Nous nous intéressons au développement, à la fonction et à l'évolution des processus de reproduction chez les insectes. Une grande partie de ce travail découle de nos travaux sur un long ARN "non codant" exprimé à l'intérieur du complexe de gènes homéotiques bithorax de la Drosophila melanogaster, qui contrôle le développement d'un type particulier de cellules de la glande accessoire mâle productrice de liquide séminal, les cellules secondaires. Chez de nombreux insectes, comme les mouches et les moustiques, on sait que le liquide séminal mâle joue un rôle essentiel dans l'apparition de changements physiologiques et comportementaux chez les femelles accouplées, connus collectivement sous le nom de réponse post-accouplement de la femelle. Grâce à nos travaux et à ceux d'autres chercheurs, nous savons maintenant que les produits des cellules secondaires sont essentiels pour prolonger la réponse post-accouplement au-delà de 24 heures chez la D. melanogaster. L'examen des cellules secondaires et des profils d'expression génique d'autres espèces de drosophiles nous a amenés à conclure que ce processus n'est pas universellement conservé, même parmi les espèces de drosophiles. En fait, il semble que cette fonction ait été récemment acquise dans le sous-groupe Sophophora des Drosophilidés. Nous étudions actuellement d'autres fonctions des cellules secondaires chez d'autres espèces afin de mieux comprendre la biologie des cellules secondaires et pourquoi elles ont été conservées chez la plupart des espèces de drosophiles.
Développement et reproduction des insectes
Subunit of Neuroscience du comportement & neurodégénerescence
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Repression of the Hox gene abd-A by ELAV-mediated Transcriptional Interference.
résumé
Intergenic transcription is a common feature of eukaryotic genomes and performs important and diverse cellular functions. Here, we investigate the iab-8 ncRNA from the Drosophila Bithorax Complex and show that this RNA is able to repress the transcription of genes located at its 3' end by a sequence-independent, transcriptional interference mechanism. Although this RNA is expressed in the early epidermis and CNS, we find that its repressive activity is limited to the CNS, where, in wild-type embryos, it acts on the Hox gene, abd-A, located immediately downstream of it. The CNS specificity is achieved through a 3' extension of the transcript, mediated by the neuronal-specific, RNA-binding protein, ELAV. Loss of ELAV activity eliminates the 3' extension and results in the ectopic activation of abd-A. Thus, a tissue-specific change in the length of a ncRNA is used to generate a precise pattern of gene expression in a higher eukaryote.
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Identification of a micropeptide and multiple secondary cell genes that modulate male reproductive success.
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Even in well-characterized genomes, many transcripts are considered noncoding RNAs (ncRNAs) simply due to the absence of large open reading frames (ORFs). However, it is now becoming clear that many small ORFs (smORFs) produce peptides with important biological functions. In the process of characterizing the ribosome-bound transcriptome of an important cell type of the seminal fluid-producing accessory gland of , we detected an RNA, previously thought to be noncoding, called (). Notably, is nested in the HOX gene cluster of the Bithorax complex and is known to contain a micro-RNA within one of its introns. We find that this RNA encodes a "micropeptide" (9 or 20 amino acids, MSAmiP) that is expressed exclusively in the secondary cells of the male accessory gland, where it seems to accumulate in nuclei. Importantly, loss of function of this micropeptide causes defects in sperm competition. In addition to bringing insights into the biology of a rare cell type, this work underlines the importance of small peptides, a class of molecules that is now emerging as important actors in complex biological processes.
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A FACS-based Protocol to Isolate RNA from the Secondary Cells of Drosophila Male Accessory Glands.
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To understand the function of an organ, it is often useful to understand the role of its constituent cell populations. Unfortunately, the rarity of individual cell populations often makes it difficult to obtain enough material for molecular studies. For example, the accessory gland of the Drosophila male reproductive system contains two distinct secretory cell types. The main cells make up 96% of the secretory cells of the gland, while the secondary cells (SC) make up the remaining 4% of cells (about 80 cells per male). Although both cell types produce important components of the seminal fluid, only a few genes are known to be specific to the SCs. The rarity of SCs has, thus far, hindered transcriptomic analysis study of this important cell type. Here, a method is presented that allows for the purification of SCs for RNA extraction and sequencing. The protocol consists in first dissecting glands from flies expressing a SC-specific GFP reporter and then subjecting these glands to protease digestion and mechanical dissociation to obtain individual cells. Following these steps, individual, living, GFP-marked cells are sorted using a fluorescent activated cell sorter (FACS) for RNA purification. This procedure yields SC-specific RNAs from ~40 males per condition for downstream RT-qPCR and/or RNA sequencing in the course of one day. The rapidity and simplicity of the procedure allows for the transcriptomes of many different flies, from different genotypes or environmental conditions, to be determined in a short period of time.
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Rab-mediated trafficking in the secondary cells of Drosophila male accessory glands and its role in fecundity.
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The male seminal fluid contains factors that affect female post-mating behavior and physiology. In Drosophila, most of these factors are secreted by the two epithelial cell types that make up the male accessory gland: the main and secondary cells. Although secondary cells represent only ~4% of the cells of the accessory gland, their contribution to the male seminal fluid is essential for sustaining the female post-mating response. To better understand the function of the secondary cells, we investigated their molecular organization, particularly with respect to the intracellular membrane transport machinery. We determined that large vacuole-like structures found in the secondary cells are trafficking hubs labeled by Rab6, 7, 11 and 19. Furthermore, these organelles require Rab6 for their formation and many are essential in the process of creating the long-term post-mating behavior of females. In order to better serve the intracellular membrane and protein trafficking communities, we have created a searchable, online, open-access imaging resource to display our complete findings regarding Rab localization in the accessory gland. This article is protected by copyright. All rights reserved.
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Using a phiC31 "Disintegrase" to make new attP sites in the Drosophila genome at locations showing chromosomal position effects.
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An engineered phiC31 "Disintegrase" able to make an attP site in Drosophila out of an attR-attL pair is described. This was used to generate attP sites at genomic locations where a mini-white (mini-w) transgene was subject to chromosomal position effects (CPE). The first step was random genomic integration of a P-element-based transposon with an insulated mini-w transgene. We then removed the upstream insulator using FLP recombinase to detect CPE. Next mini-w and the downstream insulator were "dis-integrated" leaving behind an attP site. The location is marked by a yellow+ transgene that is flanked by loxP sites, so it can also be removed. Using this system, we generated 10 new attP landing platforms. Three of these showing strong activating CPE were selected for further analysis. We show that the attP sites are functional by integrating in plasmids with attB sites. The CPE is recapitulated and can be blocked by insulators. We show that a dimerized 215 bp fragment of the 500 bp BEAF-dependent scs' insulator containing a high affinity BEAF binding site blocks the CPE, while a monomer of the sequence is less effective. This indicates that two BEAF binding sites make a stronger insulator than a single site. This system could be useful for generating attP sites at prescreened sites for other purposes, such as studying CPE in embryos or other tissues or for use with "trapped" enhancers of interest.
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The BEN Domain Protein Insensitive Binds to the Fab-7 Chromatin Boundary To Establish Proper Segmental Identity in Drosophila
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Boundaries (insulators) in the bithorax complex (BX-C) delimit autonomous regulatory domains that orchestrate the parasegment (PS)-specific expression of the BX-C homeotic genes. The boundary separates the and regulatory domains, which control Abd-B expression in PS11 and PS12, respectively. This boundary is composed of multiple functionally redundant elements and has two key functions: it blocks crosstalk between and and facilitates boundary bypass. Here, we show that two BEN domain protein complexes, Insensitive and Elba, bind to multiple sequences located in the nuclease hypersensitive regions. Two of these sequences are recognized by both Insv and Elba and correspond to a CCAATTGG palindrome. Elba also binds to a related CCAATAAG sequence, while Insv does not. However, the third Insv recognition sequences is ~100 bp in length and contains the CCAATAAG sequence at one end. Both Insv and Elba are assembled into large complexes (~420 kD and ~265-290 kD, respectively) in nuclear extracts. Using a sensitized genetic background we show that the Insv protein is required for boundary function, and that PS11 identity is not properly established in mutants. This is the first demonstration that a BEN domain protein is important for the functioning of an endogenous fly boundary.
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The lncRNA male-specific abdominal plays a critical role in Drosophila accessory gland development and male fertility.
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Although thousands of long non-coding RNAs (lncRNA) have been identified in the genomes of higher eukaryotes, the precise function of most of them is still unclear. Here, we show that a >65 kb, male-specific, lncRNA, called male-specific abdominal (msa) is required for the development of the secondary cells of the Drosophila male accessory gland (AG). msa is transcribed from within the Drosophila bithorax complex and shares much of its sequence with another lncRNA, the iab-8 lncRNA, which is involved in the development of the central nervous system (CNS). Both lncRNAs perform much of their functions via a shared miRNA embedded within their sequences. Loss of msa, or of the miRNA it contains, causes defects in secondary cell morphology and reduces male fertility. Although both lncRNAs express the same miRNA, the phenotype in the secondary cells and the CNS seem to reflect misregulation of different targets in the two tissues.
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The Female Post-Mating Response Requires Genes Expressed in the Secondary Cells of the Male Accessory Gland in Drosophila melanogaster.
Genetics 2016 Mar;202(3):1029-41. genetics.115.181644. 10.1534/genetics.115.181644. PMC4788108. 10-01-2016résumé
Seminal proteins from the Drosophila male accessory gland induce post-mating responses (PMR) in females. The PMR comprise behavioral and physiological changes that include increased egg laying, decreased receptivity to courting males, and changes in the storage and use of sperm. Many of these changes are induced by a "sex peptide" (SP) and are maintained by SP's binding to, and slow release from, sperm. The accessory gland contains two secretory cell types with distinct morphological and developmental characteristics. Products of these "main" and "secondary" cells work interdependently to induce and maintain the PMR. To identify individual genes needed for the morphology and function of secondary cells, we studied iab-6(cocu) males, whose secondary cells have abnormal morphology and fail to provide products to maintain the PMR. By RNA-seq, we identified 77 genes that are downregulated by a factor of >5× in iab-6(cocu) males. By functional assays and microscopy, we tested 20 candidate genes and found that at least 9 are required for normal storage and release of SP in mated females. Knockdown of each of these 9 genes consequently leads to a reduction in egg laying and an increase in receptivity over time, confirming a role for the secondary cells in maintaining the long-term PMR. Interestingly, only 1 of the 9 genes, CG3349, encodes a previously reported seminal fluid protein (Sfp), suggesting that secondary cells may perform essential functions beyond the production and modification of known Sfps. At least 3 of the 9 genes also regulate the size and/or abundance of secondary cell vacuoles, suggesting that the vacuoles' contents may be important for the machinery used to maintain the PMR.
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Following the intracellular localization of the iab-8ncRNA of the bithorax complex using the MS2-MCP-GFP system.
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Homeotic genes are aligned on the chromosome in the order of the segments that they specify along the antero-posterior axis of the fly. In general the genes affecting the more posterior segments repress the more anterior genes, a phenomenon known as "posterior dominance". There is however a noticeable exception to this rule in the central nervous system of Drosophila melanogaster where the posterior Abd-B gene does not repress the immediately more anterior abd-A gene. Instead, abd-A repression is accomplished by a 92 kb-long ncRNA (the iab-8ncRNA) that is transcribed from the large inter-genic region between abd-A and Abd-B. This iab-8ncRNA encodes a microRNA to repress abd-A and also a second redundant repression mechanism acting in cis and thought to be transcriptional interference with the abd-A promoter. Using in situ hybridization, a previous work suggested that the iab8ncRNA transcript forms discrete foci restricted to the nuclear periphery and that this localization may be important for its function. In order to better characterize the intra-cellular localization of the iab-8ncRNA we used the MS2-MCP system, which allows fluorescent labeling of RNA in cells and relies on the interaction between GFP-tagged MS2 coat protein (MCP-GFP) and MS2 RNA stem loops. Our results indicate that the large foci seen in previous studies correspond to the site of iab8ncRNA transcription and that the foci seen may simply be an indication of the level of transcription at the locus. We find no evidence to suggest that this localization is important for its function on abd-A repression. We discuss the idea that the iab-8ncRNA may be a relic of a more general ancient mechanism of posterior dominance during the emergence of the hox clusters that was mediated by transcriptional interference.
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The open for business model of the bithorax complex in Drosophila.
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After nearly 30 years of effort, Ed Lewis published his 1978 landmark paper in which he described the analysis of a series of mutations that affect the identity of the segments that form along the anterior-posterior (AP) axis of the fly (Lewis 1978). The mutations behaved in a non-canonical fashion in complementation tests, forming what Ed Lewis called a "pseudo-allelic" series. Because of this, he never thought that the mutations represented segment-specific genes. As all of these mutations were grouped to a particular area of the Drosophila third chromosome, the locus became known of as the bithorax complex (BX-C). One of the key findings of Lewis' article was that it revealed for the first time, to a wide scientific audience, that there was a remarkable correlation between the order of the segment-specific mutations along the chromosome and the order of the segments they affected along the AP axis. In Ed Lewis' eyes, the mutants he discovered affected "segment-specific functions" that were sequentially activated along the chromosome as one moves from anterior to posterior along the body axis (the colinearity concept now cited in elementary biology textbooks). The nature of the "segment-specific functions" started to become clear when the BX-C was cloned through the pioneering chromosomal walk initiated in the mid 1980s by the Hogness and Bender laboratories (Bender et al. 1983a; Karch et al. 1985). Through this molecular biology effort, and along with genetic characterizations performed by Gines Morata's group in Madrid (Sanchez-Herrero et al. 1985) and Robert Whittle's in Sussex (Tiong et al. 1985), it soon became clear that the whole BX-C encoded only three protein-coding genes (Ubx, abd-A, and Abd-B). Later, immunostaining against the Ubx protein hinted that the segment-specific functions could, in fact, be cis-regulatory elements regulating the expression of the three protein-coding genes. In 1987, Peifer, Karch, and Bender proposed a comprehensive model of the functioning of the BX-C, in which the "segment-specific functions" appear as segment-specific enhancers regulating, Ubx, abd-A, or Abd-B (Peifer et al. 1987). Key to their model was that the segmental address of these enhancers was not an inherent ability of the enhancers themselves, but was determined by the chromosomal location in which they lay. In their view, the sequential activation of the segment-specific functions resulted from the sequential opening of chromatin domains along the chromosome as one moves from anterior to posterior. This model soon became known of as the open for business model. While the open for business model is quite easy to visualize at a conceptual level, molecular evidence to validate this model has been missing for almost 30 years. The recent publication describing the outstanding, joint effort from the Bender and Kingston laboratories now provides the missing proof to support this model (Bowman et al. 2014). The purpose of this article is to review the open for business model and take the reader through the genetic arguments that led to its elaboration.
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Hox gene regulation in the central nervous system of Drosophila.
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Hox genes specify the structures that form along the anteroposterior (AP) axis of bilateria. Within the genome, they often form clusters where, remarkably enough, their position within the clusters reflects the relative positions of the structures they specify along the AP axis. This correspondence between genomic organization and gene expression pattern has been conserved through evolution and provides a unique opportunity to study how chromosomal context affects gene regulation. In Drosophila, a general rule, often called "posterior dominance," states that Hox genes specifying more posterior structures repress the expression of more anterior Hox genes. This rule explains the apparent spatial complementarity of Hox gene expression patterns in Drosophila. Here we review a noticeable exception to this rule where the more-posteriorly expressed Abd-B Hox gene fails to repress the more-anterior abd-A gene in cells of the central nervous system (CNS). While Abd-B is required to repress ectopic expression of abd-A in the posterior epidermis, abd-A repression in the posterior CNS is accomplished by a different mechanism that involves a large 92 kb long non-coding RNA (lncRNA) encoded by the intergenic region separating abd-A and Abd-B (the iab8ncRNA). Dissection of this lncRNA revealed that abd-A is repressed by the lncRNA using two redundant mechanisms. The first mechanism is mediated by a microRNA (mir-iab-8) encoded by intronic sequence within the large iab8-ncRNA. Meanwhile, the second mechanism seems to involve transcriptional interference by the long iab-8 ncRNA on the abd-A promoter. Recent work demonstrating CNS-specific regulation of genes by ncRNAs in Drosophila, seem to highlight a potential role for the iab-8-ncRNA in the evolution of the Drosophila Hox complexes.
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Gene expression in time and space: additive vs hierarchical organization of cis-regulatory regions.
Curr. Opin. Genet. Dev. 2011 Apr;21(2):187-93. S0959-437X(11)00033-5. 10.1016/j.gde.2011.01.021. 26-02-2011résumé
In higher eukaryotes, individual genes are often intermingled with other genes and spread out across tens to hundreds of kilobases, even though only small portions of their sequence are devoted to protein coding. Yet, in this seemingly extended and tangled mess, the cell is able to precisely regulate gene expression in both time and space. Over the past few decades, numerous elements, like enhancers, silencers and insulators have been found that shed some light on how the precise control of gene expression is achieved. Through these discoveries, an additive model of gene expression was envisioned, where the addition of the patterning details imparted by regulatory elements would create the final pattern of gene expression. Although many genes can be described using this model, recent work in the Drosophila bithorax complex suggests that this model may be somewhat simplistic and, in fact, regulatory elements sometimes seem to communicate with each other to form a functional hierarchy that is far from additive.
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Cis-regulation in the Drosophila Bithorax Complex.
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The discovery of the first homeotic mutation by Calvin Bridges in 1915 profoundly influenced the way we think about developmental processes. Although many mutations modify or deform morphological structures, homeotic mutations cause a spectacular phenotype in which a morphological structure develops like a copy of a structure that is normally found elsewhere on an organism's body plan. This is best illustrated in Drosophila where homeotic mutations were first discovered. For example, Antennapedia mutants have legs developing on their head instead of antennae. Because a mutation in a single gene creates such complete structures, homeotic genes were proposed to be key "selector genes" regulating the initiation of a developmental program. According to this model, once a specific developmental program is initiated (i.e., antenna or leg), it can be executed by downstream "realizator genes" independent of its location along the body axis. Consistent with this idea, homeotic genes have been shown to encode transcription factor proteins that control the activity of the many downstream targets to "realize" a developmental program. Here, we will review the first and perhaps, best characterized homeotic complex, the Bithorax Complex (BX-C).
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The bithorax complex of Drosophila an exceptional Hox cluster.
Curr. Top. Dev. Biol. 2009 ;88():1-33. S0070-2153(09)88001-0. 10.1016/S0070-2153(09)88001-0. 05-08-2009résumé
In his 1978 seminal paper, Ed Lewis described a series of mutations that affect the segmental identities of the segments forming the posterior two-thirds of the Drosophila body plan. In each class of mutations, particular segments developed like copies of a more-anterior segment. Genetic mapping of the different classes of mutations led to the discovery that their arrangement along the chromosome paralleled the body segments they affect along the anteroposterior axis of the fly. As all these mutations mapped to the same cytological location, he named this chromosomal locus after its founding mutation. Thus the first homeotic gene (Hox) cluster became known as the bithorax complex (BX-C). Even before the sequencing of the BX-C, the fact that these similar mutations grouped together in a cluster, lead Ed Lewis to propose that the homeotic genes arose through a gene duplication mechanism and that these clusters would be conserved through evolution. With the identification of the homeobox in the early 1980s, Lewis' first prediction was confirmed. The two cloned Drosophila homeotic genes, Antennapedia and Ultrabithorax, were indeed related genes. Using the homeobox as an entry point, homologous genes have since been cloned in many other species. Today, Hox clusters have been discovered in almost all metazoan phyla, confirming Lewis' second prediction. Remarkably, these homologous Hox genes are also arranged in clusters with their order within each cluster reflecting the anterior boundary of their domain of expression along the anterior-posterior axis of the animal. This correlation between the genomic organization and the activity along the anteroposterior body axis is known as the principle of "colinearity." The description of the BX-C inspired decades of developmental and evolutionary biology. And although this first Hox cluster led to the identification of many important features common to all Hox gene clusters, it now turns out that the fly Hox clusters are rather exceptional when compared with the Hox clusters of other animals. In this chapter, we will review the history and salient features of bithorax molecular genetics, in part, emphasizing its unique features relative to the other Hox clusters.
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Making connections: boundaries and insulators in Drosophila.
Curr. Opin. Genet. Dev. 2007 Oct;17(5):394-9. S0959-437X(07)00153-0. 10.1016/j.gde.2007.08.002. 02-10-2007résumé
In eukaryotes, enhancers must often exert their effect over many tens of kilobases of DNA with a choice between many different promoters. Given this situation, elements known as chromatin boundaries have evolved to prevent adventitious interactions between enhancers and promoters. The amenability of Drosophila to molecular genetics has been crucial to the discovery and analysis of these elements. Since these elements are involved in such diverse processes and show little or no sequence similarity between them, no single molecular mechanism has been identified that accounts for their activity. However, over the past approximately 5 years, evidence has accumulated suggesting that boundaries probably function through the formation of long-distance chromatin loops. These loops have been proposed to play a crucial role in both controlling enhancer-promoter interactions and packing DNA.
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Dissecting the regulatory landscape of the Abd-B gene of the bithorax complex.
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The three homeotic genes of the bithorax complex (BX-C), Ubx, abd-A and Abd-B control the identity of the posterior thorax and all abdominal segments. Large segment-specific cis-regulatory regions control the expression of Ubx, abd-A or Abd-B in each of the segments. These segment-specific cis-regulatory regions span the whole 300 kb of the BX-C and are arranged on the chromosome in the same order as the segments they specify. Experiments with lacZ reporter constructs revealed the existence of several types of regulatory elements in each of the cis-regulatory regions. These include initiation elements, maintenance elements, cell type- or tissue-specific enhancers, chromatin insulators and the promoter targeting sequence. In this paper, we extend the analysis of regulatory elements within the BX-C by describing a series of internal deficiencies that affect the Abd-B regulatory region. Many of the elements uncovered by these deficiencies are further verified in transgenic reporter assays. Our results highlight four key features of the iab-5, iab-6 and iab-7 cis-regulatory region of Abd-B. First, the whole Abd-B region is modular by nature and can be divided into discrete functional domains. Second, each domain seems to control specifically the level of Abd-B expression in only one parasegment. Third, each domain is itself modular and made up of a similar set of definable regulatory elements. And finally, the activity of each domain is absolutely dependent on the presence of an initiator element.
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The ABC of the BX-C: the bithorax complex explained.
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As one of two Drosophila Hox clusters, the bithorax complex (BX-C) is responsible for determining the posterior thorax and each abdominal segment of the fly. Through the dissection of its large cis-regulatory region, biologists have obtained a wealth of knowledge that has informed our understanding of gene expression, chromatin dynamics and gene evolution. This primer attempts to distill and explain our current knowledge about this classic, complex locus.
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