InteractiveFly: GeneBrief

tan: Biological Overview | References


Gene name - tan

Synonyms -

Cytological map position - 8D1-8D1

Function - enzyme

Keywords - hydrolase - abdominal pigmentation - hydrolysis of N-β-alanyl dopamine (NBAD) to dopamine during cuticular melanization - hydrolysis of carcinine to histamine in the metabolism of photoreceptor neurotransmitter

Symbol - t

FlyBase ID: FBgn0086367

Genetic map position - chrX:9,217,655-9,223,257

Classification - Hydrolase - Acyl-coenzyme A:6-aminopenicillanic acid acyl-transferase - cysteine peptidases

Cellular location - cytoplasmic



NCBI links: Precomputed BLAST | EntrezGene
Recent literature
Gibert, J. M., Mouchel-Vielh, E. and Peronnet, F. (2017). Modulation of yellow expression contributes to thermal plasticity of female abdominal pigmentation in Drosophila melanogaster. Sci Rep 7: 43370. PubMed ID: 28230190
Summary:
Phenotypic plasticity describes the ability of a given genotype to produce distinct phenotypes in different environments. This study used the temperature sensitivity of abdominal pigmentation in Drosophila melanogaster females as a model to analyse the effect of the environment on development. Previous work has shown that thermal plasticity of abdominal pigmentation in females involves the pigmentation gene tan (t). However, the expression of the pigmentation gene yellow (y) was also modulated by temperature in the abdominal epidermis of pharate females. This study investigate the contribution of y to female abdominal pigmentation plasticity. First, it was shown that y is required for the production of black Dopamine-melanin. Then, using in situ hybridization, it was shown that the expression of y is strongly modulated by temperature in the abdominal epidermis of pharate females but not in bristles. Interestingly, these two expression patterns are known to be controlled by distinct enhancers. However, the activity of the y-wing-body epidermal enhancer only partially mediates the effect of temperature suggesting that additional regulatory sequences are involved. In addition, it was shown that y and t co-expression is needed to induce strong black pigmentation indicating that y contributes to female abdominal pigmentation plasticity.
Gibert, J. M., Blanco, J., Dolezal, M., Nolte, V., Peronnet, F. and Schlotterer, C. (2017). Strong epistatic and additive effects of linked candidate SNPs for Drosophila pigmentation have implications for analysis of genome-wide association studies results. Genome Biol 18(1): 126. PubMed ID: 28673357
Summary:
The mapping resolution of genome-wide association studies (GWAS) is limited by historic recombination events and effects are often assigned to haplotype blocks rather than individual SNPs. It is not clear how many of the SNPs in the block, and which ones, are causative. Drosophila pigmentation is a powerful model to dissect the genetic basis of intra-specific and inter-specific phenotypic variation. Three tightly linked SNPs in the t-MSE enhancer have been identified in three D. melanogaster populations as major contributors to female abdominal pigmentation. This enhancer controls the expression of the pigmentation gene tan (t) in the abdominal epidermis. Two of the three SNPs were confirmed in an independent study using the D. melanogaster Genetic Reference Panel established from a North American population. This study determined the functional impact of SNP1, SNP2, and SNP3 using transgenic lines to test all possible haplotypes in vivo. All three candidate SNPs contribute to female Drosophila abdominal pigmentation. Interestingly, only two SNPs agree with the effect predicted by GWAS; the third one goes in the opposite direction because of linkage disequilibrium between multiple functional SNPs. The experimental design uncovered strong additive effects for the three SNPs, but significant epistatic effects explaining up to 11% of the total variation. These results suggest that linked causal variants are important for the interpretation of GWAS and functional validation is needed to understand the genetic architecture of traits.
BIOLOGICAL OVERVIEW

Phenotypic plasticity is the ability of a given genotype to produce different phenotypes in response to distinct environmental conditions. Phenotypic plasticity can be adaptive. Furthermore, it is thought to facilitate evolution. Although phenotypic plasticity is a widespread phenomenon, its molecular mechanisms are only beginning to be unravelled. Environmental conditions can affect gene expression through modification of chromatin structure, mainly via histone modifications, nucleosome remodelling or DNA methylation, suggesting that phenotypic plasticity might partly be due to chromatin plasticity. As a model of phenotypic plasticity, abdominal pigmentation was studied of Drosophila melanogaster females, which is temperature sensitive. Abdominal pigmentation is indeed darker in females grown at 18 °C than at 29 °C. This phenomenon is thought to be adaptive as the dark pigmentation produced at lower temperature increases body temperature. This study showed that temperature modulates the expression of tan (t) , a pigmentation gene involved in melanin production. t is expressed 7 times more at 18 °C than at 29 °C in female abdominal epidermis. Genetic experiments show that modulation of t expression by temperature is essential for female abdominal pigmentation plasticity. Temperature modulates the activity of an enhancer of t without modifying compaction of its chromatin or level of the active histone mark H3K27ac. By contrast, the active mark H3K4me3 on the t promoter is strongly modulated by temperature. The H3K4 methyl-transferase involved in this process is likely Trithorax, since it regulates t expression and the H3K4me3 level on the t promoter and also participates in female pigmentation and its plasticity. Interestingly, t was previously shown to be involved in inter-individual variation of female abdominal pigmentation in Drosophila melanogaster, and in abdominal pigmentation divergence between Drosophila species. Sensitivity of t expression to environmental conditions might therefore give more substrate for selection, explaining why this gene has frequently been involved in evolution of pigmentation (Gilbert, 2016).

Phenotypic plasticity, 'the property of a given genotype to produce different phenotypes in response to distinct environmental conditions', is a widespread phenomenon. Phenotypic plasticity can be adaptive if different but optimal phenotypes are produced by a given genotype in distinct environments. Furthermore, phenotypic plasticity could facilitate evolution. In particular, Conrad Waddington showed that changes in environmental conditions can reveal cryptic genetic variation that can be selected, allowing to fix a phenotype initially observed only in particular environmental conditions. Waddington called this process 'genetic assimilation'. Analysis of phenotypic plasticity and morphological complexity in an evolutionary framework supports indeed the idea that phenotypic plasticity increases evolutionary potential. For example, a recent study on feeding structure evolution in nematodes revealed that phenotypic plasticity correlates with morphological diversification. The question then arises whether the same genes are involved in phenotypic plasticity and in phenotypic variation within and between species. To address this question, the molecular mechanisms underlying phenotypic plasticity need to be identified. Several examples show that environmental factors can strongly affect the transcriptome, histone mark apposition. In Drosophila, female abdominal pigmentation is a plastic trait as it is darker in females grown at 18°C than at 29°C. As low temperature leads to darker pigmentation, which increases body temperature, the thermal plasticity of female abdominal pigmentation is thought to be adaptive. Abdominal pigmentation in drosophilids is a particularly appropriate model to study phenotypic plasticity, as the genes involved in abdominal pigmentation are well known. Indeed, abdominal pigmentation has been used as a model to dissect the genetic bases of sexual dimorphism and of variation within or between species. In none of these studies, which focussed on genetic factors and were performed in standard conditions (usually at 25°C), was the effect of the environment taken into account. However, Drosophila melanogaster can develop between 12°C and 30°C. As temperature varies spatially and temporally in the wild, taking it into account is paramount to understand the development and evolution of abdominal pigmentation. Using mainly genetics approaches, previous studies have shown that temperature acts on melanin production by modulating a chromatin regulator network, but the underlying molecular mechanisms were not further dissected (Gilbert, 2007). This study identifies the pigmentation gene tan (t) as the major structural gene involved in female abdominal pigmentation plasticity, and chromatin structure at this locus is shown to be modulated by temperature. Temperature dramatically modulates t expression in the female abdominal epidermis and this modulation plays a major role in female abdominal pigmentation plasticity. Temperature modulates the activity of an enhancer of t, t_MSE (Gilbert, 2007), but had no detectable effect on its chromatin structure. By contrast, the active histone mark H3K4me3 is strongly enriched on the t promoter at low temperature. The H3K4 methyl-transferase responsible for this effect is likely Trithorax (Trx). Indeed, this study shows that Trx regulates t expression and the level of H3K4me3 on the t promoter, and is involved in abdominal pigmentation as well as in its plasticity. As t has been linked to pigmentation divergence within or between Drosophila species (Gilbert, 2007; Wittkopp, 2009; Bastide, 2013; Cooley, 2012), t is listed among hotspot loci of evolution (Martin, 2013). This study therefore suggests that the sensitivity of particular genes to environmental changes could turn them into evolutionary hotspots by giving more substrate for selection (Gilbert, 2016).

This study shows that plasticity of female abdominal pigmentation in D. melanogaster involves strong modulation of the expression of the pigmentation gene t. Furthermore, the results demonstrate that this modulation plays a major role in female abdominal pigmentation plasticity. Interestingly, a previous study analysing thermal plasticity of gene expression in the whole body of three days old D. melanogaster females showed that t expression diminishes when temperature increases (Chen, 2015). However, as the abdominal pigmentation pattern is already established at this stage, it is likely that, in these experiments, other tissues contribute to the variation of t expression. As t is expressed in photoreceptors and plays a role in vision (True, 2005), it would be interesting to test whether its expression varies with temperature in adult eyes (Gilbert, 2016).

In young adults, t is the only pigmentation gene among those tested which is significantly modulated by temperature. However, a trend was observed towards a weaker e expression at 18°C than at 29°C, although not statistically significant. In pharates, several pigmentation genes, including t and e, are moderately modulated by temperature. In addition, a weaker expression of e-nEGFP was observed in A6 and A7 at 18°C than at 29°C. These findings agree with previous data showing the qualitative analysis of e expression at different temperatures using an e-lacZ transgene (Gilbert, 2007). In this previous publication, e mutants were shown to remain dark at all temperatures, and it was concluded that a functional e gene is required for the plasticity of pigmentation. The present data complete this conclusion. Indeed, this study shows that e is epistatic over t. This explains why e mutants lose abdominal pigmentation plasticity, as a functional e gene is required to observe plasticity induced by modulation of t expression (Gilbert, 2016).

Furthermore, the data show that the expression of e, DDC, y and b is modulated by temperature in pharates. This could explain the residual pigmentation plasticity observed in t mutants (Gilbert, 2016).

Lastly, spatial analysis of e expression by in situ hybridization reveals a stronger expression at 29°C than at 18°C in anterior abdominal segments. This observation suggests that the reduced but observable plasticity of these anterior segments might be due to e temperature sensitive expression (Gilbert, 2016).

The effect of temperature on t expression is mediated, at least partly, by the t_MSE enhancer. Thus, this enhancer may have particular properties making it temperature sensitive. Indeed, recent data showed that the number of redundant binding sites for a particular transcription factor in an enhancer could influence its temperature sensitivity (Crocker, 2015). Another, non-exclusive, explanation could be that temperature affects the expression or the activity of regulatory factors upstream of t. No chromatin modification of t_MSE was detected at different temperatures, possibly because this enhancer is active, although at different levels, at the temperatures tested. The level of H3K27ac could therefore be saturated and the chromatin on t_MSE decompacted at both temperatures. By contrast, the effect of temperature on t expression is correlated with the modulation of H3K4me3 deposition on the t promoter. As this histone mark correlates with active transcription, the strong accumulation of t transcripts at 18°C is more likely caused by a transcriptional response to temperature than by modulation of a post-transcriptional mechanism that would stabilize them. Interestingly, deposition of H3K4me3 can also be modulated by environmental conditions such as diet in mouse liver, drought stress in plants or chemical stress in yeast. This histone mark emerges therefore as a general mediator of environmental impact on the genome (Gilbert, 2016).

This study shows that the H3K4 methyl-transferase Trx is involved in t regulation, but also in the regulation of other pigmentation genes. As the level of H3K4me3 on the t promoter decreases when trx is inactivated, it is tempting to speculate that Trx directly regulates t. However, Trx might also indirectly control t expression through the regulation of genes upstream t. Furthermore, as Trx has no intrinsic DNA binding activity, its recruitment on t or on upstream regulators must depend on specific transcription factors. Thus, it would be interesting to identify the upstream regulators of t controlled by Trx as well as the transcription factors recruiting Trx on t or on its upstream regulators (Gilbert, 2016).

Trx also participates in the thermal plasticity of female abdominal pigmentation. This confers to Trx a very specific role as compared to other H3K4 methyl-transferases. Indeed, Set1 has been described as the main H3K4 di- and tri- methyl-transferase during Drosophila development. However, the current results demonstrate for the first time that Trx is involved in the thermal plasticity of female abdominal pigmentation (Gilbert, 2016).

Modulation of pigmentation by environmental conditions is observed in many insects. Interestingly, t expression is strongly modulated by environmental conditions in the developing wings of Junonia coenia, a butterfly with contrasting seasonal morphs (Daniels, 2014). The involvement of t in pigmentation plasticity might therefore be widespread in insects (Gilbert, 2016).

Several studies have also linked t to pigmentation variation within or between Drosophila species. Modulation of t expression through modification of t cis-regulatory sequences has been implicated in evolution of abdominal pigmentation between species (Wittkopp, 2009; Cooley, 2012). Remarkably, in D. santomea, independent mutations in t_MSE have generated three distinct loss-of-function alleles involved in the reduced pigmentation of this species. Furthermore, SNPs associated with variation of abdominal pigmentation in D. melanogaster females have been identified in t_MSE (Bastide, 2013). Interestingly, abdominal pigmentation dimorphism in female Drosophila erecta was recently shown to be caused by sequence variation in t_MSE maintained by balancing selection (Yassin, 2016). The recurrent implication of t in pigmentation evolution has led to list this gene among hotspots of evolution. In other organisms, genes sensitive to environment and involved in phenotypic plasticity are also responsible for differences within or between species. For example, in Brassicaceae, the reduced complexity locus (RCO) that participates in leaf margin dissection is modulated by temperature and has been repeatedly involved in leaf shape evolution through cis-regulatory sequence variation or gene loss. Therefore, sensitivity of particular genes to environmental conditions might turn them into evolutionary hotspots. Indeed, this broadens the range of phenotypes produced by a particular allele, providing more substrate for natural selection (Gilbert, 2016).

Ancient balancing selection at tan underlies female colour dimorphism in Drosophila erecta

Dimorphic traits are ubiquitous in nature, but the evolutionary factors leading to dimorphism are largely unclear. This study investigated a potential case of sexual mimicry in Drosophila erecta, in which females show contrasting resemblance to males. The genetic basis of this sex-limited colour dimorphism mapped to a region containing the gene tan. A striking signal of ancient balancing selection was found at the 'male-specific enhancer' of tan (t_MSE), with exceptionally high sequence divergence between light and dark alleles, suggesting that this dimorphism has been adaptively maintained for millions of years. Using transgenic reporter assays, it was confirmed that these enhancer alleles encode expression differences that are predicted to generate this pigmentation dimorphism. These results are compatible with the theoretical prediction that divergent phenotypes maintained by selection can evolve simple genetic architectures (Yassin, 2016).

The results motivate the hypothesis that the dark-female t_MSE allele of D. erecta evolved by extending the activity of an otherwise male-specific enhancer into females as well. Increasing tan expression in the female abdomen is predicted to increase production of melanic pigments (True, 2005), leading to the male-like pigmentation observed in females of dark D. erecta strains. This dark allele may therefore represent a loss of sexual dimorphism at the molecular and phenotypic levels, even as it creates a novel dimorphism among females. Curiously, this same cis-regulatory element of tan has also underlain the loss of sexual dimorphism in the related species D. santomea leading to the evolution of female-like light males (Jeong, 2008; Camino, 2015) as well as in other sexually monomorphic Drosophila species (Yassin, 2016).

In the abdomen of D. melanogaster, tan is upregulated in males by the Hox genes Abd-A and Abd-B and suppressed in females by the transcription factors bab1 and bab2 whose expressions are sexually dimorphic (Camino, 2015). Although the exact binding sites of Bab1 and Bab2 are still unknown, it is possible that the dark haplotype in D. erecta involves the loss of such sites. In another Drosophila species with monogenic female-limited colour dimorphism (D. kikkawai), the female-limited colour dimorphism (FLCD) locus is still unknown but mapping indicates that it is different from tan or bab, highlighting the complexity of this trait. Future investigations using recent advances in Drosophila molecular biology techniques as well as other species with FLCD will help the precise dissection of the genetic basis of this sexual colour dimorphism (Yassin, 2016).

Although quite rare, female-limited colour variation has been described in at least two other groups of non-drosophilid insects (Wellenreuther, 2014). Aside from the damselfly case, certain butterflies have both mimetic and non-mimetic female morphs, which vary in frequency geographically due to spatially varying selective pressures. These morphs are associated with ∼400 kb chromosomal rearrangements in Heliconius butterflies. In Papilio polytes, complex pigmentation variation correlates with ∼130 kb inversion-associated alleles of doublesex, a component of the sexual differentiation pathway. In the current study, the genetic tools and knowledge of Drosophila allowed localization of the genetic basis of a sex-limited dimorphic trait to a regulatory element of <1 kb, indicating the strong potential of this system for further insights regarding the mechanisms of sex-specific evolution and the origin of dimorphic traits (Yassin, 2016).

This study provides a rare example of the genetic basis of an ancient balanced polymorphism with clear morphological consequences. Most known examples include genes involved in immunity interactions or mate recognition such as major histocompatibility complexes in vertebrates, blood groups in primates, self-incompatibility in plants and mating-types in fungi. Balancing selection can involve different mechanisms such as heterozygous advantage, spatially or temporally variable selective pressures, or frequency-dependent selection. In the case of D. erecta FLCD, it is not clear why ecological factors would maintain discrete pigmentation morphs at similar frequencies in different populations, and preserve them for millions of years. Extensive experiments have been conducted on factors maintaining D. erecta FLCD in the laboratory. A mating preference for light females was observed when the frequency of the light allele was between 0.5 and 0.7. Although further study is called for, frequency-dependent sexual selection thus represents a plausible explanation for FLCD, especially given the morphological similarity of the current case in Drosophila with FLCD in damselflies, a prime model for frequency-dependent sexual selection (Yassin, 2016).

The monogenic nature of FLCD and frequency-dependent mating results suggest that this trait could be consistent with theoretical predictions for FDDS, which can lead to the evolution of dimorphic traits with simple genetic architectures. Disruptive selection would explain the lack of intermediate phenotypes found in nature, whereas evidence from this work is consistent with a role for balancing selection in maintaining D. erecta FLCD, potentially due to frequency-dependent sexual selection. FDDS should increase the effect of one or a few loci relative to all others. In agreement with this model, other Drosophila species have more modest and continuous pigmentation variation because of several genes including tan, whereas in the D. erecta lineage tan's role increased to shape a discrete colour dimorphism. These results reflect important steps towards understanding the evolutionary and genetic mechanisms that give rise to dimorphic traits and sex-specific variation in nature (Yassin, 2016).

The evolutionary origination and diversification of a dimorphic gene regulatory network through parallel innovations in cis and trans

The origination and diversification of morphological characteristics represents a key problem in understanding the evolution of development. Morphological traits result from gene regulatory networks (GRNs) that form a web of transcription factors, which regulate multiple cis-regulatory element (CRE) sequences to control the coordinated expression of differentiation genes. The formation and modification of GRNs must ultimately be understood at the level of individual regulatory linkages (i.e., transcription factor binding sites within CREs) that constitute the network. This study investigated how elements within a network originated and diversified to generate a broad range of abdominal pigmentation phenotypes among Sophophora fruit flies. The data indicates that the coordinated expression of two melanin synthesis enzymes, Yellow and Tan, recently evolved through novel CRE activities that respond to the spatial patterning inputs of Hox proteins and the sex-specific input of Bric-a-brac transcription factors. Once established, it seems that these newly evolved activities were repeatedly modified by evolutionary changes in the network's trans-regulators to generate large-scale changes in pigment pattern. By elucidating how yellow and tan are connected to the web of abdominal trans-regulators, this study discovered that the yellow and tan abdominal CREs are composed of distinct regulatory inputs that exhibit contrasting responses to the same Hox proteins and Hox cofactors. These results provide an example in which CRE origination underlies a recently evolved novel trait, and highlights how coordinated expression patterns can evolve in parallel through the generation of unique regulatory linkages (Camino, 2015).

This study has traced the evolutionary history of two CREs required for a novel trait, and show that they have recently evolved similar expression patterns through remarkably different architectures in a common trans-regulatory landscape. The data indicates that the tergite-wide activities of the yBE and t_MSE did not exist in the monomorphic ancestor for Sophophora, but evolved in the lineage leading to the common ancestor of the melanogaster species group. The results support a scenario where the subsequent expansion and contraction of male pigmentation pattern was driven primarily by alteration of the trans-regulators, whereas repeated losses involved both cis- and trans-evolution with respect to these CREs. Though the t_MSE and yBE drive coordinated patterns of gene expression, striking differences were found in their upstream regulators and direct regulatory linkages. These results bear on the understanding of how new gene regulatory networks form, diversify, and how coordinated regulatory activities can arise through the independent evolution of unique regulatory codes (Camino, 2015).

Hox transcription factors play a prominent role in generating the differences in serially homologous animal body parts, and the origin of novelties. The diversification of homologous parts can be driven by changes in the spatial domains of Hox protein expression, as has been shown for crustacean appendage morphology, snake limblessness, and for the water strider appendage ground plan. Changes in the downstream Hox targets are evident in cases such as the hindwings of insects, and for fruit fly tergite pigmentation. The origin of novel structures can also be traced to the co-option of Hox proteins, as exemplified by cases such as the Photuris firefly lantern and the sex combs residing on the forelegs of certain Drosophila species. For many of these evolved traits, the molecular mechanisms by which Hox expression patterns and target genes evolve remain unknown (Camino, 2015).

While mechanistic studies on the evolution of Hox-regulated CREs remain limited, several target gene CREs have been thoroughly characterized and serve as exemplars of Hox-regulation during development. Hox proteins can interact with CRE binding sites as monomers or through cooperative interactions with Hox-cofactors. The activity of these bound complexes can be further modulated through interactions with collaborating transcription factors. However, to date, few direct Hox target linkages have been traced to their evolutionary beginnings. Expression of yellow in the male A5 and A6 segments required the gain of two binding sites for Abd-B, but it remains uncertain whether these binding events require cooperative interactions with Hox cofactors and which transcription factors are acting as collaborators (Camino, 2015).

The t_MSE presented an opportunity to study how a second Hox-responsive CRE evolved in parallel to the activity at yellow. This study shows that Abd-A and Abd-B respectively are necessary and sufficient for t_MSE regulatory activity. However, the ablation of the resident Hox sites had little effect on this CRE's activity in the A5 and A6 segments, though mutations to nearby CRE sequences resulted in dramatically reduced activity. This result strongly implies that both Abd-A and Abd-B indirectly activate the t_MSE through a downstream factor or factors. While it can't be entirely ruled out that these factors are operating directly through other non-canonical Hox sites, the gel shift assays did not provide convincing evidence that such sites exist. While the Hox sites were not necessary for activation in the A5 and A6 segments, their ablation resulted in a drastic gain of regulatory activity in the A4 and A3 segments, a setting in which Abd-A is the only Hox protein present. This indicates that Abd-A is a direct repressor of t_MSE function in these anterior abdomen segments. The observed dichotomy in Abd-A function can be explained by at least two-not necessarily mutually exclusive-scenarios. First, in the A5 and A6 segments Abd-B may not act as a direct activator of the t_MSE but its occupancy of Hox sites might preclude the direct repressive effects of Abd-A. Secondly, Abd-A may interact cooperatively or collaboratively with other transcription factors in the more anterior segments to impart repression. The results with Hth support this second scenario (Camino, 2015).

The Hox co-factors Hth and Exd were prime candidates to mediate the context-dependent modulation of Abd-A activity. First, RNAi suppression of hth and exd expression each resulted in ectopic pigmentation (Rogers, 2014) and t_MSE activity in the male A4 and A3 segments. Furthermore, inspection of the t_MSE sequence revealed sites characteristic of Hth (AGACAG) and Exd (GATCAT) binding that reside in close proximity to Hox sites. This site content and arrangement is strikingly similar to that found in an abdominal-repressive module for the CRE controlling thoracic Distalless expression. Along a similar vein, this study shows that the ablation of the Hth-like site led to an anterior expansion in t_MSE activity similar to that induced by the Hox site mutations. This outcome supports the interpretation that the more recent origin of the t_MSE involved the formation of novel regulatory linkages with Hox proteins and Hox cofactors (Camino, 2015).

Morphological traits result from the activities of gene regulatory networks, in which each network is governed by a trans-regulatory tier of transcription factors and cell signaling components that ultimately regulate the expression of a set of differentiation genes. For animals, the trans-regulatory genes are remarkably conserved. It is plausible that the origin of new morphologies occurs through the formulation of new gene regulatory networks, while diversification and losses in traits would likely occur through the modification and dismantling of extant networks. The empirical evaluation of such trends of network evolution necessitates the study of trait evolution at the level of networks, CREs, and their encoded binding sites for multiple animal lineages, traits, and evolutionary time frames. The Drosophila pigmentation system is particularly well poised to make pioneering contributions to this growing body of knowledge (Camino, 2015).

The most recent common ancestor of monomorphic and dimorphic Sophophora lineages was inferred to have possessed monomorphic tergite pigmentation, in the context of an otherwise invariant morphological landscape, in which segment number and form has remained conserved at the genus level. Hence, the origin of this novel pigmentation trait may be expected to have co-opted spatial and sex-specific patterning mechanisms that shape the conserved abdomen features. Comparative analysis of orthologous yellow and tan non-coding sequences indicate that these co-option events involved the origination of novel CRE activities that connected a trans-regulatory tier of Hox, Hox-cofactors, and the Bab proteins to these key differentiation genes that encoded pigmentation enzymes (Camino, 2015).

The patterns of regulatory activity for the orthologous tan and yellow sequences support some additional inferences about the early events in this dimorphic trait's origin. While the t_MSE abdominal activity was strikingly lower in D. pseudoobscura and D. willistoni, the D. pseudoobscura yellow body element was active (albeit with expanded activity). These outcomes support at least two evolutionary scenarios. One scenario is a sequence of events where the origination of the t_MSE and y_BE in the lineage of D. pseudoobscura was followed by a secondary loss of the t_MSE. This scenario is supported by a previous observation of dimorphic Bab expression in the D. pseudoobscura abdomen, backing the notion that this species' broad pattern of monomorphic abdominal pigmentation evolved from a dimorphic ancestral state. For the other scenario, the body element-like regulatory activity of D. pseudoobscura could be due to this CRE's origin preceding that of the t_MSE. Distinguishing between these two scenarios will require a more rigorous comparison of the pigmentation phenotypes and networks within the melanogaster and obscura species groups. The outcomes would provide a more nuanced understanding of the early evolutionary history for the derived sexually dimorphic pigmentation network (Camino, 2015).

Tergite pigmentation evolution in the Sophophora subgenus has been relatively well-studied, and the accumulated results frame an extended perspective of trait evolution within a common network. Trans-evolution at the bric-à-brac (bab) locus has been found to be a major driver for the diversification of female tergite pigmentation. This study, in addition to previous studies, indicates that trans-evolution at as of yet unidentified loci may have played prominent roles in the diversification of male-limited tergite pigmentation. Regarding the repeated losses in male pigmentation, the current results are consistent with a scenario where both trans- and cis-evolution occurred, though the targets of cis-evolution have alternated between tan and yellow. While cis-evolution has been identified for a case of monomorphic gain (ebony) in tergite pigmentation, and for a case of monomorphic loss (ebony and tan), the full wealth of case studies portend to a more prominent role for evolutionary changes in the trans-regulatory tier of the pigmentation gene network. However, it is important to note that many of these case studies only assessed the activities of transgenes in D. melanogaster. While similarities in CRE activity might be indicative that expression divergence occurred through trans-evolution, it does not rule out the possibility that cis-changes occurred at other regions in the pigmentation enzyme gene loci, or that expression divergence results from combined cis- and trans-changes. In the future, it will be important to validate or reject the prominent role for trans-regulatory evolution by the reciprocal tests of CREs in species with the contrasting patterns of pigmentation. Two studies where CREs were tested in species with contrasting pigmentation phenotypes, showed that trans-regulatory evolution was a major driver for diversification of fruit fly wing spot patterns by modifying Distalless and wingless expression (Arnoult, 2013; Werner, 2010). Thus it appears the notion of a “conserved trans-landscape” requires more scrutiny (Camino, 2015).

In this study, and elsewhere, experiments indicate that pigmentation losses are associated with and perhaps result from both changes in the trans-regulatory tier and in the cis-regulatory regions of the yellow and tan genes. Interestingly, some instances of trans-regulatory modifications that cause loss of gene expression appear to leave perfectly good CREs intact. The current data provides a second instance in which loss of expression occurred without the loss of the encoded CRE. The yBE was found to be conserved in D. santomea, which diverged from D. yakuba ~400,000 years ago (Jeong, 2006). The activity for this CRE has also remained for D. ananassae since its divergence from a pigmented ancestor. In contrast, D. kikkawai has lost pigmentation while still expressing tan in the abdomen through a perfectly active t_MSE. These results suggest that these CREs were maintained within the population for long periods of time, perhaps indicating additional functions that promote the preservation of these CREs' ancestral potential. Furthermore, the observed heterogeneity of changes in cis and trans to yellow and tan were at first surprising. However, study of the binding site architecture at the yBE and t_MSE provided key clues as to why their evolution may often be uncoupled (Camino, 2015).

The coordinated expression of genes is a ubiquitous theme in developmental biology. Gene expression is finely regulated during development through the activities of CREs that are individually encoded as evolved combinations of transcription factor binding sites (regulatory logic). A compelling question is whether such synchronized expression results from the independent evolution of CREs with similar logics. This question was previously pursued for CREs of regulatory genes coordinately expressed in the developing fruit fly neurogenic ectoderm. In this case, the coordinately activated CREs are encoded by a common regulatory logic, or a so called 'cis-regulatory module equivalence class'. However, the neurogenic ectoderm CREs are deeply conserved, and arose in the distant past (over 230 million years ago) (Camino, 2015).

The recently evolved male-specific expression patterns for tan and yellow present a case in which the evolutionary formation of coordinated regulation can be observed over shorter time-scales. Though both the t_MSE and yBE0.6 drive reporter expression in the dorsal A5 and A6 segment epidermis of males during late pupal development, this study found their regulatory logic to be surprisingly dissimilar. Whereas the yBE0.6 is directly activated by Abd-B, the results indicate that the t_MSE is indirectly activated by Abd-B and Abd-A, and is directly repressed in more anterior body segments by Abd-A and seemingly Hth. Thus, this study provides an example that illustrates how coordinated expression evolved through the evolution of very different binding site architectures and logic (Camino, 2015).

The disparity of regulatory logic governing the yBE0.6 and t_MSE sheds light on the evolutionary tendencies of gene regulatory networks. The incipient stages of the dimorphic pigmentation network's origin involved the derivation of CREs that generate similar patterns through distinct combinations of binding sites. This evolutionary history establishes a 'branched' network in which several of the possible trans-regulatory alterations are incapable of generating coordinated shifts in the expression patterns for co-expressed genes. Hence, an emerging theme from the work in this system is that the differences in regulatory logic of yBE and t_MSE may necessitate changes in one CRE or the other, but is unable to be altered through a common trans regulator that influences both CRE's patterning. Future studies are needed to substantiate the occurrence and identity of the trans changes altering this network's structure. As other recently derived morphological traits are resolved to the level of binding sites within their networks, it will be instructive to see whether similar branched networks and paths of cis and trans evolution permeate their origin and diversification. The net results may reveal general principles of gene regulatory network evolution (Camino, 2015).

Genetic architecture of abdominal pigmentation in Drosophila melanogaster

Pigmentation varies within and between species and is often adaptive. The amount of pigmentation on the abdomen of Drosophila melanogaster is a relatively simple morphological trait, which serves as a model for mapping the genetic basis of variation in complex phenotypes. This study assessed natural variation in female abdominal pigmentation in 175 sequenced inbred lines of the Drosophila melanogaster Genetic Reference Panel, derived from the Raleigh, NC population. The proportion of melanization was quantified on the two most posterior abdominal segments, tergites 5 and 6 (T5, T6). Significant genetic variation was found in the proportion of melanization and high broad-sense heritabilities for each tergite. Genome-wide association studies identified over 150 DNA variants associated with the proportion of melanization on T5 (84), T6 (34), and the difference between T5 and T6 (35). Several of the top variants associated with variation in pigmentation are in tan, ebony, and bric-a-brac1, genes known to affect D. melanogaster abdominal pigmentation. Mutational analyses and targeted RNAi-knockdown showed that 17 out of 28 (61%) novel candidate genes implicated by the genome-wide association study affected abdominal pigmentation. Several of these genes are involved in developmental and regulatory pathways, chitin production, cuticle structure, and vesicle formation and transport. These findings show that genetic variation may affect multiple steps in pathways involved in tergite development and melanization. Variation in these novel candidates may serve as targets for adaptive evolution and sexual selection in D. melanogaster (Dembeck, 2016).

A genome-wide, fine-scale map of natural pigmentation variation in Drosophila melanogaster

Various approaches can be applied to uncover the genetic basis of natural phenotypic variation, each with their specific strengths and limitations. This study used a replicated genome-wide association approach (Pool-GWAS) to fine-scale map genomic regions contributing to natural variation in female abdominal pigmentation in Drosophila melanogaster, a trait that is highly variable in natural populations and highly heritable in the laboratory. Abdominal pigmentation phenotypes were examined in approximately 8000 female European D. melanogaster, isolating 1000 individuals with extreme phenotypes. Whole-genome Illumina sequencing was used to identify single nucleotide polymorphisms (SNPs) segregating in the sample, and these were tested for associations with pigmentation by contrasting allele frequencies between replicate pools of light and dark individuals. Two small regions were identified near the pigmentation genes tan and bric-a-brac 1, both corresponding to known cis-regulatory regions, which contain SNPs showing significant associations with pigmentation variation. While the Pool-GWAS approach suffers some limitations, its cost advantage facilitates replication and it can be applied to any non-model system with an available reference genome (Bastide, 2013).

Sensitivity of allelic divergence to genomic position: Lessons from the Drosophila tan gene

To identify genetic variants underlying changes in phenotypes within and between species, researchers often utilize transgenic animals to compare the function of alleles in different genetic backgrounds. In Drosophila, targeted integration mediated by the PhiC31 integrase allows activity of alternative alleles to be compared at the same genomic location. By using the same insertion site for each transgene, position effects are generally assumed to be controlled for because both alleles are surrounded by the same genomic context. This study tested this assumption by comparing the activity of tan alleles from two Drosophila species, D. americana and D. novamexicana, at five different genomic locations in D. melanogaster. The relative effects of these alleles varied among insertion sites, with no difference in activity observed between them at two sites. One of these sites simply silenced both transgenes, but the other allowed expression of both alleles that was sufficient to rescue a mutant phenotype yet failed to reveal the functional differences between the two alleles. These results suggest that more than one insertion site should be used when comparing the activity of transgenes because failing to do so could cause functional differences between alleles to go undetected (John, 2016).

The ontogeny of color: developmental origins of divergent pigmentation in Drosophila americana and D. novamexicana

Pigmentation is a model trait for evolutionary and developmental analysis that is particularly amenable to molecular investigation in the genus Drosophila. To better understand how this phenotype evolves, divergent pigmentation and gene expression over developmental time were examined in the dark-bodied D. americana and its light-bodied sister species D. novamexicana. Prior genetic analysis implicated two enzyme-encoding genes, tan and ebony, in pigmentation divergence between these species, but questions remain about the underlying molecular mechanisms. This study described stages of pupal development in both species, and this staging was used to determine when pigmentation develops and diverges between D. americana and D. novamexicana. For the developmental stages encompassing pigment divergence, mRNA expression of tan and ebony was compared over time and between species. Finally, allele-specific expression assays were used to determine whether interspecific differences in mRNA abundance have a cis-regulatory basis and find evidence of cis-regulatory divergence for both tan and ebony. cis-regulatory divergence affecting tan had a small effect on mRNA abundance and was limited to a few developmental stages, yet previous data suggests that this divergence is likely to be biologically meaningful. This study suggests that small and developmentally transient expression changes may contribute to phenotypic diversification more often than commonly appreciated. Recognizing the potential phenotypic impact of such changes is important for a scientific community increasingly focused on dissecting quantitative variation, but detecting these types of changes will be a major challenge to elucidating the molecular basis of complex traits (Cooley, 2012).

Intraspecific polymorphism to interspecific divergence: genetics of pigmentation in Drosophila

Genetic changes contributing to phenotypic differences within or between species have been identified for a handful of traits, but the relationship between alleles underlying intraspecific polymorphism and interspecific divergence is largely unknown. This study found that noncoding changes in the tan gene, as well as changes linked to the ebony gene, contribute to pigmentation divergence between closely related Drosophila species. Moreover, alleles linked to tan and ebony fixed in one Drosophila species also contribute to variation within another species, and multiple genotypes underlie similar phenotypes even within the same population. These alleles appear to predate speciation, which suggests that standing genetic variation present in the common ancestor gave rise to both intraspecific polymorphism and interspecific divergence (Wittkopp, 2009).

The evolution of gene regulation underlies a morphological difference between two Drosophila sister species

Understanding the mechanisms underlying the morphological divergence of species is one of the central goals of evolutionary biology. This study analyzes the genetic and molecular bases of the divergence of body pigmentation patterns between Drosophila yakuba and its sister species Drosophila santomea. Loss of pigmentation in D. santomea involved the selective loss of expression of the tan and yellow pigmentation genes. tan gene expression was eliminated through the mutational inactivation of one specific tan cis-regulatory element (CRE) whereas the Tan protein sequence remained unchanged. Surprisingly, three independent loss-of-function alleles of the tan CRE were identified in the young D. santomea lineage. It is submitted that there is sufficient empirical evidence to support the general prediction that functional evolutionary changes at pleiotropic loci will most often involve mutations in their discrete, modular cis-regulatory elements (Jeong, 2008).

Drosophila photoreceptors express cysteine peptidase tan

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. This study expressed Tan in Escherichia coli and confirmed by Western blotting and mass spectroscopic analyses 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 were 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).

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).

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. 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. 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).


REFERENCES

Search PubMed for articles about Drosophila Tan

Arnoult, L., Su, K. F., Manoel, D., Minervino, C., Magrina, J., Gompel, N. and Prud'homme, B. (2013). Emergence and diversification of fly pigmentation through evolution of a gene regulatory module. Science 339: 1423-1426. PubMed ID: 23520110

Bastide, H., Betancourt, A., Nolte, V., Tobler, R., Stobe, P., Futschik, A. and Schlotterer, C. (2013). A genome-wide, fine-scale map of natural pigmentation variation in Drosophila melanogaster. PLoS Genet 9: e1003534. PubMed ID: 23754958

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

Bastide, H., Betancourt, A., Nolte, V., Tobler, R., Stobe, P., Futschik, A. and Schlotterer, C. (2013). A genome-wide, fine-scale map of natural pigmentation variation in Drosophila melanogaster. PLoS Genet 9: e1003534. PubMed ID: 23754958

Camino, E. M., Butts, J. C., Ordway, A., Vellky, J. E., Rebeiz, M. and Williams, T. M. (2015). The evolutionary origination and diversification of a dimorphic gene regulatory network through parallel innovations in cis and trans. PLoS Genet 11: e1005136. PubMed ID: 25835988

Chen, J., Nolte, V. and Schlotterer, C. (2015). Temperature-Related Reaction Norms of Gene Expression: Regulatory Architecture and Functional Implications. Mol Biol Evol 32: 2393-2402. PubMed ID: 25976350

Cooley, A. M., Shefner, L., McLaughlin, W. N., Stewart, E. E. and Wittkopp, P. J. (2012). The ontogeny of color: developmental origins of divergent pigmentation in Drosophila americana and D. novamexicana. Evol Dev 14: 317-325. PubMed ID: 22765203

Crocker, J., Abe, N., Rinaldi, L., McGregor, A. P., Frankel, N., Wang, S., Alsawadi, A., Valenti, P., Plaza, S., Payre, F., Mann, R. S. and Stern, D. L. (2015). Low affinity binding site clusters confer hox specificity and regulatory robustness. Cell 160: 191-203. PubMed ID: 25557079

Daniels, E. V., Murad, R., Mortazavi, A. and Reed, R. D. (2014). Extensive transcriptional response associated with seasonal plasticity of butterfly wing patterns. Mol. Ecol. 23(24): 6123-34. PubMed ID: 25369871

Dembeck, L. M., Huang, W., Magwire, M. M., Lawrence, F., Lyman, R. F. and Mackay, T. F. (2015). Genetic architecture of abdominal pigmentation in Drosophila melanogaster. PLoS Genet 11: e1005163. PubMed ID: 25933381

Gibert, J. M., Mouchel-Vielh, E., De Castro, S. and Peronnet, F. (2016). Phenotypic plasticity through transcriptional regulation of the evolutionary hotspot gene tan in Drosophila melanogaster. PLoS Genet 12: e1006218. PubMed ID: 27508387

Gibert, J. M., Peronnet, F. and Schlotterer, C. (2007). Phenotypic plasticity in Drosophila pigmentation caused by temperature sensitivity of a chromatin regulator network. PLoS Genet 3: e30. PubMed ID: 17305433

Jeong, S., Rokas, A. and Carroll, S. B. (2006). Regulation of body pigmentation by the Abdominal-B Hox protein and its gain and loss in Drosophila evolution. Cell 125: 1387-1399. PubMed ID: 16814723

Jeong, S., Rebeiz, M., Andolfatto, P., Werner, T., True, J. and Carroll, S. B. (2008). The evolution of gene regulation underlies a morphological difference between two Drosophila sister species. Cell 132: 783-793. PubMed ID: 18329365

Martin, A. and Orgogozo, V. (2013). The Loci of repeated evolution: a catalog of genetic hotspots of phenotypic variation. Evolution 67: 1235-1250. PubMed ID: 23617905

Rogers, W. A., Grover, S., Stringer, S. J., Parks, J., Rebeiz, M. and Williams, T. M. (2014). A survey of the trans-regulatory landscape for Drosophila melanogaster abdominal pigmentation. Dev Biol 385: 417-432. PubMed ID: 24269556

True, J. R., Yeh, S. D., Hovemann, B. T., Kemme, T., Meinertzhagen, I. A., Edwards, T. N., Liou, S. R., Han, Q. and Li, J. (2005). Drosophila tan encodes a novel hydrolase required in pigmentation and vision. PLoS Genet 1: e63. PubMed ID: 16299587

Wagner, S., Heseding, C., Szlachta, K., True, J. R., Prinz, H. and Hovemann, B. T. (2007). Drosophila photoreceptors express cysteine peptidase tan. J Comp Neurol 500: 601-611. PubMed ID: 17154266

Wellenreuther, M., Svensson, E. I. and Hansson, B. (2014). Sexual selection and genetic colour polymorphisms in animals. Mol Ecol 23: 5398-5414. PubMed ID: 25251393

Wittkopp, P. J., Stewart, E. E., Arnold, L. L., Neidert, A. H., Haerum, B. K., Thompson, E. M., Akhras, S., Smith-Winberry, G. and Shefner, L. (2009). Intraspecific polymorphism to interspecific divergence: genetics of pigmentation in Drosophila. Science 326: 540-544. PubMed ID: 19900891

Werner, T., Koshikawa, S., Williams, T. M. and Carroll, S. B. (2010). Generation of a novel wing colour pattern by the Wingless morphogen. Nature 464: 1143-1148. PubMed ID: 20376004

Wittkopp, P. J., Stewart, E. E., Arnold, L. L., Neidert, A. H., Haerum, B. K., Thompson, E. M., Akhras, S., Smith-Winberry, G. and Shefner, L. (2009). Intraspecific polymorphism to interspecific divergence: genetics of pigmentation in Drosophila. Science 326: 540-544. PubMed ID: 19900891

John, A. V., Sramkoski, L. L., Walker, E. A., Cooley, A. M. and Wittkopp, P. J. (2016). Sensitivity of allelic divergence to genomic position: Lessons from the Drosophila tan gene. G3 (Bethesda) 6: 2955-2962. PubMed ID: 27449514

Yassin, A., Bastide, H., Chung, H., Veuille, M., David, J. R. and Pool, J. E. (2016). Ancient balancing selection at tan underlies female colour dimorphism in Drosophila erecta. Nat Commun 7: 10400. PubMed ID: 26778363


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date revised: 27 September 2016

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