Phenotypic plasticity is the ability of a genotype to produce contrasting phenotypes in different environments. Although many examples have been described, the responsible mechanisms are poorly understood. In particular, it is not clear how phenotypic plasticity is related to buffering, the maintenance of a constant phenotype against genetic or environmental variation. We investigate here the genetic basis of a particularly well described plastic phenotype: the abdominal pigmentation in female Drosophila melanogaster. Cold temperature induces a dark pigmentation, in particular in posterior segments, while higher temperature has the opposite effect. We show that the homeotic gene Abdominal-B (Abd-B) has a major role in the plasticity of pigmentation in the abdomen. Abd-B plays opposite roles on melanin production through the regulation of several pigmentation enzymes. This makes the control of pigmentation very unstable in the posterior abdomen, and we show that the relative spatio-temporal expression of limiting pigmentation enzymes in this region of the body is thermosensitive. Temperature acts on melanin production by modulating a chromatin regulator network, interacting genetically with the transcription factor bric-à-brac (bab), a target of Abd-B and Hsp83, encoding the chaperone Hsp90. Genetic disruption of this chromatin regulator network increases the effect of temperature and the instability of the pigmentation pattern in the posterior abdomen. Colocalizations on polytene chromosomes suggest that BAB and these chromatin regulators cooperate in the regulation of many targets, including several pigmentation enzymes. We show that they are also involved in sex comb development in males and that genetic destabilization of this network is also strongly modulated by temperature for this phenotype. Thus, we propose that phenotypic plasticity of pigmentation is a side effect reflecting a global impact of temperature on epigenetic mechanisms. Furthermore, the thermosensitivity of this network may be related to the high evolvability of several secondary sexual characters in the genus Drosophila.

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, we study abdominal pigmentation 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. We show here 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, as we show that 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.

SUMMARY Cirripedes (barnacles) are crustaceans that are characterized by a very peculiar body plan, in particular by the lack of an abdomen. To study their body plan, we searched for their engrailed gene. We found two engrailed (en.a/en.b) genes in cirripedes. The two engrailed genes of the rhizocephalan barnacle Sacculina carcini are expressed in the posterior compartment of developing segments and appendages. When the neuroectoderm differentiates into epidermis and neuroderm the expression patterns of en.a and en.b diverge dramatically. en.a expression fades in segment epidermis whereas it is subsequently detected ventrally in reiterated putative neural cells. At the same time, en.b expression increases in the epidermis, which makes it a very good segmentation marker. Five tiny en.b stripes are observed between the sixth thoracic segment and the telson. We interpret these stripes as the molecular definition of vestigial abdominal segments, being the remnant of an ancestral state in keeping with the bodyplan of maxillopod crustaceans. engrailed expression is the first molecular evidence for a segmented abdomen in barnacles.

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Members of the engrailed class encode transcription factors involved in major steps of metazoan development. Few developmental regulatory genes have been studied in such a wide range of animals. Furthermore duplications of an ancestral engrailed gene independently generated multiple engrailed paralogues in several organisms. This offers the opportunity to reconstruct the evolution of the engrailed family and to study the processes involved in the functional diversification following speciation or duplication events. The ancestral function of engrailedis very likely involved in neurogenesis. Recent studies in Drosophila and mice have shown its crucial role in neuronal connectivity and neuromuscular targeting. engrailed was probably recruited very early for a role in segmentation through intercalary evolution. Several new functions were acquired later on in specific phyla. Some duplication events have been followed by the loss of one paralogue, whereas others have led to the functional diversification of the paralogues. The Duplication-Degenerescence-Complementation model recently proposed by Force et al. seems to be the main process involved in functional diversification after duplication events. This does not exclude acquisition of new functions for one or both paralogues after duplication. The acquisition of such new functions principally involves the evolution of cis-regulatory sequences, but evolution of the coding sequence has also been revealed. However, in all engrailed duplications studied, even in ancient chromosomal duplications, the paralogues have kept redundant functions. In fact, selection seems to maintain a certain redundancy between engrailed paralogues.

Sexually dimorphic traits are often subject to diversifying selection. Genes with a male-biased gene expression also are probably affected by sexual selection and have a high rate of protein evolution. We used SAGE to measure sex-biased gene expression in Drosophila pseudoobscura. Consistent with previous results from D. melanogaster, a larger number of genes were male biased (402 genes) than female biased (138 genes). About 34% of the genes changed the sex-related expression pattern between D. melanogaster and D. pseudoobscura. Combining gene expression with protein divergence between both species, we observed a striking difference in the rate of evolution for genes with a male-biased gene expression in one species only. Contrary to expectations, D. pseudoobscura genes in this category showed no accelerated rate of protein evolution, while D. melanogaster genes did. If sexual selection is driving molecular evolution of male-biased genes, our data imply a radically different selection regime in D. pseudoobscura.

Chromodomains are found in many regulators of chromatin structure, and most of them recognize methylated lysines on histones. Here, we investigate the role of the Drosophila melanogaster protein Corto's chromodomain. The Enhancer of Trithorax and Polycomb Corto is involved in both silencing and activation of gene expression. Over-expression of the Corto chromodomain (CortoCD) in transgenic flies shows that it is a chromatin-targeting module, critical for Corto function. Unexpectedly, mass spectrometry analysis reveals that polypeptides pulled down by CortoCD from nuclear extracts correspond to ribosomal proteins. Furthermore, real-time interaction analyses demonstrate that CortoCD binds with high affinity RPL12 tri-methylated on lysine 3. Corto and RPL12 co-localize with active epigenetic marks on polytene chromosomes, suggesting that both are involved in fine-tuning transcription of genes in open chromatin. RNA–seq based transcriptomes of wing imaginal discs over-expressing either CortoCD or RPL12 reveal that both factors deregulate large sets of common genes, which are enriched in heat-response and ribosomal protein genes, suggesting that they could be implicated in dynamic coordination of ribosome biogenesis. Chromatin immunoprecipitation experiments show that Corto and RPL12 bind hsp70 and are similarly recruited on gene body after heat shock. Hence, Corto and RPL12 could be involved together in regulation of gene transcription. We discuss whether pseudo-ribosomal complexes composed of various ribosomal proteins might participate in regulation of gene expression in connection with chromatin regulators.

Drosophila body pigmentation has emerged as a major Evo-Devo model. Using two Drosophila melanogaster lines, Dark and Pale, selected from a natural population, we analyse here the interaction between genetic variation and environmental factors to produce this complex trait. Indeed, pigmentation varies with genotype in natural populations and is sensitive to temperature during development. We demonstrate that the bric à brac (bab) genes, that are differentially expressed between the two lines and whose expression levels vary with temperature, participate in the pigmentation difference between the Dark and Pale lines. The two lines differ in a bab regulatory sequence, the dimorphic element (called here bDE). Both bDE alleles are temperature-sensitive, but the activity of the bDE allele from the Dark line is lower than that of the bDE allele from the Pale line. Our results suggest that this difference could partly be due to differential regulation by AbdB. bab has been previously reported to be a repressor of abdominal pigmentation. We show here that one of its targets in this process is the pigmentation gene tan (t), regulated via the tan abdominal enhancer (t_MSE). Furthermore, t expression is strongly modulated by temperature in the two lines. Thus, temperature sensitivity of t expression is at least partly a consequence of bab thermal transcriptional plasticity. We therefore propose that a gene regulatory network integrating both genetic variation and temperature sensitivity modulates female abdominal pigmentation. Interestingly, both bDE and t_MSE were previously shown to have been recurrently involved in abdominal pigmentation evolution in drosophilids. We propose that the environmental sensitivity of these enhancers has turned them into evolutionary hotspots.