticle pigmentation, although they also exhibit altered rhythms, vision (Hotta, 1969), and courtship behavior (Kyriacou, 1978). Consistent with these phenotypes, Ebony protein can be detected in the hypodermis (which produces the cuticle), the visual system, and other brain regions (Richardt, 2002). In the fly visual system, Ebony is localized exclusively to glia including neuropile and epithelial glia (Richardt, 2002), and it is thought that Ebony functions in a metabolic pathway (Borycz, 2002; Richardt, 2003) that may terminate the action of histamine, the photoreceptor cell neurotransmitter. Based on studies of the pigmentation phenotype of ebony mutants, it was shown that Ebony protein has β-alanyl-dopamine (DA) synthase (BAS) enzymatic activity (Hovemann, 1998), and consequently mutants are lacking N-β-alanyl-dopamine (NBAD) in peripheral and neural tissues (Perez, 2004) and have elevated levels of DA and β-alanine in both types of tissues (Hodgetts, 1973; Ramadan, 1993). Recently, it was reported that the Ebony enzyme has a broader substrate specificity than anticipated from previous studies: purified Ebony can conjugate β-alanine to several different biogenic amines, including DA, serotonin (5-HT), histamine, tyramine, and octopamine (Richardt, 2003); hence, it is now considered a β-alanyl-biogenic amine synthase (Suh, 2007).

It is known that DA, 5-HT, and other biogenic amines have neuromodulatory activity in Drosophila and other insects. Together with the behavioral defects of ebony mutants, these findings suggest a model for the circadian function of Ebony, in which clock output regulates Ebony (BAS) activity, and consequent changes in biogenic amine-related signaling within a specific group of neural cells of the fly brain. This study shows that Ebony-containing glia are localized close to clock cell projections, that there is a PER/TIM-dependent control of rhythmic ebony expression within a discrete population of glial cells, and that Ebony enzymatic activity is required within glia for the clock control of locomotor activity. Cellular and molecular analyses indicate that Ebony acts downstream of the clock to control locomotor activity and that Ebony-containing glia are positioned near DA and 5-HT neurons of the larval and adult brains, consistent with the idea that these glia are required for the modulation of aminergic functions. A genetic interaction between ebony1 and an allele of the fly dopamine transporter gene (dDAT) suggests that dopaminergic transmission has a role in rhythmicity in vivo. That glia may function in rhythmicity is consistent with a genetic mosaic study that implies a role for PER/TIM-containing glia in the regulation of activity rhythms (Suh, 2007).

Studies of Ebony indicate that glia have an essential role in the orchestration of circadian locomotor activity. It is of interest that previous studies in both mammals and insects have suggested that glia might be important for the control of rhythmic physiological events. Cultured cortical astroglia that express per-luciferase transgenes, for example, show circadian rhythms of bioluminescence that may depend on diffusible signals from neurons of the suprachiasmatic nuclei; these studies suggest that such glia contain autonomous oscillators that can be reset by environmental stimuli or by interactions with clock neurons. In Drosophila, previous investigations have shown that the clock proteins PER and TIM can be detected in neurons and glia of the optic lobes and protocerebrum, and PER protein abundance fluctuates according to a circadian rhythm in both cell types. Consistent with roles for PER in neurons and glia, genetic mosaic analysis has suggested that per expression in either cell type might be sufficient for rhythmicity (albeit weak rhythmicity with glial expression). The current results indicate that Ebony is localized to glia of at least two types: those containing PER and TIM and a second class in which clock protein expression is not detectable. In the first class of cells, it seems likely that rhythmic ebony expression is controlled by an intracellular PER/TIM-based oscillator. In the latter class, ebony expression is most likely regulated by direct or indirect interactions with clock cells (Suh, 2007).

It is now an accepted axiom that neuron-glia interactions are critical for neuronal development and function. In addition to serving support roles in the mature nervous system, glial cells influence the developmental specification of neurons, migration, myelination, synapse number, and synaptic transmission. In Drosophila, studies have provided a detailed understanding of glial cell development and revealed the transcriptional mechanisms underlying the differentiation of this class of neural cells. Previous studies have shown that glial cells function in the phagocytosis of neuronal debris during development and documented roles for glia in injury-induced neuronal degeneration. Of note, a glial-specific receptor known as Draper has been described that is part of a neuron-glia signaling mechanism mediating such injury-induced responses. Insect glia have also been implicated in neurotransmitter uptake/recycling, based on studies of GABA, acetylcholine, or glutamate uptake. Finally, studies of Drosophila repo mutants have demonstrated that glial support is important for neuronal survival in insects, similar to results obtained in mammals (Suh, 2007).

Perhaps more relevant for behavior, recent studies have shown that certain types of mammalian glia (astrocytes) can regulate the excitability of neurons through the regulated release of “gliotransmitters” (glutamate, ATP, adenosine, cytokines, and growth factors), and it has become apparent that there are reciprocal neuron-glia signaling systems that regulate neuronal excitability. Although certain aspects of this dynamic communication system are beginning to be understood, clearly much remains to be learned about the specific factors that regulate neuron-glia communication. These studies have identified a glial-specific factor (Ebony) and a subpopulation of glia within the fly nervous system that function with clock neurons to regulate circadian activity rhythms. It seems likely that intercellular communication between the neuronal and glial elements of the fly circadian system is important for the temporal coordination of activity (Suh, 2007).

Does PDF release contribute to the control of rhythmic ebony expression? Colocalization studies, using antibodies for Ebony and PDF, show that greater than 80% of the Ebony-containing glia reside in close proximity to PDF neuronal somata or projections—this is the case for glia that reside in the lateral and dorsal protocerebrum and the optic medulla of the adult brain. Previous immunoelectron microscopy results show that certain PDF-containing varicosities are adjacent to glial cells of the optic medulla. Ebony-containing glia have been observed near varicosities of the PDF neuronal projections, which probably contain dense core vesicles (DCVs), and adjacent to other regions of the projections. Surprisingly, however, it appears that PDF is not essential for the regulation of the ebony rhythm. It is therefore postulated that communication between Ebony glia and clock neurons, if it occurs, is mediated by factors other than the PDF neuropeptide (Suh, 2007).

A transgene expressing an enzymatically dead form of Ebony does not provide behavioral rescue for ebony mutants; thus, BAS activity is essential for Ebony's circadian function. As indicated previously, BAS can conjugate β-alanine to many different aminergic neurotransmitters, including DA, 5-HT, histamine, octopamine, and tyramine (Richardt, 2003; Perez, 2004). Interestingly, it has been demonstrated that Ebony glia are situated near histamine release sites of photoreceptor cells in the lamina, and it has been suggested that BAS activity conjugates histamine to β-alanine to terminate action of the transmitter (Richardt, 2002). This study has shown that Ebony-containing cells are in close proximity to dopaminergic and serotonergic neurons of the larval and adult brains, suggesting a role for BAS in terminating DA and 5-HT action. A genetic interaction between e1 and DATfmn, a DAT mutant, strongly suggests that Ebony has a role in dopaminergic signaling. The rhythmic production of Ebony (BAS) may result in a circadian modulation of DA action and in turn rhythmic regulation of locomotor activity. Alternatively, circadian changes in BAS activity may result in the rhythmic production and release of N-β-alanyl-dopamine (NBAD) with high levels of NBAD driving locomotor activity. Two lines of evidence support this idea: (1) NBAD is presumably highest during the day, the time of maximal activity; (2) the e1 mutation, a protein null, eliminates NBAD, and this mutation suppresses the hyperactivity of DATfmn flies even though the double mutant is predicted to have high synaptic levels of DA (Suh, 2007).

This study has shown that Ebony glial expression is regulated in a circadian manner and that the protein is required within glia for normal behavioral rhythmicity. The localization of Ebony-containing glia near clock cells and aminergic neurons suggests an explicit model for Ebony regulation and function in the circadian system. According to this model, ebony transcription is regulated either directly by a PER/TIM-dependent oscillator within glia (for those glia containing PER and TIM) or by the release of an unidentified output factor from clock neurons. Consequently, diurnal changes in ebony-encoded and glial-localized BAS activity lead to rhythms in the conjugation of biogenic amines to β-alanine and generation of NBAA product (NBAD in glia near DA neurons). Such a diurnal modulation of amine action may help shape the temporal organization of the daily bouts of locomotor activity. This model, of course, implies the existence of a glial amine transporter that mediates the uptake of synaptic amines into glia, although such a system has not yet been identified in Drosophila (Suh, 2007).

Furthermore, it is postulated that the production of NBAD, which is high during the subjective day, serves as a bioactive compound to drive locomotor activity during the daytime. The observation that e1 mutants exhibit selective daytime deficits in locomotor activity is consistent with this idea. According to this model, NBAD is released from glia and acts on dopaminergic or other neurons to regulate excitability and/or transmitter release. There is no evidence in the literature that β-alanyl-amine conjugates have bioactivity, but this is certainly a possibility given that many other glial compounds have such activity. Obviously, NBAD may not regulate locomotor activity, by itself, as it is presumably high throughout the day, given the profile of Ebony production, whereas locomotor activity is bimodal, with bouts occurring at dawn and dusk. An alternative model for the role of Ebony in the regulation of activity is that the unconjugated amine (i.e., DA) provides excitatory drive for behavior and that its modification by BAS activity decreases such excitation. However, such a model is not consistent with the presumption that NBAD levels are highest during the daytime, the time of maximal activity, nor with the observation that the DATfmn;e1 mutant, which probably has high DA levels, is not hyperactive (Suh, 2007).

Finally, it is known that Drosophila tyrosine hydroxylase (TH) RNA is transcribed according to a circadian rhythm, with high abundance occurring during the subjective day, and it is thus a good assumption that TH enzymatic activity and DA production is maximal during the day. Such a profile of DA production may explain why a high constitutive expression of Ebony (BAS) in glia can restore rhythmic behavior. Because TH production is presumably still rhythmic in ebony mutants, high NBAD levels would be expected to occur in such flies only during the daytime, thus permitting behavioral rhythmicity (Suh, 2007).


REGULATION

tan and ebony genes regulate a novel pathway for transmitter metabolism at fly photoreceptor terminals

In Drosophila melanogaster, ebony and tan, two cuticle melanizing mutants, regulate the conjugation (ebony) of β-alanine to dopamine or hydrolysis (tan) of the β-alanyl conjugate to liberate dopamine. β-alanine biosynthesis is regulated by black. ebony and tan also exert unexplained reciprocal defects in the electroretinogram, at ON and OFF transients attributable to impaired transmission at photoreceptor synapses, which liberate histamine. Compatible with this impairment, both mutants have reduced histamine contents in the head, as measured by HPLC, and have correspondingly reduced numbers of synaptic vesicles in their photoreceptor terminals. Thus, the histamine phenotype is associated with sites of synaptic transmission at photoreceptors. When they receive microinjections into the head, wild-type Sarcophaga bullata (in whose larger head such injections are routinely possible) rapidly (<5 sec) convert exogenous [3H]histamine into its β-alanine conjugate, carcinine, a novel metabolite. Drosophila tan has an increased quantity of [3H]carcinine, the hydrolysis of which is blocked; ebony lacks [3H]carcinine, which it cannot synthesize. Confirming these actions, carcinine rescues the histamine phenotype of ebony, whereas β-alanine rescues the carcinine phenotype of black;tan double mutants. The equilibrium ratio between [3H]carcinine and [3H]histamine after microinjecting wild-type Sarcophaga favors carcinine hydrolysis, increasing to only 0.5 after 30 min. These findings help resolve a longstanding conundrum of the involvement of tan and ebony in photoreceptor function. It is suggested that reversible synthesis of carcinine occurs in surrounding glia, serving to trap histamine after its release at photoreceptor synapses; subsequent hydrolysis liberates histamine for reuptake (Borycz, 2002).

These findings indicate that a novel histamine phenotype underlies the reciprocal actions of ebony and tan. Unlike other aspects of their phenotypes, however, the mutants do not act reciprocally; both reduce the histamine contents of the head. Associated with reduced histamine in both mutants are parallel decreases in the number and packing of synaptic vesicles in the photoreceptor terminals, implying that the histamine phenotype is at least partly of photoreceptor origin. HPLC data are consistent with the conversion into [3H]carcinine of exogenous [3H]histamine, either taken up by or injected into the fly's head. Such uptake has been demonstrated previously in mutant hdc flies, which are unable to synthesize histamine and lack vision. The amount of [3H]carcinine converted in ebony and tan is consistent with the action of ebony in regulating β-alanyl conjugation of histamine and of tan in regulating the hydrolysis of carcinine back to histamine. The equilibrium between the actions of both enzymes evidently favors the hydrolysis of carcinine to liberate histamine, so that the wild-type carcinine content is normally low. Confirming this pathway, ebony flies fed carcinine have increased head histamine, as does the wild type. In addition, feeding β-alanine to double-mutant black;tan flies rescues their ability to synthesize carcinine by providing β-alanine as a substrate. Finally, evidence is provided for the existence of alternative metabolic pathways, with metabolites that convert back to histamine only slowly, if at all (Borycz, 2002).

The evidence for in vivo carcinine biosynthesis is novel for the visual system but has previously been reported biochemically for CNS extracts from the crab Carcinus maenas, which accumulates carcinine in the heart. Not all Carcinus tissues are able to metabolize carcinine, however, suggesting that the hydrolysis pathway represented by tan in Drosophila is either lacking or of low activity. The universal histamine immunoreactivity of, and likely prevalence of histaminergic transmission at, arthropod photoreceptors suggests that a carcinine biosynthesis pathway could be widely used at this site. Carcinine was not sought in a previous study of insect histamine metabolites but is certainly not restricted to arthropods. It is also a minor metabolite in mammals, in which it exerts a positive inotropic action at the heart (Borycz, 2002).

The simultaneous action of both Ebony and Tan proteins in wild-type flies indicates that carcinine forms rapidly. Within 5 sec of injecting Sarcophaga, [3H]histamine gains access to Ebony and is already converted to carcinine. The independent regulation of synthase and hydrolase activity means that the rates for carcinine biosynthesis and hydrolysis can be independently regulated by differential transcription under different physiological conditions. For example, ebony transcription exhibits a circadian modulation (Claridge-Chang, 2001). The significance of carcinine as a metabolite may in fact lie not as much in the identity of the metabolite itself as in the rates of its biosynthesis and reversible hydrolysis, which the [3H]histamine evidence indicates are normally adjusted to a 2:1 equilibrium ratio in favor of hydrolysis. Although it is clear that Ebony acts rapidly, the methods used do not allow assessment of whether it contributes to the termination of histamine action at the cleft. Resolution of this question is crucial to understand how insects, especially fast-flying diurnal flies, are able to use the high-temporal resolution of their photoreceptors. The latter depends on the rapid clearance of released histamine at photoreceptor terminals (Borycz, 2002).

Exact sites of histamine metabolism in the lamina and distal medulla are still not clear. The photoreceptor terminals R1-R6 that surround the axons of L1 and L2 within a cartridge are wrapped in turn by three epithelial glial cells. These are well placed to metabolize histamine from the synaptic cleft and thereby regulate its postsynaptic action at sites on L1 and L2. Moreover, the epithelial glia do indeed express Ebony strongly (Richardt, 2002). Carcinine biosynthesis by Ebony in the epithelial glia would remove histamine released into the lamina, presumably from the synaptic cleft, but would store histamine in a form that can then rapidly liberate it by hydrolysis. The site of that hydrolysis is unknown in detail, but mosaic studies indicate that tan acts in or close to the eye, compatible with its action in the photoreceptors (Borycz, 2002).

The accumulation of [3H]carcinine in tan, but its lack in ebony, can be explained by the reciprocal regulation of β-alanyl conjugation of histamine in the two mutants. However, this still fails to explain how a reduction in head histamine results from the reciprocal action of the two genes. It is proposed that histamine content is reduced in tan because of the failure to liberate histamine from accumulated carcinine, a function that is autonomous to the mutant eye, but is reduced in ebony because carcinine fails to trap histamine after it is released, leaving the histamine free to diffuse away from the compound eye. The fate of histamine after diffusion is unclear but could finally be loss, to the thorax, thence by excretion. It is proposed that this loss is the primary reason for the reduced head content of histamine in ebony. In the absence of functional Ebony protein, mutant flies also fail to trap exogenous [3H]histamine, much of which is likewise lost by excretion. As a result, not only is total head histamine reduced but also the amount of 3H incorporation. In tan, a reciprocal effect occurs, with [3H]histamine incorporation increasing with respect to wild type. It is believed that this may signify increased efficiency in the histamine uptake mechanisms in response to the greater reduction in the head histamine of tan. In Drosophila gynandromorphs with a single mutant ebony eye, the defect in the ERG transients is nonautonomous (R. Hodgetts, personal communication to Borycz, 2002). One interpretation of this difference from tan is that a mutant ebony lamina is unable to convert released histamine to carcinine, so that the histamine remains extracellular and may be free to diffuse to other sites, including the other eye, where it is converted to carcinine by functional Ebony. The fact that such sites can rescue the ERG defect in the mutant eye suggests that the lack of transients when both eyes are mutant for ebony could reflect the presence in the synaptic cleft of residual histamine, even that released in the dark. It is proposed that carcinine that accumulates by the action of functional Ebony in a mutant tan eye is localized initially to the epithelial glia and is not free to diffuse. Therefore, it sequesters much of the histamine pool. In that case, the ERG defect in the mutant tan eye may be attributed to insufficient release of histamine. Thess findings thus help shed light on the involvement in lamina function of tan and ebony and offer a possible explanation for why both, albeit for different reasons, result in the loss of the ON transients of the ERG. An alternative interpretation, offered without reference to histamine metabolism and possibly an independent effect, is that the loss of the lamina transients of ERG in tan could result from the decreased availability of dopamine during larval development (Neckameyer, 2001), with ebony showing reciprocal defects to those shown by tan. Still left to be resolved is whether the carcinine pathway operates at other sites. These include (1) terminals of head mechanoreceptors, which also contain histamine and in which function is both lost and rescued by exogenous histamine in flies mutant for hdc; (2) wide-field histamine-like immunoreactive neurons in the central brain; and (3) dopaminergic neurons in the brain (Borycz, 2002).

The production of carcinine is not the sole metabolic pathway for photoreceptor histamine. In the horseshoe crab Limulus, histamine is also a putative photoreceptor transmitter, and an additional or alternative metabolic pathway involves gamma-glutamyl histamine, a means of histamine inactivation reported previously in the opisthobranch Aplysia. It is not clear what additional metabolites might also exist in Drosophila, but the presence of 3H peaks with HPLC retention times shorter than that for carcinine allows a number of candidates, possibly up to three. It was not possible to detect acetyl-histamine or imidazol-4-acetic acid, but a 3H peak with the same retention time as γ-glutamyl histamine exists, so this metabolite could be present. Other metabolites probably exist as well. For example, the separate actions of pargyline and deprenyl and of clorgyline could indicate a role for monoamine oxidases. Therefore, it is surprising that the monoamine oxidase gene appears to have been lost from the Drosophila genome, making the identity of these metabolites a topic for future clarification as well. Moreover, insensitivity to semicarbazide and hydroxylamine could indicate the lack of SSAO action (Borycz, 2002).

In addition to their activities at one time and in one genetic background, the relative activities of the metabolic pathway for carcinine (regulated by ebony and tan) and for alternative metabolites indicate that each pathway can be differentially regulated. Regulation is seen in black;tan double mutants, which have a large early retention peak suggesting that, in the congenital absence of a capacity to synthesize carcinine, histamine metabolism switches into another pathway, possibly for γ-glutamyl histamine. That pathway is not increased in the single mutant tan, possibly because tan is able to store released histamine as carcinine. Such shifts can also apparently occur in the short term, as for example in Sarcophaga injected with pargyline and deprenyl. Under the influence of these drugs, the early HPLC retention peaks are diminished, and the histamine peak is larger, suggesting that histamine metabolism via carcinine is upregulated (Borycz, 2002).

Ebony, a novel nonribosomal peptide synthetase for beta-alanine conjugation with biogenic amines in Drosophila

Using Ebony protein either expressed in Escherichia coli or in Schneider S2 cells, evidence is provided for its substrate specificity and reaction mechanism. Ebony activates beta-alanine to aminoacyladenylate by an adenylation domain and covalently attaches it as a thioester to a thiolation domain in a nonribosomal peptide synthetase (NRPS) related mechanism. In a second reaction, biogenic amines act as external nucleophiles on beta-alanyl-S-pantetheine-Ebony, thereby releasing in a fast reaction the dipeptide (peptidoamine) in a process that is novel in higher eucaryotes. Therefore, Ebony is defined as a beta-alanyl-biogenic amine synthetase. Insight into the reaction mechanism stems from mutational analysis of an invariant serine that disclosed Ebony as a multienzyme with functional analogy to the starting modules of NRPSs. In light of a putative biogenic amine-deactivating capacity, Ebony function in the nervous system must be reconsidered. It is proposed that in the Drosophila eye Ebony is involved in the transmission process by inactivation of histamine through beta-alanyl conjugation (Richardt, 2003).

Ebony similar to NRPSs belongs to the large family of aminoacyladenylate-forming enzymes. A relation to the thioesterification process, however, is in addition to NRPSs only present in the group of acyl carrier proteins including polyketide synthases and fatty acid synthases. The homology between polyketide and fatty acid synthases and Ebony is limited to the core sequence element of the thiolation domain, which contains the invariant serine, the P-pant cofactor-mediated acyl carrier. Acyl carrier proteins, NRPSs, and Ebony need to be activated by P-pant cofactor transfer, which requires a corresponding transferase activity. Searching for two conserved amino acid sequence motifs detected in previously sequenced P-pant transferases, a reading frame was indeed identified in the Drosophila data base that showed a considerable homology to this conserved region. Expression of the corresponding putative P-pant transferase cDNA in E. coli gave rise to a protein that in vitro enhanced low level phosphantetheinylated S2 cell-derived Ebony activity depending on the presence of CoA comparable with the phosphopantetheinyltransferase (Richardt, 2003).

Drosophila has preserved an amino acid activation mechanism that until now was considered to be specific for microbial NRPSs. Ebony combines this unique feature with a functional domain that allows peptide bond formation with a structurally constrained group of amines. However, a connection between two or more NRPS-like modules that enable the activation of amino acids and the formation of dipeptides has not yet been detected in higher eucaryotes even though genuine dipeptides such as β-alanyl-histidine (carnosine) have been shown to exist in vertebrates. Given the existence of a single NRPS-like activation domain in Ebony, nonribosomal synthesis of dipeptides in higher eucaryotes cannot generally be excluded. However, it would require two amino acid activation modules in addition to a functional domain for condensation of the two activated amino acids as well as a thioesterase activity for peptide release. Evidence that this complex structure of multimodular NRPS activity has been preserved through evolution to higher eucaryotes is still lacking (Richardt, 2003).

Ebony uses a novel two-step reaction mechanism including amino acid activation and binding followed by peptide bond formation. The procedure of amino acid activation and binding resembles that of NRPSs. Peptide bond formation and product release require a nucleophilic attack of an incoming primary amine that must meet the observed structural prerequisites. This is different in multimodular NRPSs in two ways. 1) The nature of peptide bond forming amino acid is predetermined by the specificity of a second adenylation domain within the multimodular enzyme, and 2) in NRPSs, a condensation domain located between the modules is essential for peptide bond catalysis. Such a condensation domain is missing in Ebony. Instead, a C-terminal domain with a yet unknown function seems to be responsible for catalyzing the nucleophilic attack of the primary amines (with relaxed substrate specificity) on the activated carboxyl thioester group of β-alanine. The mechanism of this reaction and that of dipeptide product release are still unknown (Richardt, 2003).

Ebony is expressed in diverse tissues at different times during development. Nervous system activity has been predicted from the behavioral and visual phenotype of the mutant but was only recently confirmed by activity staining of an ebony-lacZ fusion gene transformant and by immunocytochemistry. The puzzling fact that evidence for dopaminergic neurons in the lamina was lacking led to experiments that revealed that Ebony is involved in beta-alanyl-histamine formation in the eye. The capacity of capturing the biogenic amines histamine, dopamine, tyramine, octopamine (see Tyramine β hydroxylase), and serotonin that clearly fulfill different functions in Drosophila to β-alanine might reflect a key function of Ebony at specific sites of the body (Richardt, 2003).

This study provides evidence that Ebony is indeed capable of binding biogenic amines including histamine to β-alanine. Therefore, it is plausible to assign to Ebony a function in histamine neurotransmitter metabolism at the photoreceptor synapse of the eye. Because histamine synthesis as well as metabolic degradation in the eye is relatively slow, the almost infinite transmitter supply must be maintained by a fast re-uptake system. Therefore, at the synapse where transmitter removal excites the postsynaptic cell by disinhibition, a mechanism of fast retraction of histamine from the synaptic cleft is essential. Interestingly, in illuminated barnacle photoreceptor preparations, [3H]histamine was concentrated over the photoreceptor terminals, whereas after incubation in the dark, the label was found at the glia. This observation lends support to the concept that at darkening a fast clearance of transmitter out of the synaptic cleft would be achieved by transport of histamine into the surrounding glia where it could be trapped by Ebony via β-alanine binding. The model requires that β-alanine is sufficiently loaded in the glia to prime Ebony for histamine capture and a biochemical pathway that allows the subsequent reuse of the withdrawn histamine in the photoreceptor. Although both histamine transport into photoreceptor as well as into glia has been reported previously, it remains to be investigated whether a mechanism exists that darkening and concomitant reduction of histamine release shifts uptake toward glia followed by immediate inactivation by β-alanine binding (Richardt, 2003).

Fast histamine removal from the synaptic cleft is essential for the function of arthropod photoreceptor synapses that operate with tonic release of histamine. In vitro product formation from [β-alanine-loaded Ebony with histamine or any of the other biogenic amine substrates was already completed within 10 s, the shortest time point that could be determined under standard assay conditions. This time point is still far away from the reaction velocity expected for a function in neurotransmitter inactivation. Beyond this point, reaction velocity may differ among the biogenic amines serving as substrate. Determination of Vmax and Km values of individual biogenic amines requires specific analytical methods operating in the millisecond range. They will disclose whether Ebony can fulfill the kinetic prerequisites for neurotransmitter inactivation (Richardt, 2003).

Schizophrenia susceptibility gene dysbindin regulates glutamatergic and dopaminergic functions via distinctive mechanisms in Drosophila

The dysfunction of multiple neurotransmitter systems is a striking pathophysiological feature of many mental disorders, schizophrenia in particular, but delineating the underlying mechanisms has been challenging. This study shows that manipulation of a single schizophrenia susceptibility gene, dysbindin, is capable of regulating both glutamatergic and dopaminergic functions through two independent mechanisms, consequently leading to two categories of clinically relevant behavioral phenotypes. Dysbindin has been reported to affect glutamatergic and dopaminergic functions as well as a range of clinically relevant behaviors in vertebrates and invertebrates but has been thought to have a mainly neuronal origin. This study found that reduced expression of Drosophila dysbindin (dysb) in presynaptic neurons significantly suppresses glutamatergic synaptic transmission and that this glutamatergic defect is responsible for impaired memory. However, only the reduced expression of Dysb in glial cells is the cause of hyperdopaminergic activities that lead to abnormal locomotion and altered mating orientation. This effect is attributable to the altered expression of a dopamine metabolic enzyme, Ebony, in glial cells. Thus, Dysb regulates glutamatergic transmission through its neuronal function and regulates dopamine metabolism by regulating Ebony expression in glial cells (Shao, 2011).

The current study investigated functions of Ddysb to explore how the altered expression of a single schizophrenia susceptibility gene relates to the pathophysiology and clinically relevant phenotypes. The function of this gene is highly conserved from Drosophila to vertebrates and even to humans. The observed pattern of dysb expression in the Drosophila brain is very similar to that reported in the vertebrate brain: widespread and enriched in neurons. Loss-of-function mutations and RNAi knockdown of dysb in Drosophila produced phenotypes similar to those observed in the sandy mouse, including attenuated glutamatergic transmission, hyperdopaminergic activity, memory defects, and locomotor hyperactivity. Moreover, the human DTNBP1 gene was capable of rescuing dysb1 mutant phenotypes in Drosophila. With the help of genetic tools exclusively available in Drosophila, however, surprising insights were gained (Shao, 2011).

First, although Dysb is widely expressed in the brain, restoring Dysb in glutamatergic neurons alone was sufficient to rescue hypoglutamatergic transmission and memory defects. Second, Dysb's functions in glial cells are essential for normal dopaminergic activity and associated behaviors, including locomotion and mating orientation. Third, all observed pathophysiological and behavioral phenotypes were rescued with acute genetic or pharmacological treatments in adults (Shao, 2011).

Special attention was devoted to validating the phenotypes observed, including maintaining an isogenic background for all genotypes, balancing the behavioral assays, and confirming the manifested phenotypes by different genetic manipulations (mutations, genetic rescuing, and RNAi knockdown). (Shao, 2011).

An increasing number of studies suggest that genetic variation in DTNBP1 in normal human populations affects verbal and visual memories as well as working memory. This association is supported by studies on the sandy mouse, which is defective in a range of memory tasks, including spatial memory, novel object recognition, and contextual fear conditioning . However, the physiological causes of such memory defects are not clearly defined (Shao, 2011).

This study showed that altered dysb function in glutamatergic neurons alone is responsible for attenuated glutamatergic transmission and for the memory defect. It is interesting that this memory defect is not a developmental phenotype and could be rescued acutely both by feeding flies with the NMDA receptor agonist glycine and by expressing dysb only in glutamatergic neurons. Such a result is consistent with reports showing that NMDA receptors in the Drosophila brain are involved in memory formation (Shao, 2011).

Before the current study, the expression and function of dysbindin were considered to occur primarily, if not exclusively, in neurons. However, recent reports have demonstrated that in mouse and rat brains the expression level of dysbindin in glia is comparable with, if not higher than, its expression in neurons, although its glial functions remained to be determined. Genetic tools available for Drosophila allowed definition of the function of dysbindin in glia but also gaining of insight into the underlying mechanisms (Shao, 2011).

Anatomically, it was shown that immunohistochemical signals of Dysb were detected in glial cells labeled by GFP-tagged membrane proteins, with sparse Dysb distribution in cell bodies and the majority of glial Dysb signals in glial processes or in thin layers surrounding individual neuronal cell bodies. This observation was supported by the distribution pattern of VFP-tagged Dysb in GFP-labeled glial cells (Shao, 2011).

Evidence supporting a functional role of Dysb in glia is very strong. The escalated dopamine level in the dysb1 mutant could be rescued by targeted expression of the dysb or human DTNBP1 transgene only in glial cells but not in neurons. In addition, the hyperdopaminergia-elicited behaviors, including locomotor hyperactivity and mating disorientation, were rescued only through targeted glial expression of dysb or human DTNBP1 transgenes. More convincingly, knocking down dysb universally or in glia but not in neurons resulted in embryonic or pupal lethality, respectively (Shao, 2011).

Further investigation suggests that mutations of dysb cause hyperdopaminergic activity by down-regulating the expression of Ebony. The biochemical data profiling mRNA and protein expression corroborated well with genetic observations, supporting the idea that Ebony plays critical role in mediating the effects of Dysb in glial cells. It is likely that this Dysb/Ebony-produced hyperdopaminergic activity somehow leads to reduced TH and Tan expression in neurons through a negative feedback mechanism for maintaining the homeostasis of dopaminergic activity (Shao, 2011).

How Dysb regulates expression of Ebony remains to be determined. One possibility comes from reports that human dysbindin can function as a nucleocytoplasmic shuttling protein that regulates the transcription of several genes either directly or by binding with other transcription-related factors. This study analyzed the Dysb protein sequence with the PSORT II Prediction WWW Server and found that the probability that Dysb localizes to the nucleus is 94.1%. Thus, it is plausible that Dysb in glia plays a role in regulating gene transcription (Shao, 2011).

Alternatively, Dysb might regulate the dopamine level in glial cells by affecting the stability of the Ebony protein. The dysbindin-containing BLOC-1 complex is a component of the endosomal protein sorting and compartmental machinery. Abnormalities in Ebony protein sorting may lead to abnormalities in ubiquitylation, protein instability, or malfunction of the enzyme (Shao, 2011).

Although the possibility of generating fly models of schizophrenia has been raised recently, the intent of this study is not to model schizophrenia in Drosophila. Instead, it was of interest to discover whether and how a single mild genetic alteration, similar to those observed in cases of schizophrenia, gives rise to complex phenotypes at the neurotransmitter regulation and behavioral levels. This study led to two interesting observations (Shao, 2011).

First, it was surprising to see that a rather mild 30%-40% reduction in dysb expression led to significant alterations in both glutamatergic transmission and dopaminergic activity. Most schizophrenia susceptibility genes reported to date are identified not from mutations but from single-nucleotide polymorphisms or haplotypes, which are believed to produce only mild alterations at the gene expression level. It therefore is debatable how strong the contribution of an individual genetic variant is and whether multiple genetic components acting in concert are needed for the effects. This study shows that a mild reduction of at least one of the susceptibility genes is sufficient to cause complex changes in multiple neurotransmitter systems through very different mechanisms. These findings suggest that these susceptibility genes might play such critical roles in neurotransmitter regulation that a mild change in expression is sufficient to cause detectable behavioral phenotypes (Shao, 2011).

Second, although a developmental role of dysbindin has been reported earlier and is supported, as mentioned above, both neurotransmitter and behavioral phenotypes examined in this study could be rescued through acute treatments. Schizophrenia is considered a neurodevelopmental disorder, a notion that is supported by animal model studies of development and by genetic mouse models of neurodevelopmental candidate genes and susceptibility genes. However, this study suggests that, to some extent, some of the genetically relevant phenotypes are reversible or could be treated in adults (Shao, 2011).

The metabolism of histamine in the Drosophila optic lobe involves an ommatidial pathway: β-alanine recycles through the retina

Flies recycle the photoreceptor neurotransmitter histamine by conjugating it to β-alanine to form β-alanyl-histamine (carcinine). The conjugation is regulated by Ebony, while Tan hydrolyses carcinine, releasing histamine and β-alanine. In Drosophila, β-alanine synthesis occurs either from uracil or from the decarboxylation of aspartate but detailed roles for the enzymes responsible remain unclear. Immunohistochemically detected β-alanine is present throughout the fly's entire brain, and is enhanced in the retina especially in the pseudocone, pigment and photoreceptor cells of the ommatidia. HPLC determinations reveal 10.7 ng of β-alanine in the wild-type head, roughly five times more than histamine. When wild-type flies drink uracil their head β-alanine increases more than after drinking l-aspartic acid, indicating the effectiveness of the uracil pathway. Mutants of black, which lack aspartate decarboxylase, cannot synthesize β-alanine from l-aspartate but can still synthesize it efficiently from uracil. The findings of this study demonstrate a novel function for pigment cells, which not only screen ommatidia from stray light but also store and transport β-alanine and carcinine. This role is consistent with a β-alanine-dependent histamine recycling pathway occurring not only in the photoreceptor terminals in the lamina neuropile, where carcinine occurs in marginal glia, but vertically via a long pathway that involves the retina. The lamina's marginal glia are also a hub involved in the storage and/or disposal of carcinine and β-alanine (Borycz, 2012).

The histamine recycling pathway in photoreceptors faces two major physiological demands. First, histamine is released at high rates, which if unopposed would deplete the eye within seconds. Second, the demands on histamine recycling vary greatly from moment to moment, at least within the time frame of 100 ms, depending on the light stimulus conditions that result from the fly's own activity and changes in its ambient light conditions. To maintain a constant supply of histamine may therefore require not only a fast recycling pathway via carcinine but also storage sites for the neurotransmitter, as well as β-alanine and their conjugate carcinine. These storage sites seem to be the marginal and fenestrated glia and, in the retina, the pigment cells. The fenestrated glia have already been recognized as such a site, and three interrelated candidate functions, recycling, spillover and reserve, have been identified. It is imagined that a rapid reuptake pathway is processed via epithelial glia and their capitate projections in the lamina. The additional storage sites in the ommatidium and cartridge are possibly responsible for the slower supply of histamine to photoreceptors for re-release, the supply of β-alanine for synthesis of carcinine in the epithelial and proximal satellite glia, or the return of carcinine to the photoreceptor for the liberation of both (Borycz, 2012).

Drosophila Ebony: a novel type of nonribosomal peptide synthetase related enzyme with unusually fast peptide bond formation kinetics

Drosophila Ebony is a beta-alanyl biogenic amine synthetase with proven function in cuticle and in glia of the nervous system. It is closely related to nonribosomal peptide synthetases (NRPSs), which typically consist at least of an adenylation-, a peptidyl carrier protein- and a peptide bond forming condensation domain. Besides its role in cuticle formation, Ebony is in most glia of the brain thought to convert biogenic amines to beta-alanyl conjugates. If the metabolization of the neurotransmitter histamine to beta-alanyl histamine requires a fast reaction in visual signal transduction, Ebony must be able to fulfill this requirement. Since NRPSs are in general slowly acting multi-modular protein machineries, the enigma of how Ebony quickly facilitates this inactivation remained a key-question for understanding its role in vision. To quantitatively analyze the reaction kinetics, phosphopantetheinylated holo-Ebony prepared from Baculovirus infected Sf9 cells was used. Kinetic parameters for the loading reaction, e.g. the formation of beta-alanyl-Ebony thioester, complied with those of slow NRPSs. In contrast, single-turnover analysis of the last reaction step, peptide bond formation between pre-activated beta-alanyl Ebony thioester and histamine, revealed a very rapid conjugation reaction. This bi-phasic nature of activity identifies Ebony as a novel type of NRPS-related molecule that combines a slow amino acid activation phase with a very fast product formation step (Hartwig, 2014).

Genetic convergence in the evolution of male-specific color patterns in Drosophila

Convergent evolution provides a type of natural replication that can be exploited to understand the roles of contingency and constraint in the evolution of phenotypes and the gene networks that control their development. For sex-specific traits, convergence offers the additional opportunity for testing whether the same gene networks follow different evolutionary trends in males versus females. Thus study used an unbiased, systematic mapping approach to compare the genetic basis of evolutionary changes in male-limited pigmentation in several pairs of Drosophila species that represent independent evolutionary transitions. A strong evidence for repeated recruitment of the same genes to specify similar pigmentation in different species was found. At one of these genes, ebony, convergent evolution of sexually dimorphic and monomorphic expression through cis-regulatory changes was observed. However, this functional convergence has a different molecular basis in different species, reflecting both parallel fixation of ancestral alleles and independent origin of distinct mutations with similar functional consequences. These results show that a strong evolutionary constraint at the gene level is compatible with a dominant role of chance at the molecular level (Signor, 2016).

Recent studies have led to an emerging consensus that convergent phenotypes often reflect evolutionary changes in the same genes (the 'evolutionary hotspot' model). However, most case studies supporting the hotspot model relied heavily on candidate gene approaches, which results in a positive ascertainment bias and some difficulty in interpreting the results. Although genetic mapping is more laborious than candidate gene analysis, it offers several key advantages: it is unbiased in that the loci implicated in other species are no more likely to be discovered than any other genes, it produces direct evidence of a gene's causative role in trait evolution (and, equally importantly, allows other genes to be ruled out), and it provides a quantitative estimate of the relative importance of each gene to the overall genetic architecture of a phenotype. This study applied this approach to the ananassae species subgroup, in which multiple species have independently evolved similar male-specific color patterns. The convergent phenotypes were found ti be controlled by overlapping, but not identical, sets of genes in different evolutionary contrasts. The only gene that was implicated in all three high-resolution mapping crosses and could not be ruled out in the fourth, low-resolution cross (D. m. malerkotliana/D. bipectinata) was ebony. Moreover, ebony is the strongest QTL in all contrasts, contributing 35% to 80% of overall divergence. ebony has previously been implicated in both intraspecific and interspecific differences in pigmentation in several other Drosophila species. Clearly, ebony fits the definition of an evolutionary hotspot. ebony encodes an enzyme, β-alanyl-dopamine synthase, that synthesizes light pigment precursors so that higher ebony expression causes lighter pigmentation. Like other pigmentation genes, it has additional roles in other tissues, such as control of circadian locomotor activity in brain glial cells. Every time ebony has been implicated in the evolution of pigmentation, cis-regulatory rather than coding mutations were involved. Presumably, this pattern reflects the ability of cis-regulatory mutations to overcome pleiotropic constraints by uncoupling gene functions in different cell types (Signor, 2016).

Repeated involvement of the same gene in multiple phenotypic transitions could potentially result from different evolutionary processes: recurrent de novo mutation, lineage sorting of ancestral variation, or interspecific introgression. Adaptation from standing variation is likely to be faster than awaiting a new mutation because potentially beneficial alleles are available immediately and are likely to be present at higher frequencies than alleles arising de novo. Cases of parallel fixation appear to be fairly common, most notably when a reservoir of newly adaptive alleles is available to colonizing populations. Similarly, introgressive hybridization has been implicated, among other traits, in the evolution of wing color patterns in Heliconius butterflies and high-altitude adaptation in humans. Genome-wide analyses suggest that interspecific introgression may play a more important role in evolution than previously thought. This study found that in D. malerkotliana, D. bipectinata, and D. parabipectinata, at least some of the putative causative SNPs at the ebony locus are shared across species, most likely reflecting ancestral lineage sorting. However, these variants are not found either in D. pseudoananassae or in the ercepeae species complex, suggesting a role for independent ebony mutations in these taxa. The region of the ebony locus associated with the evolution of color patterns in the bipectinata complex evolves so rapidly that it cannot be aligned with other taxa. Outside of the ananassae subgroup, evolutionary changes in ebony expression that lead to divergent pigmentation map to a different, more upstream region of the gene; population-genetic analysis rules out this region in the bipectinata complex. It can be concluded that the widely convergent involvement of ebony in the evolution of color patterns is not due solely to fixation of pre-existing variation, but reflects independent origin of distinct, though functionally similar, cis-regulatory mutations (Signor, 2016).

Cuticle color depends not just on the level of ebony, but on the balance between ebony, tan, yellow, and potentially other enzymes. For example, tan encodes a β-alanyl-dopamine hydrolase, which reverses the chemical reaction catalyzed by ebony. Although tan is implicated in the evolution of pigmentation in several species, it has been ruled out in many other studies including this one (Table S5). Interestingly, the evolutionary changes that were found to involve tan are never male limited; for example, tan controls a female-limited color polymorphism in D. erecta, and the secondary loss of pigmentation in D. santomea affects both sexes. It is possible that ebony could be favored as the male hotspot due to its dosage sensitivity and chromosomal location. ebony, but not tan or yellow, has a semi-dominant loss-of-function phenotype, suggesting that cis-regulatory mutations in ebony could be more readily visible to directional selection. At the same time, yellow and tan are X-linked, whereas ebony is autosomal, suggesting that it could harbor higher levels of standing genetic variation when not under directional selection. Interestingly, in D. melanogaster, D. americana, and the bipectinata species complex, the role of ebony in phenotypic evolution appears to derive from a combination of pre-existing and de novo mutations. Thus, chromosomal sex, the topology of the regulatory network, the kinetics of the pigment synthesis pathway, and population-genetic factors may all contribute to the evolutionary hotspot status of ebony (Signor, 2016).

In an accompanying paper (Yassin, 2016), a similarly unbiased and systematic approach was taken to map the genetic basis of natural variation in female-specific abdominal pigmentation in multiple species of the Drosophila montium species subgroup, which is closely related to the ananassae lineage. This variation was found to map to the pdm3 transcription factor in several distantly related species. Moreover, convergent involvement of pdm3 appears to reflect independent mutations in this gene in different species. In contrast, ebony does not contribute to color pattern variation in any of the four montium-subgroup species examined. Why do evolutionary hotspots differ in closely related lineages? Is this a matter of historical contingency or different gene network topology in different clades? Or are different genes within a shared network favored for cis-regulatory evolution in different sexes, resulting in sex-specific evolutionary hotspots? Intriguingly, in the only montium subgroup species in which ebony was implicated in color pattern variation, the pigmentation phenotype is male specific, supporting the latter hypothesis (Signor, 2016).

One can envision two principal mechanisms for the gain and loss of sex-specific traits. First (the instructive model), the genes responsible for phenotypic changes may be the same genes that are regulated in a dimorphic manner to generate sex-specific phenotypes, as was observed for ebony. Alternatively (the permissive model), the causative genes could be monomorphic, while sexual dimorphism is encoded in parallel to or downstream of these genes. For example, ebony could have been expressed equally between sexes in all species, while a different, 'gatekeeper' gene is sexually dimorphic in all species. In the latter scenario, high levels of ebony expression in both sexes would mask the dimorphic phenotypes promoted by the sex-specific gatekeeper gene, while low ebony levels would uncover the underlying dimorphism. Thus, ebony would be the causative gene responsible for differences between lineages, while the gatekeeper gene is responsible for sexual dimorphism (Signor, 2016).

These two models suggest very different mechanisms for the evolution of sexual dimorphism. Under the instructive model, gain and loss of sex-specific traits is caused by frequent changes in sex-specific gene regulation. Under the permissive model, the targets of the sex determination pathway can remain static over long evolutionary distances, while the underlying sexual dimorphism is revealed or obscured by sexually monomorphic genetic changes that occur elsewhere in the developmental pathway (Signor, 2016).

The current results argue for the instructive model: in all evolutionary contrasts, sex-specific pigmentation is associated with sex-specific ebony expression, and sexually monomorphic pigmentation is associated with monomorphic expression. Thus, sex-specific transcriptional regulation of ebony has been gained and/or lost several times within the ananassae species subgroup. A similar pattern has been observed in the expression of desat-F, a hydrocarbon desaturase enzyme involved in the synthesis of cuticular pheromones. It appears that sex-specific gene regulation can be gained and lost quite easily over short evolutionary timescales and that the evolution of sexually dimorphic traits is more likely to follow the instructive model (Signor, 2016).

Although the study of color pattern evolution in Drosophila has largely been dominated by candidate gene analyses, it has now been enriched by several unbiased, high-resolution genetic mapping studies, including this work. These studies have gone beyond spotlighting individual genes to provide a more holistic picture of the genetic architecture of evolutionary changes and have confirmed the predominance of cis-regulatory mutations in phenotypic evolution. Although no single gene is involved in all cases, the number of players appears to be limited, and most genes have been implicated repeatedly in multiple taxa. Collectively, parallel genetic analyses in multiple species suggest a 'toolkit model' of convergent evolution. For any trait, there are a limited number of genes that can potentially evolve to produce phenotypic changes. Within that toolkit, the relative likelihood of each gene's involvement may depend on its position in the regulatory network that controls the development of that trait, on the historical contingencies specific to each evolving lineage, and, potentially, on sex. Together, these trends result in a pattern where convergent phenotypes have distinct yet overlapping genetic basis in different species (Signor, 2016).


DEVELOPMENTAL BIOLOGY

Adult

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).

Circadian regulation of gene expression systems in the Drosophila head

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).

Drosophila photoreceptors express cysteine peptidase Tan and Ebony

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).


EFFECTS OF MUTATION

Expression of Ebony exclusively in glia rescues the behavioral phenotype of ebony mutants

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).

Catecholamines in Drosophila melanogaster (wild type and ebony mutant) decuticalarized retinas and brains

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).

Reciprocal functions of the Drosophila yellow and ebony proteins in the development and evolution of pigment patterns

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).

Drosophila pigmentation evolution: divergent genotypes underlying convergent phenotypes

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).

Drosophila tan encodes a novel hydrolase required in pigmentation and vision: ebony and cuticle pigmentation

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).

The genetic basis of adaptive pigmentation variation in Drosophila melanogaster

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).

Natural variation of ebony gene controlling thoracic pigmentation in Drosophila melanogaster

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 role of carcinine in signaling at the Drosophila photoreceptor synapse

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).

Activity and coexpression of Drosophila black with ebony in fly optic lobes reveals putative cooperative tasks in vision that evade electroretinographic detection

Drosophila mutants black and ebony show pigmentation defects in the adult cuticle, which disclose their cooperative activity in β-alanyl-dopamine formation. In visual signal transduction, Ebony conjugates beta-alanine to histamine, forming β-alanyl-histamine or carcinine. Mutation of ebony disrupts signal transduction and reveals an electroretinogram (ERG) phenotype. In contrast to the corresponding cuticle phenotype of black and ebony, there is no ERG phenotype observed when black expression is disrupted. This discrepancy calls into question the longstanding assumption of Black and Ebony interaction. The purpose of this study was to investigate the role of Black and Ebony in fly optic lobes. A presynaptic histamine uptake pathway was excluded, and histamine recycling via carcinine formation in glia was confirmed. β-Alanine supply for this pathway is independent of enzymatic synthesis by Black and β-alanine synthase Pyd3. Two versions of Black are expressed in vivo. Black is a specific aspartate decarboxylase with no activity on glutamate. RNA in situ hybridization and anti-Black antisera localized Black expression in the head. Immunolabeling revealed expression in lamina glia, in large medulla glia, in glia of the ocellar ganglion, and in astrocyte-like glia below the ocellar ganglion. In these glia types, Black expression is strictly accompanied by Ebony expression. Activity, localization, and strict coexpression with Ebony strongly indicate a specific mode of functional interaction that, however, evades ERG detection (Ziegler, 2013).


REFERENCES

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

Borycz, J., Borycz, J. A., Edwards, T. N., Boulianne, G. L. and Meinertzhagen, I. A. (2012). The metabolism of histamine in the Drosophila optic lobe involves an ommatidial pathway: β-alanine recycles through the retina. J Exp Biol 215: 1399-1411. PubMed ID: 22442379

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

Hartwig, S., Dovengerds, C., Herrmann, C. and Hovemann, B. T. (2014). Drosophila Ebony: a novel type of nonribosomal peptide synthetase related enzyme with unusually fast peptide bond formation kinetics. FEBS J [Epub ahead of print]. PubMed ID: 25229196

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

Shao, L., et al. (2011). Schizophrenia susceptibility gene dysbindin regulates glutamatergic and dopaminergic functions via distinctive mechanisms in Drosophila. Proc. Natl. Acad. Sci. 108(46): 18831-6. PubMed Citation: 22049342

Signor, S.A., Liu, Y., Rebeiz, M. and Kopp, A. (2016). Genetic convergence in the evolution of male-specific color patterns in Drosophila. Curr Biol 26(18):2423-33.. PubMed ID: 27546578

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

Yassin, A., Delaney, E. K., Reddiex, A. J., Seher, T. D., Bastide, H., Appleton, N. C., Lack, J. B., David, J. R., Chenoweth, S. F., Pool, J. E. and Kopp, A. (2016). The pdm3 locus is a hotspot for recurrent evolution of female-limited color dimorphism in Drosophila. Curr Biol 26: 2412-2422. PubMed ID: 27546577

Ziegler, A. B., Brusselbach, F. and Hovemann, B. T. (2013). Activity and coexpression of Drosophila black with ebony in fly optic lobes reveals putative cooperative tasks in vision that evade electroretinographic detection. J Comp Neurol 521: 1207-1224. PubMed ID: 23124681


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