Jean-Michel Gibert

Insect pigmentation is an ecologically critical trait influencing many physiological processes. In Drosophila melanogaster , abdominal pigmentation is sexually dimorphic: males have fully pigmented posterior segments, while females exhibit a posterior melanin stripe. Pigmentation relies on the expression of pigmentation genes that encode enzymes involved in pigment synthesis. These genes are tightly regulated during pupal and young adult stages. To expand the gene regulatory network of pigmentation genes, we conducted an RNAi screen using the yellow-Gal4 driver, expressed during the pupal stage in abdominal epidermis. One of the candidates from this screen, Kdm5 , encodes a histone demethylase erasing the H3K4me3 histone mark catalyzed by the histone methyl-transferase Trithorax (Trx). We show that Kdm5 down-regulation reduces abdominal pigmentation, mimicking trx down-regulation. Kdm5 activates melanin production through regulation of the pigmentation gene tan . Transcriptomic analyses reveal that Kdm5 and Trx share many targets in pupal abdominal epidermis, including piRNA pathway components such as piwi and aubergine . These piRNA components, originally associated with transposon silencing in the germline, also function in some somatic tissues such as the nervous system, the fat body or the gut. We demonstrate that Piwi and Aubergine participate in female abdominal pigmentation establishment, without evident piRNA production. We also show that Kdm5 and Piwi act not only in pupal abdominal epidermis but also in pupal fat body. This study therefore expands the regulatory network of pigmentation genes. It identifies a new somatic function for Kdm5 and Piwi and reveals a role for pupal fat body in female abdominal pigmentation regulation. Article summary: We investigated Drosophila female abdominal pigmentation to enrich the regulatory network of pigmentation genes. Using a Gal4 driver expressed in abdominal epidermis, we conducted an RNAi screen. We discovered that the histone demethylase Kdm5 activates the pigmentation gene tan . Using transcriptomic analysis and genetics experiments, we showed that the piRNA genes piwi and aub act downstream of Kdm5 to regulate tan expression, without evident piRNA production. Moreover, Kdm5 and piwi act not only in the pupal epidermis but also in the pupal fat body, thus showing for the first time a role of this tissue in abdominal pigmentation regulation.

Despite long-lasting interest and research efforts, the genetic bases of developmental stability—the robustness to developmental noise—and its commonly used estimator, fluctuating asymmetry (FA), remain poorly understood. The Drosophila melanogaster Cyclin G gene (CycG) encodes a transcriptional cyclin that regulates growth and the cell cycle. Over-expression of a potentially more stable isoform of the protein (deletion of a PEST-rich domain, hereafter called CycGΔP) induces extreme wing size and shape FA (i.e. high developmental noise), indicating a major disruption of developmental stability. Previous attempts to identify the genetic bases of FA have been impeded by the constitutively low level of developmental noise, limiting the power to detect any effect. Here, we leverage the extreme developmental instability induced by overexpression of CycGΔP to explore the genetic bases of FA: we perform a genome-wide screen for deficiencies that enhance or reduce CycGΔP-induced wing FA. 495 deficiencies uncovering 90% of the euchromatic genome were combined with a recombinant chromosome expressing CycGΔP. We identified 13 and 16 deficiencies that respectively enhance and decrease FA. Analysis of mutants for some genes located in these deficiencies shows that Cyclin G ensures homogeneous growth of organs in synergy with the major morphogens of the wing, Dpp and Wg, as well as the Hippo and InR/TOR pathways. They also reveal that CycGΔP-induced FA involves Larp, a potential direct interactor of Cyclin G that regulates translation at the mitochondrial membrane. This opens up new research perspectives for understanding developmental stability, suggesting a significant role for mitochondrial activity.

How developing organisms respond to a changing environment is a fundamental question. Pollutants and temperature are major environmental factors. Using the bristle patterning of Drosophila as a model system, we observed that cold temperature and methotrexate, a medical drug that contaminates wastewaters, increase dorsocentral (DC) bristle number, a trait normally robust. The patterning of bristles is well understood and involves the achaete-scute (ac-sc) proneural genes. Modular enhancers activate ac-sc expression in groups of cells, called proneural clusters, from which bristle precursors are selected by lateral inhibition, a process involving Notch signalling and ac-sc auto-activation. In addition, ac-sc basal expression is controlled by a cocktail of repressive factors. We observed that the deletion of the DC enhancer prevents the induction of ectopic DC bristles by methotrexate but does not stop low temperature to induce DC bristles. Indeed, we show that methotrexate has a strong synergy with mutants of factors that regulate the DC enhancer and extends the zone of activity of this enhancer. In contrast, temperature interacts with repressors of ac-sc basal expression. Thus, methotrexate and temperature both affect DC bristle patterning but by distinct mechanisms, methotrexate on the DC enhancer and cold independently of this enhancer.

Despite long-lasting interest and research efforts, the genetic bases of developmental stability the robustness to developmental noise and its most commonly used estimator, fluctuating asymmetry (FA), remain poorly understood. The Drosophila melanogaster Cyclin G gene ( CycG ) encodes a transcriptional cyclin that regulates growth and the cell cycle. Over-expression of a potentially more stable isoform of the protein (deleted of a PEST-rich domain, hereafter called CycG ^P ) induces extreme wing size and shape FA ( i.e. high developmental noise), indicating a major disruption of developmental stability. Previous attempts to identify the genetic bases of FA have been impeded by the constitutively low level of developmental noise, strongly limiting the power to detect any effect. Here, we leverage the extreme developmental instability induced by overexpression of CycG ^P to explore the genetic bases of FA: we perform a genome-wide screen for deficiencies that enhance or reduce CycG ^P -induced wing FA. 499 deficiencies uncovering 90% of the euchromatic genome were combined with a recombinant chromosome expressing CycG ^P . We identified 13 and 16 deficiencies that enhance and decrease FA, respectively. Analysis of mutants for some genes located in these deficiencies shows that Cyclin G ensures homogeneous growth of organs in synergy with the major morphogens of the wing, Dpp and Wg, as well as the Hippo and InR/TOR pathways. They also reveal that CycG ^P -induced FA involves Larp, a potential direct interactor of Cyclin G, that regulates translation at the mitochondrial membrane. This opens up new research perspectives for understanding developmental stability, suggesting a significant role for mitochondrial activity.

Temperature sensitivity of abdominal pigmentation in Drosophila melanogaster females allows to investigate the mechanisms underlying phenotypic plasticity. Thermal plasticity of pigmentation is due to modulation of tan and yellow expression, encoding pigmentation enzymes. Furthermore, modulation of tan expression by temperature is correlated to the variation of the active histone mark H3K4me3 on its promoter. Here, we test the role of the DotCom complex, which methylates H3K79, another active mark, in establishment and plasticity of pigmentation. We show that several components of the DotCom complex are involved in the establishment of abdominal pigmentation. In particular, Grappa, the catalytic unit of this complex, plays opposite roles on pigmentation at distinct developmental stages. Indeed, its down-regulation from larval L2 to L3 stages increases female adult pigmentation, whereas its down-regulation during the second half of the pupal stage decreases adult pigmentation. These opposite effects are correlated to the regulation of distinct pigmentation genes by Grappa: yellow repression for the early role and tan activation for the late one. Lastly, reaction norms measuring pigmentation along temperature in mutants for subunits of the DotCom complex reveal that this complex is not only involved in the establishment of female abdominal pigmentation but also in its plasticity.

Cuticle pigmentation was shown to be associated with body temperature for several relatively large species of insects, but it was questioned for small insects. Here we used a thermal camera to assess the association between drosophilid cuticle pigmentation and body temperature increase when individuals are exposed to light. We compared mutants of large effects within species (Drosophila melanogaster ebony and yellow mutants). Then we analyzed the impact of naturally occurring pigmentation variation within species complexes (Drosophila americana/Drosophila novamexicana and Drosophila yakuba/Drosophila santomea). Finally we analyzed lines of D. melanogaster with moderate differences in pigmentation. We found significant differences in temperatures for each of the four pairs we analyzed. The temperature differences appeared to be proportional to the differently pigmented area: between Drosophila melanogaster ebony and yellow mutants or between Drosophila americana and Drosophila novamexicana, for which the whole body is differently pigmented, the temperature difference was around 0.6 °C ± 0.2 °C. By contrast, between D. yakuba and D. santomea or between Drosophila melanogaster Dark and Pale lines, for which only the posterior abdomen is differentially pigmented, we detected a temperature difference of about 0.14 °C ± 0.10 °C. This strongly suggests that cuticle pigmentation has ecological implications in drosophilids regarding adaptation to environmental temperature.

Cuticle pigmentation has been clearly demonstrated to impact body temperature for several relatively large species of insects, but it was questioned for small insects. Here we used a thermal camera to assess the impact of drosophilid cuticle pigmentation on body temperature when individuals are exposed to light. We compared mutants of large effects within species ( Drosophila melanogaster ebony and yellow mutants). Then we analyzed the impact of naturally occurring pigmentation variation within species complexes ( Drosophila americana/Drosophila novamexicana and Drosophila yakuba/Drosophila santomea ). Finally we analyzed lines of D. melanogaster with moderate differences in pigmentation. We found significant differences in temperatures for each of the four pairs we analyzed. The temperature differences appeared to be proportional to the differently pigmented area: between Drosophila melanogaster ebony and yellow mutants or between Drosophila americana and Drosophila novamexicana , for which the whole body is differently pigmented, the difference in temperatures was around 0.6°C ±0.2°C. By contrast, between D. yakuba and D. santomea or between Drosophila melanogaster Dark and Pale lines, for which only the posterior abdomen is differentially pigmented, we detected a temperature difference of about 0.14°C ±0.10°C. This demonstrates that cuticle pigmentation has ecological implications in drosophilids regarding adaptation to environmental temperature.

L’évolution à petite échelle ou microévolution concerne l’évolution au niveau intra-spécifique ou entre espèces proches. Au niveau intra-spécifique, elle permet d’analyser les forces évolutives en action : mutation, dérive génétique, migration et sélection. De plus, en raison de ce temps évolutif court, il est plus facile d’identifier les bases génétiques des différences phénotypiques observées. La plupart des études porte sur des populations actuelles mais de plus en plus de travaux analysent l’ADN ancien. Ces derniers apportent non seulement des informations importantes pour retracer l’histoire des populations mais permettent également de reconstituer les phénotypes d’individus disparus depuis plusieurs milliers d’années. Dans cette courte revue, je présente des travaux montrant comment se mettent en place des barrières pré-zygotiques ou post-zygotiques impliquées dans la formation d’espèces, avec l’exemple de la barrière géographique due à la formation de l’isthme de Panama et celui de la divergence de l’hétérochromatine chez les drosophilidés. Par ailleurs, à propos de cas bien établis, je décris les différentes approches qui ont été utilisées pour identifier les bases génétiques de variations phénotypiques : approche gène-candidat pour ce qui concerne le mélanisme chez les félins, cartographie QTL (Quantitative trait loci) pour la variation du nombre de plaques osseuses latérales chez les épinoches, étude d’association pour la pigmentation chez la coccinelle asiatique. Enfin, j’illustre le rôle de la sélection naturelle avec l’exemple iconique de l’évolution du bec des pinsons des Galapagos et l’implication de certains gènes du développement dans sa diversification morphologique.

Drosophila melanogaster has played a paramount role in epigenetics, the study of changes in gene function inherited through mitosis or meiosis that are not due to changes in the DNA sequence. By analyzing simple phenotypes, such as the bristle position or cuticle pigmentation, as read-outs of regulatory processes, the identification of mutated genes led to the discovery of major chromatin regulators. These are often conserved in distantly related organisms such as vertebrates or even plants. Many of them deposit, recognize, or erase post-translational modifications on histones (histone marks). Others are members of chromatin remodeling complexes that move, eject, or exchange nucleosomes. We review the role of D. melanogaster research in three epigenetic fields: Heterochromatin formation and maintenance, the repression of transposable elements by piRNAs, and the regulation of gene expression by the antagonistic Polycomb and Trithorax complexes. We then describe how genetic tools available in D. melanogaster allowed to examine the role of histone marks and show that some histone marks are dispensable for gene regulation, whereas others play essential roles. Next, we describe how D. melanogaster has been particularly important in defining chromatin types, higher-order chromatin structures, and their dynamic changes during development. Lastly, we discuss the role of epigenetics in a changing environment.