Dr Robert Maeda

has been a pioneering model system for investigations into the genetic bases of behaviour. Studies of circadian activity were some of the first behaviours investigated in flies. The Activity Monitoring (DAM) system by TriKinetics played a key role in establishing the fundamental feedback loop of the circadian clock. Although this method has many times proven to be extremely useful, it suffers from its simplification of activity to the interruption of an infrared (IR) beam. It is blind to fly movements not disrupting the beam and any modifications to this assay to achieve better resolution often requires the purchase of new and expensive modules. We required a relatively high-throughput system to explore the potential post-mating activity changes of larger species. Rather than investing in a larger and more complex DAM system, we designed a new monitoring system that is more versatile, economic and sensitive than DAM. This new system, called DrosoVAM ( Video-assisted Activity Monitoring), is simple to implement and cost efficient, using a Raspberry Pi-controlled IR, digital video system to record multiple chambers and Python scripts that drive the deep learning software DeepLabCut, to track fly activity over multiple days.

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.

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.

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.

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.

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.

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.

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.

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.

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.