InteractiveFly: GeneBrief

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

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 DAT^fmn, 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 DAT^fmn 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 DAT^fmn;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).

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 Hdc^P218 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).

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 V_max and K_m 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).

Modular cis-regulatory logic of yellow gene expression in silkmoth larvae

Color patterns in butterflies and moths are crucial traits for adaptation. Previous investigations have highlighted genes responsible for pigmentation (i.e., yellow and ebony). However, the mechanisms by which these genes are regulated in lepidopteran insects remains poorly understood. To elucidate this, molecular studies involving dipterans have largely analyzed the cis-regulatory regions of pigmentation genes and have revealed cis-regulatory modularity. This study used well-developed transgenic techniques in Bombyx mori and demonstrated that cis-regulatory modularity controls tissue-specific expression of the yellow gene. First, which body parts are regulated by the yellow gene via black pigmentation were edentified. Then, three discrete regulatory elements driving tissue-specific gene expression in three regions of B. mori larvae were identified. Finally, it was found that there is no apparent sequence conservation of cis-regulatory regions between B. mori and Drosophila melanogaster, and no expression driven by the regulatory regions of one species when introduced into the other species. Therefore, the trans-regulatory landscapes of the yellow gene differ significantly between both taxa. The results of this study confirm that lepidopteran species use cis-regulatory modules to control gene expression related to pigmentation, and represent a powerful cadre of transgenic tools for studying evolutionary developmental mechanisms (Suzuki, 2019).

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

Suzuki, T. K., Koshikawa, S., Kobayashi, I., Uchino, K. and Sezutsu, H. (2019). Modular cis-regulatory logic of yellow gene expression in silkmoth larvae. Insect Mol Biol. PubMed ID: 30737958

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

date revised: 28 December 2023

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