Podcasts about drosophila melanogaster

BUFFALO, NY- June 19, 2024 – A new research paper was published in Aging (listed by MEDLINE/PubMed as "Aging (Albany NY)" and "Aging-US" by Web of Science) Volume 16, Issue 11, entitled, “Dietary sucrose determines the regulatory activity of lithium on gene expression and lifespan in Drosophila melanogaster.” The amount of dietary sugars and the administration of lithium both impact the lifespan of the fruit fly Drosophila melanogaster. It is noteworthy that lithium is attributed with insulin-like activity as it stimulates protein kinase B/Akt and suppresses the activity of glycogen synthase kinase-3 (GSK-3). However, its interaction with dietary sugar has largely remained unexplored. In this new study, researchers Katharina Jans, Kai Lüersen, Jakob von Frieling, Thomas Roeder, and Gerald Rimbach from the University of Kiel investigated the effects of lithium supplementation on known lithium-sensitive parameters in fruit flies, such as lifespan, body composition, GSK-3 phosphorylation, and the transcriptome, while varying the dietary sugar concentration. “Based on this proposed overlapping bioactivity of dietary sugar and lithium in the female fruit fly, we decided to investigate the extent of these similarities and whether a joint mechanism lies at their root.” For all these parameters, the researchers observed that the efficacy of lithium was significantly influenced by the sucrose content in the diet. Overall, they found that lithium was most effective in enhancing longevity and altering body composition when added to a low-sucrose diet. Whole-body RNA sequencing revealed a remarkably similar transcriptional response when either increasing dietary sucrose from 1% to 10% or adding 1 mM LiCl to a 1% sucrose diet, characterized by a substantial overlap of nearly 500 differentially expressed genes. “Hence, dietary sugar supply is suggested as a key factor in understanding lithium bioactivity, which could hold relevance for its therapeutic applications.” DOI - https://doi.org/10.18632/aging.205933 Corresponding author - Katharina Jans - jans@foodsci.uni-kiel.de Video short - https://www.youtube.com/watch?v=HaxZU4Gd5Z4 Sign up for free Altmetric alerts about this article - https://aging.altmetric.com/details/email_updates?id=10.18632%2Faging.205933 Subscribe for free publication alerts from Aging - https://www.aging-us.com/subscribe-to-toc-alerts Keywords - aging, lithium, longevity, glycogen synthase kinase 3, fruit fly, trace element About Aging (Aging-US): Aging aims to promote 1) treatment of age-related diseases by slowing down aging, 2) validation of anti-aging drugs by treating age-related diseases, and 3) prevention of cancer by inhibiting aging. (Cancer and COVID-19 are age-related diseases.) Aging is indexed by PubMed/Medline (abbreviated as “Aging (Albany NY)”), PubMed Central, Web of Science: Science Citation Index Expanded (abbreviated as “Aging‐US” and listed in the Cell Biology and Geriatrics & Gerontology categories), Scopus (abbreviated as “Aging” and listed in the Cell Biology and Aging categories), Biological Abstracts, BIOSIS Previews, EMBASE, META (Chan Zuckerberg Initiative) (2018-2022), and Dimensions (Digital Science). Please visit our website at https://www.Aging-US.com​​ and connect with us: Facebook - https://www.facebook.com/AgingUS/ X - https://twitter.com/AgingJrnl Instagram - https://www.instagram.com/agingjrnl/ YouTube - https://www.youtube.com/@AgingJournal LinkedIn - https://www.linkedin.com/company/aging/ Pinterest - https://www.pinterest.com/AgingUS/ Spotify - https://open.spotify.com/show/1X4HQQgegjReaf6Mozn6Mc MEDIA@IMPACTJOURNALS.COM

As sailors use constellations, wind direction, and current to determine their heading, so, too, do animals process diverse sensory information to set their course. Via this sensory processing, the animal's brain develops a sense of direction, a prerequisite for navigating between points. To understand how the sense of direction is generated in the brain, we interrogate neurons in the brain of the fruit fly, Drosophila melanogaster. With numerous tools that allow observing the neural network structures and activities and perturbing them, we begin to understand how the brain transforms sensory information into a sense of direction. Series: "GRIT Talks" [Science] [Show ID: 39332]

As sailors use constellations, wind direction, and current to determine their heading, so, too, do animals process diverse sensory information to set their course. Via this sensory processing, the animal's brain develops a sense of direction, a prerequisite for navigating between points. To understand how the sense of direction is generated in the brain, we interrogate neurons in the brain of the fruit fly, Drosophila melanogaster. With numerous tools that allow observing the neural network structures and activities and perturbing them, we begin to understand how the brain transforms sensory information into a sense of direction. Series: "GRIT Talks" [Science] [Show ID: 39332]

As sailors use constellations, wind direction, and current to determine their heading, so, too, do animals process diverse sensory information to set their course. Via this sensory processing, the animal's brain develops a sense of direction, a prerequisite for navigating between points. To understand how the sense of direction is generated in the brain, we interrogate neurons in the brain of the fruit fly, Drosophila melanogaster. With numerous tools that allow observing the neural network structures and activities and perturbing them, we begin to understand how the brain transforms sensory information into a sense of direction. Series: "GRIT Talks" [Science] [Show ID: 39332]

As sailors use constellations, wind direction, and current to determine their heading, so, too, do animals process diverse sensory information to set their course. Via this sensory processing, the animal's brain develops a sense of direction, a prerequisite for navigating between points. To understand how the sense of direction is generated in the brain, we interrogate neurons in the brain of the fruit fly, Drosophila melanogaster. With numerous tools that allow observing the neural network structures and activities and perturbing them, we begin to understand how the brain transforms sensory information into a sense of direction. Series: "GRIT Talks" [Science] [Show ID: 39332]

Dr Erica McAlister uncovers a treasure trove of remarkable insights from the insect world including the innocuous flies that are Drosophila melanogaster. More is known about these flies than any other animal on the planet, as a model for human genetics. And the hoverfly that arguably undergoes the biggest transformation of any animal and how insect metamorphosis could be a tool to track future climate change.

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.26.550642v1?rss=1 Authors: Fabian, B., Grabe, V., Beutel, R. G., Hansson, B. S., Sachse, S. Abstract: Drosophila melanogaster encounters a variety of odor cues signaling potentially harmful threats throughout its life, which are detected by highly specialized olfactory circuits enabling the animal to avoid them. We studied whether such crucial neuronal pathways are hard-wired or can be modulated by experience. Using long-term exposure to high concentrations of geosmin, an indicator of potentially lethal microorganisms, we demonstrate at the single-cell level that the underlying neuronal circuitry undergoes structural changes in the antennal lobe, while higher brain centers remain unaffected. In particular, second-order neurons show neurite extensions and synaptic remodeling after the exposure period, whereas olfactory sensory neurons and glia cells remain unaffected. Flies that were exposed to geosmin tolerate this innately aversive odorant in general choice and oviposition assays. We show that even a highly specific olfactory circuit is plastic and adaptable to environmental changes. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.20.549932v1?rss=1 Authors: Deal, S. L., Bei, D., Gibson, S. B., Delgado-Seo, H., Fujita, Y., Wilwayco, K., Seto, E. S., Yamamoto, S. Abstract: The dopaminergic system has been extensively studied for its role in behavior in various animals as well as human neuropsychiatric and neurological diseases. However, we still know little about how dopamine levels are tightly regulated in vivo. In order to identify novel regulators of dopamine levels, we utilized Drosophila melanogaster cuticle pigmentation as a readout, where dopamine is used as a precursor to melanin. First, we measured dopamine from classical mutants of genes that are known to be important for cuticle pigmentation to understand the relationship between dopamine levels and cuticle color. We then performed an RNAi-based screen to identify novel regulators of cuticle pigmentation. We identified 153 genes that were not only enriched for conserved homologs and disease-associated genes, but were unexpectedly enriched for multiple developmental signaling pathways and mitochondria-associated proteins. Upon measuring dopamine from 35 prioritized candidates from this cohort, we found 10 that caused significant reduction in dopamine in the head while one caused an increase. While most of these 11 genes are expressed in the fly brain, only two of them, clu and mask, altered dopamine levels specifically in the brain upon knockdown in dopaminergic cells. Interestingly, mask may act as a hub of dopamine regulation since it is associated with three nodes found in our screen (Receptor tyrosine kinase/EGF signaling, Hippo signaling, and mitochondrial dynamics). Further examination suggests that Mask likely acts on dopamine synthesis by regulating transcription of the rate-limiting dopamine synthesis enzyme, tyrosine hydroxylase. In conclusion, this screen identified 11 new regulators of dopamine levels and provides molecular handles to investigate how dopamine levels are controlled in vivo, as well as revealed an unexpected relationship between fly pigmentation genes, developmental signaling and human neurological and neurodevelopmental disease genes. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.06.26.546511v1?rss=1 Authors: Corthals, K., Andersson, V., Churcher, A. M., Reimegaard, J., Enjin, A. Abstract: The ability of animals to perceive and respond to sensory information is essential for their survival in diverse environments. While much progress has been made in understanding various sensory modalities, the sense of hygrosensation, which involves the detection and response to humidity, remains poorly understood. In this study, we focused on the hygrosensory, and closely related thermosensory, systems in the vinegar fly Drosophila melanogaster to unravel the molecular profile of the cells of these senses. Using a transcriptomic analysis of over 37,000 nuclei, we identified twelve distinct clusters of cells corresponding to temperature-sensing arista neurons, humidity-sensing sacculus neurons, and support cells relating to these neurons. By examining the expression of known and novel marker genes, we validated the identity of these clusters and characterized their gene expression profiles. We found that each cell type could be characterized by a unique expression profile of ion channels, GPCR signaling molecules, synaptic vesicle cycle proteins, and cell adhesion molecules. Our findings provide valuable insights into the molecular basis of hygro- and thermosensation. Understanding the mechanisms underlying hygro- and thermosensation may shed light on the broader understanding of sensory systems and their adaptation to different environmental conditions in animals. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.04.21.537850v1?rss=1 Authors: Qin, J., Yang, T., Li, K., Liu, T., Zhang, W. Abstract: As the first step of food ingestion, the swallow is under rigorous sensorimotor control. Nevertheless, the mechanisms underlying swallow control at a molecular and circuitry level remain largely unknown. Here, we find that mutation of the mechanotransduction channel genes nompC, Tmc, or piezo impairs the regular pumping rhythm of the cibarium during feeding of the fruit fly Drosophila melanogaster. A group of multi-dendritic mechanosensory neurons, which co-express the three channels, wrap the cibarium and are crucial for coordinating the filling and emptying of the cibarium. Inhibition of them causes difficulty in food emptying in the cibarium, while their activation leads to difficulty in cibarium filling. Synaptic and functional connections are detected between the pharyngeal mechanosensory neurons and the motor circuit that controls swallow. This study elucidates the role of mechanosensation in swallow, and provides insights for a better understanding of the neural basis of food swallow. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.03.27.534462v1?rss=1 Authors: Tan, S. L., Hewson, L. J., Mustaffar, N. F., He, Q. Q., Wimmer, N., Trim, P. J., King, B. M., Snel, M. F., Hemsley, K. M., Ferro, V., O'Keefe, L. V., Lau, A. A. Abstract: Sanfilippo syndrome, or mucopolysaccharidosis (MPS) types A, B, C or D, are neurodegenerative lysosomal storage disorders resulting from the lack of a specific enzyme involved in heparan sulfate (HS) catabolism. Several treatments are under evaluation for these conditions including substrate reduction therapy, with the most studied compound of this class being the isoflavone genistein. However, recent outcomes from a Phase III clinical trial have shown that high dose oral genistein does not significantly improve neurodevelopmental outcomes in MPS III patients. Here, we have tested an N-acetylglucosamine (GlcNAc) analogue inhibitor, 4-deoxy-GlcNAc peracetate, at reducing HS accumulation in cells from patients with Sanfilippo syndrome as a novel substrate reduction therapy. We then confirmed the capacity of this compound to modulate substrate accumulation in vivo in a Sanfilippo Drosophila model. Treatment with this compound significantly reduced HS in cultured MPS IIIA patient fibroblasts in a time-dependent manner. Neuronal and ubiquitous knockdown Drosophila models of MPS IIIC displaying elevated heparan sulfate and behavioural defects exhibited reduced HS burden relative to vehicle-treated controls following oral feeding with the GlcNAc analogue inhibitor. These findings indicate that this compound may be beneficial in slowing the accumulation of HS and may represent a novel therapeutic for Sanfilippo syndrome. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.03.04.531088v1?rss=1 Authors: Kumar, M., Has, C., Lam-Kamath, K., Ayciriex, S., Dewett, D., Bashir, M., Poupault, C., Schuhmann, K., Knittelfelder, O., Raghuraman, B. K., Ahrends, R., Rister, J., Shevchenko, A. Abstract: The Drosophila melanogaster eye is a popular model to elucidate the molecular mechanisms that underlie the structure and function of the eye as well as the causes of retinopathies. For instance, the Drosophila eye has been used to investigate the impacts of ageing and environmental stresses such as light-induced damage or dietary deficiencies. Moreover, large-scale screens have isolated genes whose mutation causes morphological and functional ocular defects, which includes key components of the phototransduction cascade. However, the proteome of the Drosophila eye is poorly characterized. Here, we used GeLC-MS/MS to quantify 3516 proteins he adult Drosophila melanogaster eye and provide a generic and expandable resource for further genetic, pharmacological, and dietary studies. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.01.08.523160v1?rss=1 Authors: Landis, J. E., Sungu, K., Sipe, H., Copeland, J. M. Abstract: RNAi targeting the electron transport chain has been proven to prolong life span in many different species, and experiments specifically with Drosophila melanogaster and Caenorhabditis elegans have shown a distinct role for neurons. To determine which subset of neurons is implicated in this life span extension, we used the GAL4/UAS system to activate RNAi against genes of Complex I and Complex V. We found life span extension of 18 -- 24% with two glutamate neuron (D42 and VGlut) GAL4 lines. We used the GAL80 system to determine if the overlapping set of glutamate neurons in these two GAL4 lines imparts the life span extension. Limiting GAL4 activity to non-VGlut glutamate neurons in the D42 background failed to extend life span, suggesting that glutamate neurons have a unique role in aging. Interestingly, RNAi of the electron transport chain in D42 glutamate neurons also caused an increase in daytime and nighttime sleep and a decrease in nighttime locomotor activity. Changes to sleep patterns and prolonged life span were not accompanied by any changes in female fertility or response to starvation. Our findings demonstrate that a small subset of neurons can control life span, and further studies exploring the role of the electron transport chain in aging can be focused on the activity of glutamate neurons. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.12.15.519769v1?rss=1 Authors: Jones, H., Willis, J. A., Firth, L. C., Giachello, C. N. G., Gilestro, G. F. Abstract: Understanding how the brain encodes behaviour is the ultimate goal of neuroscience and the ability to objectively and reproducibly describe and quantify behaviour is a necessary milestone on this path. Recent progresses in machine learning and computational power have pushed the development and adoption of powerful systems leveraging on high-resolution videorecording to track pose and describe behaviour in all four dimensions, however the temporal and spatial resolution of these systems come as a compromise with their throughput and accessibility. Here we describe coccinella, an open source reductionist framework that allows for high-throughput analysis of behaviour using real-time tracking on a distributed mesh of microcomputers followed by resource-lean statistical learning. We show that a reductionist system outperforms state-of-the-art alternatives when exploring the pharmacobehaviour in Drosophila melanogaster. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.12.06.519343v1?rss=1 Authors: Vieira, D. V., Carlota, R. R., de-Carvalho, J., Telley, I. A. Abstract: In cells, mRNA can be associated with various proteins, forming ribonucleoprotein complexes (RNPs) which take part in spatiotemporal control of translation. In the Drosophila melanogaster developing egg chamber, a set of RNPs is transported from the nurse cells to the oocyte and targeted selectively to specific cellular locations. This mRNA sorting process leads to the final oocyte polarization pre-defining the body axes of the future embryo. However, how mRNA is encoded for selection and directed transport is mechanistically not well understood. A master mRNA involved in body axes formation is bicoid, which localizes anterolaterally and is essential for head and thorax definition of the embryo. A protein that was identified essential for bicoid anterior localization is Exuperantia (Exu). Here, we use a live imaging-based pulse-chase approach, which reveals selective transport dynamics of Exu from nurse cells to the oocyte during mid to late-stage oogenesis. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.11.18.517027v1?rss=1 Authors: Narwal, S., Rai, A., Iyer, S. M., Tare, K. N., Tare, M. Abstract: Parkinsons Disease (PD) is the second most common neurodegenerative disease affecting 1-2% of the global population with no cure to date. PD is characterized by accumulation of Lewy Bodies (LBs), which are caused due to aggregation of incorrectly folded -Synuclein, (SNCA). Another form of PD manifestation is characterized by loss of function of parkin, which encodes an E3 ubiquitin ligase. Despite extensive research, the cause for onset and progression of PD remains unknown and current therapeutics mainly help manage the disease. An alternative line of treatment can be useful. In this study, we have employed two different genetic models of Drosophila to screen for Ayurvedic compounds and found an Ayurvedic mercury based organo-metallic drug Ras-Sindoor has neuroprotective function. Our data indicate that characteristic locomotory dysfunction phenotype of PD is restored upon administration of the compound. Interestingly, RS fed flies also exhibit reduced transcript levels of initiator caspase dronc, which possibly prevents cell death in dopaminergic neurons. Additionally, RS fed PD model flies exhibit an enhanced life span. Our studies emphasize beneficial use of traditional Ayurvedic compounds as a holistic cure for PD like multifactorial diseases. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.10.20.513010v1?rss=1 Authors: Li, K., Tsukasa, Y., Kurio, M., Maeta, K., Tsumadori, A., Baba, S., Nishimura, R., Murakami, A., Onodera, K., Morimoto, T., Uemura, T., Usui, T. Abstract: Appropriate modulation of escape behaviors in response to potentially damaging stimuli is essential for survival. Although nociceptive circuitry has been studied, it is poorly understood how genetic contexts affect the relevant escape responses. Using an unbiased genome-wide association analysis, we identified a Ly6/-neurotoxin family protein, Belly roll (Bero), which negatively regulates Drosophila nociceptive escape behavior. We show that Bero is expressed in abdominal leucokinin-producing neurons (ABLK neurons) and bero knockdown in ABLK neurons resulted in enhanced escape behavior. Furthermore, we demonstrated that ABLK neurons responded to the activation of nociceptors and initiated the behavior. Notably, bero knockdown reduced the persistent neuronal activity and increased the evoked nociceptive responses in ABLK neurons. Our findings reveal that Bero modulates an escape response by regulating distinct neuronal activities in ABLK neurons. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.10.13.512092v1?rss=1 Authors: Scholz, H., Berger, M., Yapıcı, B. Abstract: Drosophila melanogaster larvae develop on fermenting fruits with increasing ethanol concentrations. To address the relevance of ethanol in the behavioral response of the larvae, we analyzed the function of ethanol in the context of olfactory associative behavior in Canton S and w1118 larvae. The motivation of larvae to move toward or out of an ethanol-containing substrate depends on the ethanol concentration and the genotype. Ethanol in the substrate reduces the attraction to odorant cues in the environment. Relatively short repetitive exposures to ethanol, which are comparable in their duration to reinforcer representation in olfactory associative learning and memory paradigms, result in positive or negative association with the paired odorant or indifference to it. The outcome depends on the order in which the reinforcer is presented during training, the genotype and the presence of the reinforcer during the test. Independent of the order of odorant presentation during training, Canton S and w1118 larvae do not form a positive or negative association with the odorant when ethanol is not present in the test context. When ethanol is present in the test, w1118 larvae show aversion to an odorant paired with a naturally occurring ethanol concentration of 5%. Our results provide insights into the parameters influencing olfactory associative behaviors using ethanol as a reinforcer in Drosophila larvae and indicate that short exposures to ethanol might not uncover the positive rewarding properties of ethanol for developing larvae. Copy rights belong to original authors. Visit the link for more info Podcast created by PaperPlayer

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.09.30.510181v1?rss=1 Authors: Gruber, L., Cantera, R., Pleijzier, M. W., Hansson, B. S., Rybak, J. Abstract: To manage the great complexity of detecting and identifying olfactory cues, the insect olfactory system has evolved two main strategies: combinatorial coding and specialized, narrowly tuned olfactory pathways. In combinatorial coding, odorants are encoded by activation of multiple, broadly tuned olfactory sensory neurons that innervate distinct sets of glomeruli. In specialized olfactory pathways, information regarding a single or a few odorants is processed in a discrete, narrowly tuned circuit within a dedicated glomerulus. Here, we compared the narrowly tuned glomerulus DA2 with the broadly tuned glomerulus DL5 at the ultrastructural level, by using volume based focused ion beam scanning electron microscopy. We provide a detailed analysis of neuronal innervation, synaptic composition as well as a circuit diagram of the major glomerular cell types: olfactory sensory neurons (OSNs), uniglomerular projection neurons (uPNs) and multiglomerular neurons (MGNs). By comparing our data with a previously mapped narrowly tuned glomerulus (VA1v), we disclose putative generic features of narrowly tuned glomerular circuits: a high density of neuronal fibers and synapses, a low degree of sensory lateralization, strong axo-axonic connections between OSNs as well as dendro-dendritic connections between uPNs, and a low degree of presynaptic inhibition at the OSN axons. We also show a unique property of the large uPN dendrite in DL5, which forms substantial amount of autapses. Copy rights belong to original authors. Visit the link for more info Podcast created by PaperPlayer

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.09.30.510382v1?rss=1 Authors: Umezaki, Y., Hidalgo, S., Nguyen, E., Nguyen, T., Suh, J., Uchino, S. S., Chiu, J. C., Hamada, F. N. Abstract: Animals are motivated to eat based on their internal need. When animals are hungry, the sensory detection of food elicits bursts of physiological changes in their bodies. This is the cephalic phase response (CPR), which prepares animals to properly digest food before nutrients enter their bodies. Despite animals uniformly exhibiting a strong CPR, to what extent and how the internal state influences CPR is largely unclear. Here, we demonstrate in Drosophila that tasting food triggers CPR, which is strongly influenced by the internal state. We found that feeding an artificial sweetener or gustatory excitation by optogenetics trigger CPR, which is a rapid and partial recovery from the starved state. While clock genes and hunger signals profoundly drive CPR, they are not required for the process of response after nutrient intake. Therefore, we propose that CPR is the critical layer of regulatory mechanisms representing internal energy homeostasis and metabolism. Copy rights belong to original authors. Visit the link for more info Podcast created by PaperPlayer

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.09.27.509663v1?rss=1 Authors: Thane, M., Paisios, E., Stöter, T., Krüger, A.-R., Gläss, S., Dahse, A.-K., Scholz, N., Gerber, B., Lehmann, D. J., Schleyer, M. Abstract: Neuronally orchestrated muscular movement and locomotion are defining faculties of multicellular animals. Due to its numerically simple brain and neuromuscular system and its genetic accessibility, the larva of the fruit fly Drosophila melanogaster is an established model to study these processes at tractable levels of complexity. However, although the faculty of locomotion clearly pertains to the individual animal, present studies of locomotion in larval Drosophila mostly use group assays and measurements aggregated across individual animals. The alternative is to measure animals one at a time, an extravagance for larger-scale analyses. In principle or in practice, this in particular rules out grasping the inter- and intra-individual variability in locomotion and its genetic and neuronal determinants. Here we present the IMBA (Individual Maggot Behaviour Analyser) for tracking and analysing the behaviour of individual larvae within groups. Using a combination of computational modelling and statistical approaches, the IMBA reliably resolves individual identity across collisions. It does not require specific hardware and can therefore be used in non-expert labs. We take advantage of the IMBA first to systematically describe the inter- and intra-individual variability in free, unconstrained locomotion in wild-type animals. We then report the discovery of a novel, complex locomotion phenotype of a mutant lacking an adhesion-type GPCR. The IMBA further allows us to determine, at the level of individual animals, the modulation of locomotion across repeated activations of dopamine neurons. Strikingly, IMBA can also be used to analyse 'silly walks', that is patterns of locomotion it was not originally designed to investigate. This is shown for the transient backward locomotion induced by brief optogenetic activation of the brain-descending 'mooncrawler' neurons, and the variability in this behaviour. Thus, the IMBA is an easy-to-use toolbox allowing an unprecedentedly rich view of the behaviour and behavioural variability of individual Drosophila larvae, with utility in multiple biomedical research contexts. Copy rights belong to original authors. Visit the link for more info Podcast created by PaperPlayer

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.09.23.509131v1?rss=1 Authors: Kim, W. J., Lee, S. G., Kang, C., Saad, B., Nguyen, K.-N. H., Guerra-Phalen, A., Bui, D., Abbas, A.-H., Trinh, B., Malik, A., Zeghal, M., Auge, A.-C., Islam, M. E., Wong, K., Stern, T., Lebedev, E., Sun, D., Miao, H., Wu, Z., Sherratt, T. N. Abstract: Males have finite resources to spend on reproduction. Thus, males rely on a 'time investment strategy' to maximize their reproductive success. For example, male Drosophila melanogaster extends their mating duration when surrounded by conditions enriched with rivals. Here we report a novel form of behavioral plasticity whereby male fruit flies exhibit a shortened duration of mating when they are sexually experienced; we refer to this plasticity as 'shorter-mating-duration (SMD)'. SMD is a plastic behavior and requires sexually dimorphic taste neurons. We identified several neurons in the male foreleg and midleg that express specific sugar, pheromone and mechanosensory receptors. Using a cost-benefit model and behavioral experiments, we further show that SMD behavior exhibits adaptive behavioral plasticity in male flies. Thus, our study delineates the molecular and cellular basis of the sensory inputs required for SMD; this represents a plastic interval timing behavior that could serve as a model system to study how multisensory inputs converge to modify interval timing behavior for improved adaptation. Copy rights belong to original authors. Visit the link for more info Podcast created by PaperPlayer

En las células, la transmisión de la información genética no se da de manera continua, sino de manera “estocástica” o aleatoria, por intermitencias. Este fenómeno, conocido como "ruido celular o de expresión", desempeña un papel fundamental en muchos procesos biológicos. Investigadores del IGMM, el Instituto de Genética Molecular de Montpellier, al sur de Francia, buscaron conocer mejor este mecanismo gracias al estudio de embriones de la mosca Drosophila. Las imágenes obtenidas son fascinantes. El ruido celular está implicado en muchos procesos biológicos, por ejemplo, en el rebote de la carga viral al interrumpirse la triterapia contra el VIH, pero también en el desarrollo embrionario. Un equipo de investigadores del Instituto de Genética Molecular de Montpellier, el IGMM, al sur de Francia, quiso comprender mejor algunos aspectos de este ruido celular. Los resultados fueron publicados en dos artículos, uno más centrado en la biología y el otro en modelos matemáticos, en la revista Nature Communications. Las investigaciones fueron hechas por un equipo transversal, entre biólogos, matemáticos y físicos. El italiano Antonio Trullo es analista de imágenes y forma parte del equipo del laboratorio dirigido por la bióloga Mounia Lagha del IGMM. Antonio Trullo conversó con RFI sobre estas investigaciones. Escuche aquí la entrevista en su formato largo:     Los investigadores observaron al microscopio y captaron en imágenes las primeras horas del desarrollo de un embrión de la mosca Drosophila Melanogaster (embriogénesis). Las imágenes son muy gráficas y parecieran muchos puntos que se mueven y se dividen a su ritmo, casi como un ballet. Los pequeños puntos amarillos son los genes al activarse. Dos modelos para un mecanismo Lo novedoso de esta investigación es que, para comprender mejor este fenómeno aleatorio de expresión genética, se basaron en dos modelos, uno usando un etiquetado fluorescente del ARN mensajero implementado por la bióloga Virginia Pimmet y otro basándose en modelos matemáticos, gracias a la colaboración del equipo del matemático Ovidiu Radulescu de la Universidad de Montpellier. En particular, los científicos se interesaron por el proceso de "pausa" de la transcripción genética, que corresponde a una pausa temporal del ARN polimerasa II, mejor conocido como ARN Pol II, una enzima que asegura la transcripción de los genes, y en particular estudiaron el llamado promotor, una parte del ARN responsable de iniciar o “empujar” la transcripción de un gen. Los investigadores demostraron que "la modulación del ruido de expresión en las células infectados por el VIH y en los embriones de Drosophila utiliza un mecanismo común la llamada “pausa proximal” en el promotor del ARN Pol II." Sobre el laboratorio de Mounia Lagha En este laboratorio dirigido por la bióloga Mounia Lagha (medalla de bronce CNRS 2017), se estudian los mecanismos que regulan la expresión de los genes implicados en el desarrollo embrionario, en concreto, el desarrollo temprano de la mosca Drosophila, en las primeras 4 horas de embriogénesis. Mounia Lagha, ingeniera agrónoma y formada en biología del desarrollo, realizó su doctorado sobre la formación del tejido muscular en el embrión de ratón en el Instituto Pasteur. Para desarrollar enfoques mecanicistas, la joven bióloga cambió de organismo modelo durante su formación posdoctoral: se incorporó a la Universidad de Berkeley en 2010 para trabajar en el embrión de la mosca de la fruta Drosophila, interesándose en el proceso de "pausa" de la transcripción, que corresponde a una detención temporal de la polimerasa, la enzima que asegura la transcripción de los genes. Desde su contratación en el CNRS en 2014, año en que recibió el Premio Paoletti, ha continuado su trabajo en el Instituto de Genética Molecular de Montpellier. Un ambicioso proyecto de investigación, apoyado por una beca del ERC Starting Grant del Consejo Europeo de Investigación y llevado a cabo por jóvenes científicos de diferentes disciplinas. Entrevistado: el físico Antonio Trullo, analista de imágenes del laboratorio de Mounia Lagha, del Institute de Génétique Moléculaire de Montpellier (IGMM) del CNRS. Otros temas que le pueden interesar: Descubren nuevo componente de la sangre: las mitocondrias Científicos descubren nuevo hallazgo sobre cómo se afianzan nuestros recuerdos

British scientists discover fruit flies get hangry (who knew!), Delhi teen creates affordable air purifier, Spain's robot chef creates a stir, London's sky-pool gives onlookers the heebeegeebees!

In this episode of the Epigenetics Podcast, we caught up with Monica Dus from the University of Michigan to talk about her work on nutriepigenetics and the effects of diet on behavior. The focus of Monica Dus and her team is to study the effect of sugar on the brain and how diet has an effect on behavior. The Dus lab takes a multidisciplinary approach to answer questions like "What causes animals to overeat if they consume foods rich in sugar, salt, and fat?" and "How does such a diet alter the basic physiology and biochemistry of the brain to promote food intake and weight gain?" By doing this, they showed recently that the Polycomb Repressive Complex 2 (PRC2) plays a role in reprogramming the sensory neurons of Drosophila Melanogaster, reducing sweet sensation and hence promoting obesity when flies are fed a high sugar diet. In response to that diet the binding of PRC2 to chromatin in sweet gustatory neurons is altered and reshapes the developmental transcriptional network. In this episode we discuss how flies taste food and sugar, how sugar modulates taste, and how a high sugar diet influences the taste and amount of food flies eat.   References Monica Dus, SooHong Min, … Greg S. B. Suh (2011) Taste-independent detection of the caloric content of sugar in Drosophila (Proceedings of the National Academy of Sciences of the United States of America) DOI: 10.1073/pnas.1017096108 Christina E. May, Anoumid Vaziri, … Monica Dus (2019) High Dietary Sugar Reshapes Sweet Taste to Promote Feeding Behavior in Drosophila melanogaster (Cell Reports) DOI: 10.1016/j.celrep.2019.04.027 Daniel Wilinski, Jasmine Winzeler, … Monica Dus (2019) Rapid metabolic shifts occur during the transition between hunger and satiety in Drosophila melanogaster (Nature Communications) DOI: 10.1038/s41467-019-11933-z Anoumid Vaziri, Morteza Khabiri, … Monica Dus (2020) Persistent epigenetic reprogramming of sweet taste by diet (Science Advances) DOI: 10.1126/sciadv.abc8492 How to Science Podcast NeuroEpic Podcast   Related Episodes Transcription and Polycomb in Inheritance and Disease (Danny Reinberg) The Role of Small RNAs in Transgenerational Inheritance in C. elegans (Oded Rechavi) Epigenetic Origins Of Heterogeneity And Disease (Andrew Pospisilik)   Contact Active Motif on Twitter Epigenetics Podcast on Twitter Active Motif on LinkedIn Active Motif on Facebook Email: podcast@activemotif.com

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.10.30.361899v1?rss=1 Authors: Tomita, J., Ban, G., Kato, Y. S., Kume, K. Abstract: The central complex is one of the major brain regions that control sleep in Drosophila, but the circuitry details of sleep regulation have yet to be elucidated. We attempted to identify a novel sleep-regulating neuronal circuit in the central complex. no-bridgeKS49 mutant flies with an anatomical defect in the protocerebral bridge (PB) significantly decreased sleep. By transient activation of a subset of the PB neurons using 14 Gal4 drivers, we found the sleep-promoting R59E08-Gal4 and the wake-promoting R52B10-Gal4. The R59E08-Gal4 expressing PB neurons were identified as the PB interneurons according to their morphological features. On the other hand, using genetic mosaic analysis, we showed that activation of neurons projecting from the PB to the FB and contralateral NO (PFN neurons) in the R52B10-Gal4 driver reduced sleep. A targeted GFP reconstitution across synaptic partners (t-GRASP) analysis demonstrated synaptic contacts between the sleep-promoting PB neurons and the wake-promoting PFN neurons. Furthermore, we found that activation of a pair of dopaminergic neurons with axons projecting the PB (T1 DA neurons) significantly decreased sleep. The wake-promoting T1 DA neurons were more likely to physically associate with the sleep-promoting PB interneurons. Dopamine 2-like receptor (Dop2R) knockdown in the R59E08-Gal4 expressing PB interneurons significantly increased sleep. These results indicated that the neuronal circuit in the PB regulated by dopamine signaling mediates sleep-wakefulness. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.10.09.333831v1?rss=1 Authors: Tsiarli, M. A., Conard, A. M., Xu, L., Nguyen, E., Larschan, E. N. Abstract: Neural stem cell (NSC) differentiation is controlled by cell-intrinsic and external signals from the stem cell niche including niche surface glia (SG). However, the mechanisms by which transcription factors drive NSC differentiation within the niche remain largely unknown. Here, we show that the transcription factor, Chromatin-linked adaptor for MSL proteins (CLAMP) is required for NSC differentiation. CLAMP promotes transcription of genes involved in stemness, proliferation, and glial development and represses transcription of genes involved in neurogenesis and niche survival. Consistent with transcriptional changes, CLAMP promotes NSC proliferation and SG production. Furthermore, glial-specific knock-down of clamp causes similar phenotypes to clamp null mutants. CLAMP motifs are present at many target genes including the glial-determining gene, glial cells missing, and Notch, a key regulator of neurogenesis. Collectively, our results suggest that CLAMP regulates a transcriptional program which drives NSC proliferation and differentiation via cell-intrinsic and niche-dependent mechanisms that involve niche glia. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.09.30.320200v1?rss=1 Authors: Schumann, I., Triphan, T. Abstract: The post-embryonal development of arthropod species, including crustaceans and insects, is characterized by ecdysis or molting. This process defines growth stages and is controlled by a conserved neuroendocrine system. Each molting event is divided in several critical time points, such as pre-molt, molt and post-molt, and leaves the animals in a temporarily highly vulnerable state while their cuticle is re-hardening. The molting events occur in an immediate ecdysis sequence within a specific time window during the development. Each sub-stage takes only a short amount of time, which is generally in the order of minutes. To find these relatively short behavioral events, one needs to follow the entire post-embryonal development over several days. As the manual detection of the ecdysis sequence is time consuming and error prone, we designed a monitoring system to facilitate the continuous observation of the post-embryonal development of the fruit fly Drosophila melanogaster. Under constant environmental conditions we are able to observe the life cycle from the embryonic state to the adult, which takes about ten days in this species. Specific processing algorithms developed and implemented in Fiji and R allow us to determine unique behavioral events on an individual level - including egg hatching, ecdysis and pupation. In addition, we measured growth rates and activity patterns for individual larvae. Our newly created RPackage PEDtracker can predict critical developmental events and thus offers the possibility to perform automated screens that identify changes in various aspects of larval development. In conclusion, the PEDtracker system presented in this study represents the basis for automated real-time staging and analysis not only for the arthropod development. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.09.29.319475v1?rss=1 Authors: SINGH, R. K., Jonely, M., Leslie, E., Rejali, N. A., Noriega, R., Bass, B. L. Abstract: Drosophila melanogaster Dicer-2 (dmDcr-2) differentially processes dsRNA with blunt or 2 nucleotide 3{per thousand}-overhanging termini. We investigated the transient kinetic mechanism of these reactions using a rapid reaction stopped-flow technique and time-resolved fluorescence spectroscopy. We found that ATP binding to dmDcr-2{per thousand}s helicase domain impacts the kinetics of dsRNA binding and dissociation in a termini-dependent manner, emphasizing the termini-dependent discrimination of dsRNA on a biologically-relevant time-scale. ATP-hydrolysis mediates local unwinding of dsRNA, and directional translocation on unwound single-stranded RNA, which is concurrent with a slow rewinding prior to dsRNA cleavage. Time-resolved fluorescence anisotropy reveals a nucleotide-dependent change in conformational dynamics of the helicase and Platform.PAZ domains in the nanosecond timescale that is correlated with termini-dependent dsRNA cleavage. Our study delineates kinetic events and transient intermediates for a Dicer-catalyzed reaction, thus establishing a framework for understanding other Dicers and how accessory factors modulate the reaction. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.09.25.312967v1?rss=1 Authors: Braco, J. T., Saunders, C. J., Nelson, J. M., Johnson, E. C. Abstract: In insects, Adipokinetic hormone is the primary hormone responsible for the mobilization of stored energy. While a growing body of evidence has solidified AKHs role in modulating the physiological and behavioral responses to metabolic stress, little is known about the upstream endocrine circuit that directly regulates AKH release. We evaluated the AKH-expressing cell transcriptome to identify potential regulatory elements controlling AKH cell activity, and found that a number of receptors show consistent expression levels, including all known dopamine receptors, dopamine ecdysone receptor (DopEcR), Dopamine 2-like receptor (D2R), Dopamine 1-like receptor 2 (DopR2), DopR, and the Pigment Dispersing Factor (PDFR). We tested the consequences of targeted genetic knockdown and found that RNAi elements targeting each dopamine receptor caused a significant reduction in survival under starvation. In contrast, PDFR knockdown significantly extended lifespan under starvation whereas expression of a tethered PDF in AKH cells resulted in a significantly shorter lifespan during starvation. These manipulations also caused various changes in locomotor activity under starvation. Specifically, there were higher amounts of locomotor activity in dopamine receptor knockdowns, in both replete and starved states. PDFR knockdown resulted in increased locomotion during replete conditions and locomotion levels that were comparable to wild-type during starvation. Expression of a membrane-tethered PDF led to decreased locomotion under baseline and starvation. Next, we used live-cell imaging to evaluate the acute effects of the ligands for these receptors (dopamine, ecdysone, and Pigment Dispersing Factor) on AKH cell activation. Dopamine application led to a transient increase in intracellular calcium in a sugar-dependent manner. Furthermore, we found that co-application of dopamine and ecdysone led to a complete loss of this response, suggesting that these two hormones are acting antagonistically. We also found that PDF application directly led to an increase in cAMP in AKH cells, and that this response was dependent on expression of the PDFR in AKH cells. Together these results suggest a complex circuit in which multiple hormones act on AKH cells to modulate metabolic state. Copy rights belong to original authors. Visit the link for more info

(This episode is in Serbian)Why do we sleep? The answer can seem obvious to everyone - because sleep helps us to rest, regenerate, preserve energy for the day ahead… However, the process that is so familiar to us remains among the most mysterious phenomena in biology – as a matter of fact, everything we just stated still remains at the level of the hypothesis. So why is sleep necessary for life? Dragana Rogulja teaches neurobiology at Harvard Medical School Blavatnik Institute of Neurobiology, has dedicated her career to researching the molecular mechanisms that govern sleep. For this purpose, Dragana and a team of scientists from Harvard examined Drosophila Melanogaster, because sleep in the fruit fly is remarkably similar to mammalian sleep. Dragana revealed to us that flies, in the absence of sleep, behave similarly to humans - lack of sleep leads to numerous disorders and diseases, even death. Through a conversation with Dragana, we learned not only about similarities between flies and humans, but we also heard in more details about her long-term research, which for the first time offered to science something more than hypotheses when it comes to question of our need to sleep. We encourage you to learn more about Dragana Rogulja's research at the following links:Rogulja Lab: http://roguljalab.hms.harvard.eduArticle: "Sleep Loss Can Cause Death through Accumulation of Reactive Oxygen Species in the Gut: https://www.dropbox.com/s/08f6yh5o9792kzf/Sleep%20ROS%20Gut%20Alex%20Yossi%20Keishi.pdf?dl=0 

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.09.04.281022v1?rss=1 Authors: Bilz, F., Gilles, M.-M., Schatton, A., Pflueger, H.-J., Schubert, M. Abstract: Activation and modulation of sensory-guided behaviors by biogenic amines assure appropriate adaptations to changes in an insect's environment. Given its genetic tool kit Drosophila melanogaster represents an excellent model organism to study larger networks of neurons by optophysiological methods. Here, we studied stationary crawling movements of 3rd instar larvae and revealed how the octopaminergic VUM neuron system reacts during crawling behavior and tactile stimulations. We conducted calcium imaging experiments on dissections of the isolated nervous system (missing all sensory input) and found spontaneous rhythmic wave pattern of neuronal activity in VUM neuron clusters over the range of thoracic and abdominal neuromeres in the VNC. In contrast, in vivo preparations (semi-intact animals, receiving sensory input) did not reveal such spontaneous rhythmic pattern. However, tactile stimulations activated different clusters of the VUM neuron system simultaneously in these preparations. The activation intensity of VUM neurons in the VNC was correlated with the location and degree of body wall stimulation. While VUM neuron cluster near the respective location of body wall stimulation were less activated more distant cluster showed stronger activation. Repeated gentle touch stimulations led to decreased response intensities, repeated harsh stimulations resulted in increasing intensities over trials. Optophysiological signals correlated highly with crawling behavior in freely moving larvae stimulated similarly. We conclude that the octopaminergic system is strongly coupled to the neuronal pattern generator of crawling movements and that it is simultaneously activated by physical stimulation, rather intensity than sequential coded. We hope that our work raises the interest in whole biogenic network activity and shows that octopamine release does not only underlie "the more the better" principle but instead has a more complex function in control and modulation of insect's locomotion. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.08.07.241208v1?rss=1 Authors: Dapergola, E., Menegazzi, P., Raabe, T., Hovhanyan, A. Abstract: Endogenous clocks enable organisms to adapt their physiology and behavior to daily variation in environmental conditions. Metabolic processes in cyanobacteria to humans are effected by the circadian clock, and its dysregulation causes metabolic disorders. In mouse and Drosophila were shown that the circadian clock directs translation of factors involved in ribosome biogenesis and synchronizes protein synthesis. However, the role of clocks in Drosophila neurogenesis and the potential impact of clock impairment on neural circuit formation and function is less understood. Here we demonstrate that light stimuli or circadian clock causes a defect in neural stem cell growth and proliferation accompanied by reduced nucleolar size. Further, we define that light and clock independently affect the InR/TOR growth regulatory pathway due to the effect on regulators of protein biosynthesis. Altogether, these data suggest that alterations in growth regulatory pathways induced by light and clock are associated with impaired neural development. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.06.29.177675v1?rss=1 Authors: Shin, M., Copeland, J. M., Venton, B. J. Abstract: Drosophila melanogaster, the fruit fly, is an exquisite model organism to understand neurotransmission. Dopaminergic signaling in the Drosophila mushroom body (MB) is involved in olfactory learning and memory, with different compartments controlling aversive learning (corner) vs appetitive learning (medial tip). Here, the goal was to develop techniques to measure endogenous dopamine in compartments of the MB for the first time. We compared three stimulation methods: acetylcholine (natural stimulus), P2X2 (chemogenetics), and CsChrimson (optogenetics). Evoked dopamine release was measured with fast-scan cyclic voltammetry in isolated adult Drosophila brains. Acetylcholine stimulated the largest dopamine release (0.40 M), followed by P2X2 (0.14 M), and CsChrimson (0.07 M). With the larger acetylcholine and P2X2 stimulations, there were no regional or sex differences in dopamine release. However, with CsChrimson, dopamine release was significantly higher in the corner than the medial tip, and females had more dopamine than males. Michaelis-Menten modeling of the single-light pulse revealed no significant regional differences in Km, but the corner had a significantly lower Vmax (0.12 M/s vs. 0.19 M/s) and higher dopamine release (0.05 M vs. 0.03 M). Optogenetic experiments are challenging because CsChrimson is also sensitive to blue light used to activate green fluorescent protein, and thus, light exposure during brain dissection must be minimized. These experiments expand the toolkit for measuring endogenous dopamine release in Drosophila, introducing chemogenetic and optogenetic experiments for the first time. With a variety of stimulations, different experiments will help improve our understanding of neurochemical signaling in Drosophila. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.05.17.090001v1?rss=1 Authors: Leung, A., Cohen, D., van Swinderen, B., Tsuchiya, N. Abstract: The physical basis of consciousness remains one of the most elusive concepts in current science. One influential conjecture is that consciousness is to do with some form of causality, measurable through information. The integrated information theory of consciousness (IIT) proposes that conscious experience, filled with rich and specific content, corresponds directly to a hierarchically organised, irreducible pattern of causal interactions; i.e. an integrated informational structure among elements of a system. Here, we tested this conjecture in a simple biological system (fruit flies), estimating the information structure of the system during wakefulness and general anesthesia. We found that causal interactions among populations of neurons during wakefulness collapsed to isolated clusters of interactions during anesthesia. We used classification analysis to quantify the accuracy of discrimination between wakeful and anesthetised states, and found that informational structures inferred conscious states with greater accuracy than a scalar summary of the structure, a measure which is generally championed as the main measure of IIT. Spatially, we found that the information structures collapsed rather uniformly across the fly brain. Our results speak to the potential utility of the novel concept of an 'informational structure' as a measure for level of consciousness, above and beyond simple scalar values. Copy rights belong to original authors. Visit the link for more info

In this episode from the survey of genomes, Kaitlyn Morse from the 2019 Hiram College Genetics course moves us into the world of eukaryotic genomes. Her focus is on the first model animal for genetic analysis - Drosophila melanogaster or as most people know it, the fruit fly.

Aram Megighian, University of Padova, Italy speaks on "Drosophila melanogaster: from neurophysiology to behavior". This movie has been recorded by ICGEB Trieste at "Drosophila melanogaster models for neurodegenerative diseases" Course, 17 - 20 April 2018, Trieste, Italy.

At our place we don’t remember this many fruit flies from years past, so I guess in a way this has been a good year, at least for the fruit flies.

The genetic basis underlying adaptive evolution is still largely unknown. Adaptive evolution is facilitated by natural selection that acts on the genetic variation present in a population. Favoring some genetic variants over others, natural selection eventually produces adaptations that allow populations to survive in changing or new environments. Populations colonizing new habitats that differ from their original habitat are often confronted with a multitude of novel ecological constraints to which they need to adapt. A well-annotated genome and a diverse genetic toolkit make the fruit fly Drosophila melanogaster an ideal model system for studying the genetics underlying adaptation. As a cosmopolitan species, D. melanogaster has adapted to a wide range of thermal environments. Despite having a tropical origin in southern-central Africa, it has successfully settled in temperate environments around the world. Thermal adaptations that have helped to deal with the greater range and variability in temperature as well as low-temperature extremes have been required to prosper in temperate environments. Chromatin-based gene regulation is known to be disrupted by varying temperatures. Variation in the temperature, at which flies live, result in varying expression levels of Polycomb group (PcG) regulated genes with higher expression at lower temperatures. Chapter 1 and 2 of this thesis aim to answer the question whether this thermosensitivity of PcG regulation has been detrimental for colonizing temperate environments and thus needed to be buffered by natural selection. Thermosensitivity of PcG regulation was observed in different natural populations of D. melanogaster. A lower degree of thermosensitive expression was consistently found for populations from temperate climates when compared to those from the tropics. In Chapter 1, evidence is presented for positive selection acting on the polyhomeotic (ph) gene region to reduce thermosensitivity of PcG regulation in temperate populations from Europe. The targets of selection appear to be single nucleotide polymorphisms (SNPs) in a relatively small cis-regulatory region between the two PcG target genes polyhomeotic proximal (ph-p) and CG3835 that are highly differentiated between European and African populations. Using reporter gene assays, it was demonstrated that these SNPs influence gene expression and that the European alleles confer reduced thermosensitivity of expression in contrast to the African alleles. In Chapter 2, thermosensitivity of another PcG target gene, vestigial (vg), was investigated in six natural populations including four temperate populations from high-altitude Africa and central to high-latitude Europe, and two tropical populations from the ancestral species range. All four temperate populations exhibited a lower degree of thermosensitive expression than the two tropical populations. The underlying mechanisms of increased buffering, however, seem to differ between these temperate populations. Thermal adaptation to temperate environments also includes dealing with low-temperature extremes. Severe cold stress is a main limiting factor imposed on D. melanogaster by temperate climates. Increased cold tolerance in temperate populations is thought to have evolved by natural selection. Cold tolerance is a quantitative trait that appears to be highly polygenic and has been mapped to different quantitative trait loci (QTL) in the genome. In Chapter 3, such a QTL region was fine-mapped to localize causal genes for increased cold tolerance in temperate flies. As a result, brinker (brk) was identified as a new candidate gene putatively involved in cold stress adaptation.

Wed, 19 Nov 2014 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/17863/ https://edoc.ub.uni-muenchen.de/17863/1/Barth_Teresa.pdf Barth, Teresa ddc:570, ddc:

Focusing at the fly visual system I am addressing the identity and function of neurons accomplishing two fundamental processing steps required for survival of most animals: neurons of peripheral circuits underlying colour vision as well neurons of higher order circuits underlying visual memory. Colour vision is commonly assumed to rely on photoreceptors tuned to narrow spectral ranges. In the ommatidium of Drosophila, the four types of so-called inner photoreceptors express different narrow-band opsins. In contrast, the outer photoreceptors have a broadband spectral sensitivity and are thought to exclusively mediate achromatic vision. Using computational models and behavioural experiments, I here demonstrate that the broadband outer photoreceptors contribute to colour vision in Drosophila. A model of opponent processing that includes the opsin of the outer photoreceptors scores the best fit to wavelength discrimination behaviour of flies. To experimentally uncover the contribution of individual photoreceptor types, I used blind flies with disrupted phototransduction (norpA-) and rescued norpA function in genetically targeted photoreceptors and receptor combinations. Surprisingly, dichromatic flies with only broadband photoreceptors and one additional receptor type can discriminate different colours, indicating the existence of a specific output comparison of outer and inner photoreceptors. Furthermore, blocking interneurons postsynaptic to the outer photoreceptors specifically impairs colour but not intensity discrimination. These findings show that outer receptors with a complex and broad spectral sensitivity do contribute to colour vision and reveal that chromatic and achromatic circuits in the fly share common photoreceptors. Higher brain areas integrate sensory input from different modalities including vision and associate these neural representations with good or bad experiences. It is unclear, however, how distinct sensory memories are processed in the Drosophila brain. Furthermore, the neural circuit underlying colour/intensity memory in Drosophila remained so far unknown. In order to address these questions, I established appetitive and aversive visual learning assays for Drosophila. These allow contrasting appetitive and aversive visual memories using neurogenetic methods for circuit analysis. Furthermore, the visual assays are similar to the widely used olfactory learning assays and share reinforcing stimuli (sugar reward and electric shock punishment), conditioning regimes and methods for memory assessment. Thus, a direct comparison of the cellular requirements for visual and olfactory memories becomes feasible. I found that the same subsets of dopamine neurons innervating the mushroom body are necessary and sufficient for formation of both sensory memories. Furthermore, expression of D1-like Dopamine Receptor (DopR) in the mushroom body is sufficient to restore the memory defect of a DopR null mutant (dumb-). These findings and the requirement of the mushroom body for visual memory in the used assay suggest that the mushroom body is a site of convergence, where representations of different sensory modalities may undergo associative modulation.

The fixation of beneficial variants leaves genomic footprints characterized by a reduction of genetic variation at linked neutral sites and strong, localized allele frequency differentiation among subpopulations. In contrast, for phenotypic evolution the effect of adaptation on the genes controlling the trait is little understood. Theoretical work on polygenic selection suggests that fixations of beneficial alleles (causing selective sweeps) are less likely than small-to-moderate allele frequency shifts among subpopulations. This thesis encompasses three projects in which we have experimentally addressed the issue of selective sweeps vs. allele frequency shifts in the context of polygenic adaptation. We studied three X-linked QTL underlying variation in chill coma recovery time (CCRT), a proxy for cold tolerance, in Drosophila melanogaster from temperate (European) and tropical (African) environments. The analysis of these QTL was performed by means of selective sweep mapping and quantitative complementation tests coupled with expression assays. While the results of the selective sweep mapping approach identified a gene (CG4491) that is unlikely to be affecting CCRT, quantitative and gene expression analyses revealed two linked candidate genes (brk and CG1677) that appear to differ in their evolutionary histories. We found that the difference in expression of the gene brk between populations affects CCRT variation. Cold tolerant flies from the temperate zone have a lower expression of this gene than cold sensitive flies from the tropics. We found that a likely cause of this difference is variation in a cis-regulatory element in the brk 5’ enhancer region. Sequence variants in this element exhibit moderate frequency differences between populations from temperate and tropical environments, forming two latitudinal clines: one from the equator to the north and another one in opposite direction to the south. In contrast, the other gene within the same QTL (CG1677), which is linked to brk, showed no measurable effect on cold tolerance but is a likely target of strong positive selection leading to a selective sweep in the European population. These results are consistent with the aforementioned theoretical predictions about footprints of selection in polygenic adaptation. They are also proof of the conceptual bias incurred when identifying candidate genes within a QTL via selective sweep mapping, at least in naturally evolving populations. The challenge for the evolutionary genetics community in the coming years is to develop statistical tools that are as powerful and robust as those already available to map selective sweeps to identify sites in the genome where allele frequency shifts have occurred due to adaptive evolution at the phenotypic level. Finally, the last section of the results is a report of a new population genetics dataset. It consists of a collection of 80 inbred lines from a natural D. melanogaster population in Sweden and 19 full genome sequences derived from this sample. We hope this material will provide us with further insight into the processes underlying adaptation to novel and stressful environments.

The central goal of this dissertation is to understand the genetic and functional aspects of how populations adapt to new or changing environments. Genetic variation within a population, either at protein coding genes or at regulatory elements, provides the substrate upon which natural selection can act to drive adaptation. There is considerable evidence that changes in gene expression account for a large proportion of morphological, physiological and behavioral variation between and within species that can contribute to adaptation and speciation. Due to the major role that gene expression changes can have in shaping phenotypes, the first three chapters of this dissertation deal with the study of how changes in gene expression can facilitate adaptation. I use Drosophila melanogaster from ancestral and derived regions of the species' range as a model system for studying local adaptation. In chapter 1, I perform high-throughput RNA-sequencing (RNA-seq) of brain tissue of flies from an ancestral (Zimbabwe) and a derived (the Netherlands) population. The whole brain transcriptome was assayed for differences in gene expression between African and European flies in order to understand how differences in brain expression may lead to local adaptation. I found over 300 candidate genes that differed significantly in expression between the populations, including a cluster of genes on chromosome arm 3R that showed reduced expression in Europe and genetic evidence for positive selection. Other candidate genes involved in stress response, olfaction and detoxification were also identified. Additionally, I compared brain gene expression between males and females and found an enrichment of sex-biased genes on the X chromosome. Chapter 2 presents a detailed study of one of the candidate genes identified in chapter 1. The metallothionein gene, MtnA, shows over four-fold higher expression in the brain of European flies than of African flies. I found a derived deletion in the 3’ untranslated region (UTR) of MtnA that segregates at high frequency within the Dutch population, but is absent from the Zimbabwean population. The presence of the deletion was perfectly associated with the observed variation in MtnA expression. When additional populations of D. melanogaster were screened for the presence of the deletion, I found that it showed a clinal distribution that was significantly correlated with latitude and temperature. Furthermore, using population genetic data and a selective sweep analysis I show that the MtnA locus is evolving under positive selection. In Chapter 3 I report a population genetic analysis of the enhancer region of CG9505, a gene that shows significantly higher expression in European than in African populations of D. melanogaster. A previous study found that there was very low nucleotide polymorphism in the enhancer region of CG9509 in flies from the Netherlands, a pattern that is consistent with a selective sweep. I analyzed an additional set of five populations from Zambia, Egypt, Malaysia, France and Germany in order to gain a better understanding of how selection has affected the evolution of this enhancer. I found that there is a depletion of nucleotide diversity in all of the non-sub-Saharan populations (Egypt, Malaysia, France and Germany), which share a common high-frequency derived haplotype. Population genetic analyses suggest that a selective sweep took place in the enhancer region of CG9509 just after D. melanogaster migrated out of sub-Saharan Africa. Finally, in chapter 4 I performed in situ hybridizations to examine the expression of tissue-specific reporter genes in the D. melanogaster testis. In the male germline of D. melanogaster, reporter genes that reside on the X chromosome show a reduction in expression relative to those located on the autosomes. This phenomenon was demonstrated by randomly inserting reporter gene constructs on the X chromosome and the autosomes. By doing in situ hybridizations on testis of flies having reporter gene insertions on the X chromosome and autosomes, I could show that the expression difference mainly occurs in meiotic and post-meiotic cells. For most constructs, expression was very low or absent in the testis apex, which is enriched with pre-meiotic cells. These results suggest that the suppression of X-linked gene expression in the Drosophila male germline occurs through a different mechanism than the MSCI (meiotic sex chromosome inactivation) known to occur in mammals.

Mon, 30 Jun 2014 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/17122/ https://edoc.ub.uni-muenchen.de/17122/1/Braecker_Lasse_B.pdf Bräcker, Lasse Björn ddc:570, ddc:500, Fakultät für Biologie

When confronted with a large-field stimulus rotating around the vertical body axis, flies display a following behaviour of the head and steer in the direction of motion. As neural control elements for this so-called 'optomotor response', the large tangential horizontal cells (HS-cells) of the lobula plate have been the prime candidates for long. When HS-cells are surgically damaged or genetically removed, flies display reduced optomotor responses. To provide a better understanding of the role of HS-cells in the control of optomotor behaviour three approaches were taken. First, experiments were designed to investigate which of the HS-cells could be participating in head yaw movements in fixed flies and yaw turning behaviour during tethered flight. Horizontal motion at different elevations was presented to the flies. Comparison of the optomotor responses with HS-cell receptive fields suggests that HSN and HSE participate in head yaw movements whereas all three HS-cells are used to control yaw turning behaviour during flight. Second, to test whether HS-cells are sufficient to elicit yaw optomotor responses, a bi-stable Channelrhodopsin-2 variant 'ChR2 (C128S)' was expressed in HS-cells using the Gal4 / UAS-system. Combining a blue light stimulus with ChR2(C128S) allowed to activate HS-cells without presenting a visual stimulus to the eye of the fly. These experiments revealed that blue light was sufficient to evoke robust head yaw movement in fixed flies as well as turning behaviour in tethered flying flies, thus, mimicking front-to-back visual stimulation on the stimulated side. Third, the role of the receptive field layouts of HS-cells for optomotor responses was studied. Flies with a gain-of-function of a single Dscam1 isoform in all HS-cells (Dscam gain-of-function (D(GOF)) flies) were tested. Compared with HS-cells of control flies, HS-cells of D(GOF) flies show reduced sensitivity to horizontal motion in the frontal and enhanced sensitivity to motion in the lateral part of visual space. The optomotor response of tethered flying flies were analyzed. Compared with control flies, D(GOF) flies responded significantly weaker to visual stimuli extending over the entire azimuth extension of HS-cell receptive fields. Stimulating flies with additional motion in the rear part of visual space significantly reduced optomotor responses of control flies, whereas (GOF) flies responded to both visual stimuli with about equal strength. Although D(GOF) HS-cells had dramatically reduced sensitivity to motion in the frontal part of visual space, D(GOF) flies responded robustly to motion in this region of visual space. D(GOF) and control flies also showed differences in head yaw movements. These behavioural differences did not correlate with the difference in the receptive fields of D(GOF) and control HS-cells. The experiments indicate that HS-cells are sufficient to trigger yaw turns of the head and whole body. All three HS-cells control body turns during flight and head yaw turns are controlled by HSN- and HSE-cells. The layout of HS-cell receptive fields, however, does not correlate 1 : 1 with optomotor responses. During flight, flies rely additionally on cells sensitive in the frontal part of visual space. Furthermore, the layout of the HS-cell receptive fields is important for incorporating motion information in the rear part of visual space to the optomotor responses.

The main goal of this thesis was to develop demographic models of the fruit fly Drosophila melanogaster using Approximate Bayesian Computation and Next Generation Sequencing Data. These models were used to reconstruct the history of African, European, and North American populations. Chapter 1 deals with the demographic history of North American D. melanogaster. This project was motivated by the release of full-genome sequences of a North American population, which showed greater diversity than European D. melanogaster although the introduction of the fruit fly to North America dates back to only �200 years ago. Here, we tested di�erent demographic models involving populations of Zimbabwe, The Netherlands, and North Carolina (North America). Among the tested models we included variants with and without migration, as well as a model involving admixture between the population of Africa and Europe that generated the population of North America. We found that the admixture model �ts best the observed data and we estimated the proportion of European and African admixture in the North American population. This population has 85% European and 15% African ancestry. We also estimated other population parameters including population sizes (current and ancestral) and divergence times. Con�cerning previous studies we also estimated the divergence between African and European populations to be around 19,000 years ago. Chapter 2 deals with gene flow of D. melanogaster between African and European populations. Gene flow in D. melanogaster is well acknowledged but has not been quanti�ed using DNA sequence data. Previous studies from the late 80's based on allozymes found that the number of migrants per generation (Nm) was around 2 between several populations distributed worldwide. Here we used ABC methods and full-genome sequences to estimate the rate of migration between a population from Rwanda in Africa and a population from France. We found that Nm is around 10, which may imply there was a signi�cant increase of gene flow in the last few decades. Our estimates show that the migration rate between these two populations is not necessarily symmetrical, with migration from Europe to Africa being higher than the opposite, although the di�erence does not seem to be significant. The study of gene flow is relevant because it constitutes an important force in population genetics. Theoretical studies have shown that, under neutrality, it is enough to have one migrant per generation to stop two populations from diverging and speciating, and if migration is strong enough it can also overcome the e�ect of selection. Chapter 3 focuses on the sequencing of 130 full genomes of D. melanogaster from Africa and 9 from France. This project made use of haploid embryos, a new technique introduced in 2011 that allows the development of haploid D. melanogaster, which is then used for sequencing. The main goal of this project was to characterize these populations in terms of their diversity, admixture, and di�erentiation. We found that the most diverse population comes from Zambia, which is now thought to be much closer to D. melanogaster 's center of origin. We also found a signi�cant amount of non-cosmopolitan admixture in several African populations, meaning that there exists a signi�cant amount of back migration from Europe to Africa (corroborating the fi�ndings of chapter 2). In order to identify admixture tracts a new method was developed for this purpose, which uses a hidden Markov model to locate admixed regions along the genome. Admixed regions, as well as regions showing high levels of identity by descent were masked for downstream population genetics analyses. These full genomes constitute the second e�ort of the Drosophila Population Genomics Project (DPGP 2) and are now available for the scienti�c community.

Drosophila melanogaster is a model organism to study innate immunity in invertebrates. Temperate and tropical D. melanogaster populations, being exposed to different environments, potentially face distinct parasites and parasite pressure. Indeed, there is experimental evidence suggesting that tropical D. melanogaster populations survive longer than temperate ones to infection by the fungal parasite Beauveria bassiana. In the present work we test the generality of this conclusion and investigate if host populations differ in their molecular response to infection. We first exposed to B. bassiana infection two tropical (from Africa and South-East Asia) and two temperate (from Europe and North America) D. melanogaster out-crossed populations. We consistently found a significant effect of B. bassiana on Drosophila mortality, but we were not able to identify a significant difference in survival to infection among populations. These results indicate that tropical populations may not always survive better than temperate ones, and suggest that other environmental factors, such as humidity or local species richness may be more accurate predictors of immune competence. Subsequently, we recorded transcriptional response to B. bassiana in all D. melanogaster populations, both by microarray and RNA sequencing. To our knowledge this is the first time that transcriptional response to fungal infection has been determined in multiple D. melanogaster out-crossed populations. We found few or no genes significantly induced 8 hours after infection. On the other hand, we identified between 200 and 1,300 genes induced 24 hours after infection depending on the population. This means that transcriptional response to B. bassiana begins between 8 and 24 hours after infection. We reveal here that host populations respond differently at the molecular level, as shown by the large variation in the number of induced genes. We report that gene ontology categories related to translation, biosynthesis and reproduction are enriched in genes down-regulated upon infection, suggesting a metabolic cost of mounting the defence response. Next, we wanted to assess the selective pressures acting on induced candidate genes. We compared the genes induced in all populations to the ones induced specifically in each population and computed population genetic statistics for a subset of genes in each category. We noticed higher conservation at non-synonymous sites for commonly induced genes compared to population specific ones. This hints that common genes are under stronger selective constraints. Another topic we addressed in the present work is the effect of endosymbionts and trans-generational immune priming on D. melanogaster survival to B. bassiana. We tested for a protective effect of the endosymbiont Wolbachia pipientis in two D. melanogaster inbred lines. We did not find an effect of Wolbachia on survival to infection in two independent experimental replicates. In absence of infection, flies bearing Wolbachia had a lower fitness than cured ones. Therefore W. pipientis appears to have a negative effect on Drosophila general vigour, but no effect on mortality upon infection. Finally, we tested if flies whose parents were exposed to B. bassiana were less susceptible when infected by the same parasite. This would imply a transfer of immune memory from parents to offspring, which is called trans-generational immune priming. However, no evidence of immune transfer for two D. melanogaster out-crossed populations could be found. Yet, as trans-generational immune priming depends on host and parasite genotype, more experiments are needed to determine its generality in the D. melanogaster – B. bassiana system.

Ken Irvine and Gongping Sun explain how JNK signaling leads to nuclear accumulation of the transcription factor Yorkie through phosphorylation of an adaptor protein.

Calcium signaling is required for long-term memory in fruit flies.

Tue, 26 Mar 2013 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/16940/ https://edoc.ub.uni-muenchen.de/16940/1/Mirkovic-Hoesle_Milijana.pdf Mirkovic-Hösle, Milijana ddc:540, ddc:500, Fakultät

Fri, 15 Jun 2012 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/14568/ https://edoc.ub.uni-muenchen.de/14568/1/Hartl_Marion.pdf Hartl, Marion ddc:540, ddc:500, Fakultät für Chemie und Pharmaz

Thu, 26 Apr 2012 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/14413/ https://edoc.ub.uni-muenchen.de/14413/1/Ohler_Stephan.pdf Ohler, Stephan ddc:570, ddc:500, Fakultät für Biologie

Grazia D. Raffa, Dipartimento di Biologia e Biotecnologie, Universita’ “La Sapienza”, Rome, ITALY speaks on "Organization and evolution of Drosophila melanogaster telomeres". This seminar has been recorded by ICGEB Trieste

A long-standing question in evolutionary biology concerns the molecular causes underlying adaptive evolution. These can either stem from structural changes in proteins or from changes in the expression patterns of proteins or mature RNAs. Over the last decade, many studies have shown that gene expression changes can have a huge impact on the phenotype of an organism and play an important role in adaptive evolution. A major prerequisite for adaptive evolution to occur at the gene expression level is the presence of expression variation among members of a population. This variation serves as the raw material for adaptive evolution. The genetic causes underlying changes in expression patterns can either be located in cis-regulatory regions of the affected gene, such as transcription factor binding sites, or in trans-regulatory regions, such as transcription factors. Mutations in cis-regulatory elements have relatively few pleiotropic effects and their effects are often additive, thus, cis-regulatory changes are thought to be especially well-suited targets of selection. A major factor influencing gene expression is the sex of an organism. The sex-bias of a gene also influences the pace at which proteins evolve, such that male-biased genes often show more rapid evolution than female-biased or unbiased genes between Drosophila species. Here, we investigated genome-wide gene expression variation in adult females of two populations of D. melanogaster, one from the ancestral species range (Zimbabwe) and one from the derived species range (the Netherlands). We found relatively little expression polymorphism present within the populations and high expression divergence between the populations. More than 500 genes were expressed differentially between the populations. These are candidate genes for those that have undergone adaptive regulatory evolution to the new, derived environment. When comparing our study of female adults to a study investigating male adult flies of the same populations, we found that there is significantly less expression polymorphism in females within the populations but significantly more expression divergence between the populations. Further, there was little overlap in genes that differ in expression between the populations in males and females. This suggests that general differences exist between the sexes in gene expression regulation and that regulatory evolution has been mainly sex-specific. Our findings show that extensive gene expression variation exists in D. melanogaster and further highlight the importance of accounting for sex when investigating gene expression. In order to elucidate the genetic and evolutionary mechanisms that underlie differential gene expression between the populations, we employed a candidate gene approach. Analysis of molecular variation in the coding and upstream regions of several differentially expressed genes in both populations revealed evidence for a recent selective sweep in the European population for the gene CG34330. In the putative promoter region of the gene, there is one indel and one SNP where a derived variant is fixed in the European population, but at low frequency in the African population. These are candidates for those variants that control the expression level of the gene. For another gene, Jon99Ciii, we found evidence for recurrent structural protein evolution acting since the split of D. melanogaster from D. simulans and D. sechellia. However, no evidence for recent regulatory evolution could be found for this gene. Motivated by findings that male-biased genes often evolve faster than both female- and unbiased genes between Drosophila species, we examined the molecular evolution of sex-biased genes and their contribution to within-population polymorphism, between-population divergence and between-species divergence in D. melanogaster and D. ananassae. This was studied on both the DNA-sequence level and the expression level. We found strong purifying selection limiting protein sequence variation within species. In contrast, a high proportion of divergence could be attributed to positive selection. In D. melanogaster, male-biased genes showed the highest fraction of adaptive substitutions, a pattern that was especially pronounced on the X chromosome. In contrast, male-biased genes did not show higher variation within or between populations, suggesting that inter-species divergence is not just a simple extension of inter-population divergence and intra-population variation. For D. ananassae, we did not observe a higher rate of adaptive evolution for male-biased genes, a finding that suggests that the type or strength of selection acting on sex-biased genes differs between lineages. Similarly, on the expression level, we found that sex-biased genes show high expression divergence between species, but low divergence between populations.

Background: Changes in gene regulation are thought to be crucial for the adaptation of organisms to their environment. Transcriptome analyses can be used to identify candidate genes for ecological adaptation, but can be complicated by variation in gene expression between tissues, sexes, or individuals. Here we use high-throughput RNA sequencing of a single Drosophila melanogaster tissue to detect brain-specific differences in gene expression between the sexes and between two populations, one from the ancestral species range in sub-Saharan Africa and one from the recently colonized species range in Europe. Results: Relatively few genes (

Mon, 7 Nov 2011 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/13794/ https://edoc.ub.uni-muenchen.de/13794/1/Esslinger_Stephanie.pdf Esslinger, Stephanie Maria ddc:540, ddc:500, Fakultät für Chem

A combination of functional and proteomics approaches identifies regulators of ERK signaling in Drosophila.

Summary The present work is focused on the identification of positively selected genes that are involved in local adaptation in European Drosophila melanogaster. This species is globally distributed as human commensal and occupies almost every ecozone. The ancestral range, however, is afrotropical and the questions arise how and when the fruit fly managed to invade new environments that differed in environmental parameters. One phenotype that might have been of crucial importance for the cosmopolitan distribution of an insect species is cold tolerance. Using fly samples from the ancestral range of tropical Africa and the derived range of temperate Europe we compared their cold tolerance by measuring the time they need to recover from a cold induced chill coma. We picked the most divergent African and European fly lines as parental lines and created a huge population of X chromosomal recombinants. We searched their X chromosome for quantitative trait loci (QTL) that caused phenotypic divergence between the parental lines and identified several loci that were associated with chill coma recovery time. Subsequently, we went back to the original population samples from Africa and Europe and characterized a European selective sweep that was co-localized with one QTL. We established a novel colonization model to tackle the question when D. melanogaster spread from Africa and invaded new environments, such as Europe and Asia. We sequenced ~280 fragments of the X and third chromosome of an Asian population sample and aligned them with the corresponding fragments of the African and European sample that were already sequenced before. By means of Approximate Bayesian Computation (ABC) we found one common ancestor of European and Asian D. melanogaster that left Africa around 16,800 years ago. We reject an ancient colonization event from Africa to Asia, which could have led to the strongly divergent Asian phenotype of the ‘Far East Race’. A formerly performed genome scan of X-linked genetic variation of the European and African sample revealed interesting candidates of European-specific adaptation. To analyze one candidate region more closely we conducted a follow-up study and sequenced the entire candidate in both population samples. We found multiple European specific genetic variants one of which was an insertion/deletion polymorphism that generates a new transcript of the flotillin-2 gene. This transcript (Flo-2-C) is unique to D. melanogaster and encodes a truncated version of flotillin, a membrane-anchoring scaffolding protein. An expression analysis revealed that the Flo-2-C transcript is expressed in most fly lines independent of the gene structure in third instar larvae. Thus, a disordered gene structure does not prevent the process of transcription and might reflect a young gene variant.

Thu, 26 May 2011 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/13137/ https://edoc.ub.uni-muenchen.de/13137/1/Aumiller_Verena_N.pdf Aumiller, Verena Nicole ddc:540, ddc:500, Fakultät für Chemie

Wed, 18 May 2011 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/13089/ https://edoc.ub.uni-muenchen.de/13089/1/Kemkener_Claus.pdf Kemkemer, Claus Stefan Oliver ddc:570, ddc:500, Fakultät für

A protein required for light sensation also plays a role in thermosensing in the fruit fly.

The goal of this study was to gain a deeper understanding of the selective sweep models and the statistical and computational methods that disentangle selective sweeps from neutrality. In the Introduction of the thesis I review the literature on the main approaches that have been developed in the last decade to separate selective sweeps from neutral demographic scenarios. Methods on complete and incomplete selective sweeps are reviewed as well as selective sweeps on structured populations. Further, I analyze the effects of past demographic events, especially bottlenecks, on the genealogies of a sample. Finally, I demonstrate that the ineffectiveness of separating selective sweeps from bottlenecks stems from the lack of robust statistics, and most importantly from the similar genealogies that bottlenecks and selective sweeps may generate locally on a recombining chromosome. In the first chapter I introduce a method that combines statistical tests in a machine learning framework, in order to disentangle selective sweeps from neutral demographic scenarios. The approach uses support vectormachines to learn examples from neutral scenarios and scenarios with selection. I demonstrate that the novel approach outperforms previously published approaches for a variety of demographic scenarios. The main reason for the performance difference is the usage of the scenarios with selection, that are not analyzed by classical statistical methods. In the second chapter of the thesis I present an application of the methods on detecting a selective sweep in the African population of D. melanogaster. Demographic history and ascertainment bias schemes have been taken into account. Results pinpoint to the HDAC6 gene as a target of recent positive selection. This study demonstrates the variable threshold approach, which remedies the tendency of some neutrality tests to detect selective sweeps at the edges of the region of interest. In the third chapter I present the results of the analysis of selective sweeps in multi-locus models. I assume that a phenotypic trait evolves under stabilizing or directional selection. In contrast to the classical models of selective sweeps, the evolutionary trajectory of an allele that affects the trait might belong to one of the three categories: it either fixes, disappears or remains polymorphic. Thereafter, I analyze the properties of coalescent trees and neutral polymorphism patterns that are generated from each of the three categories. I show that for the majority of simulated datasets selection cannot be detected unless the trajectory is either fixed or close to fixation.

The aim of this thesis was to use the genealogical information contained in genetic variation profiles of natural populations to describe the evolution of a particular species. In the first project we analysed the colonization process that brought Drosophila melanogaster from Africa to Asia. Southeast Asian populations of the fruit fly D. melanogaster differ from ancestral African and derived European populations by several morphological characteristics. It has been argued that this morphological differentiation could be the result of an early colonization of Southeast Asia that predated the migration of D. melanogaster to Europe after the last glacial period (around 10,000 years ago). To investigate the colonization process of Southeast Asia, we collected nucleotide polymorphism data for 200 X-linked and 50 autosomal loci from a population of Malaysia. We analysed this new SNP dataset jointly with already existing data from an African and a European population by employing an Approximate Bayesian Computation (ABC) approach. By contrasting different demographic models of these three populations, we do not find any evidence for an early divergence between the African and the Asian populations. Rather, we show that Asian and European populations of D. melanogaster share a non-African most recent common ancestor (MRCA) that existed about 2500 years ago. The second project of my PhD thesis is an analysis of the importance of seed dormancy at the population level in two wild tomato species. Seed banks, that is, plant seeds remaining in soils for several generations before germination, are of practical importance in conservation biology because they diminish the immediate ecological impact of habitat fragmentation and prevent species extinction. From an evolutionary perspective, seed banks increase the genetic diversity of plant populations and buffer the effect of varying climatic conditions by magnifying the effects of good years and by dampening the effects of bad years. In this study we estimate the germination rates for two wild tomato species (Solanum chilense and Solanum peruvianum) found in western South-America in a wide range of habitats by using DNA sequences coupled to a coalescent model in combination with ecological data. We develop an ABC framework to integrate ecological information on above ground population census sizes, in order to estimate seed bank and metapopulation parameters for each species. We provide the first evidence that it is possible to disentangle the effect of the metapopulation structure from that of the seed bank on the effective population size and to obtain accurate estimates of germination rates based on a coalescent model. The third and last project of this thesis is related to the development of a computational tool that facilitates the analysis of nucleotide polymorphism datasets in an ABC framework. With the availability of whole-genome sequence data, biologists are able to test hypotheses regarding the demography of populations. Furthermore, the advancement of the ABC methodology allows the demographic inference to be performed in a simple framework using summary statistics. We present here msABC, a coalescent-based software that facilitates the simulation of multi-locus data, suitable for an ABC analysis. msABC is based on Hudson's ms algorithm, which is used extensively for simulating neutral demographic histories of populations. The flexibility of the original algorithm has been extended so that sample size may vary among loci, missing data can be incorporated in simulations and calculations, and a multitude of summary statistics for single or multiple populations is generated. The source code of msABC is available at http://bio.lmu.de/~pavlidis/msabc.

Background: Variation at the level of gene expression is abundant in natural populations and is thought to contribute to the adaptive divergence of populations and species. Gene expression also differs considerably between males and females. Here we report a microarray analysis of gene expression variation among females of 16 Drosophila melanogaster strains derived from natural populations, including eight strains from the putative ancestral range in sub-Saharan Africa and eight strains from Europe. Gene expression variation among males of the same strains was reported previously. Results: We detected relatively low levels of expression polymorphism within populations, but much higher expression divergence between populations. A total of 569 genes showed a significant expression difference between the African and European populations at a false discovery rate of 5%. Genes with significant overexpression in Europe included the insecticide resistance gene Cyp6g1, as well as genes involved in proteolysis and olfaction. Genes with functions in carbohydrate metabolism and vision were significantly over-expressed in the African population. There was little overlap between genes expressed differently between populations in females and males. Conclusions: Our results suggest that adaptive changes in gene expression have accompanied the out-of-Africa migration of D. melanogaster. Comparison of female and male expression data indicates that the vast majority of genes differing in expression between populations do so in only one sex and suggests that most regulatory adaptation has been sex-specific.

Visual motion detection is of major importance for flies as they use the optic flow generated by their self-motion to control their course during flight. This so called optomotor behavior is thought to be controlled by a set of large-field motion-sensitive cells in the optic lobes called lobula plate tangential cells (LPTCs). LPTCs come in different variants and are tuned to different preferred directions. Their responses can be explained by assuming input from an array of local motion detectors of the correlation-type. In addition, they receive input from other LPTCs from both the ipsi- and the contralateral hemisphere. Response properties of LPTCs have been extensively described in large fly species. However, information about the presynaptic circuits that constitute the local motion detectors is still largely missing. Research on the fruit fly Drosophila promises to close this gap as it allows for combining physiological recordings from motion-sensitive cells with a genetic manipulation of the system. In that way the function of neurons too small for electrophysiological recordings can also be analyzed. Here, I provide important steps towards elucidating the cellular implementation of the correlation-type motion detector in the fly brain. First, I tested different genetically encoded Calcium indicators (GECIs) expressed in LPTCs by stimulating the neurons with potassium chloride. These experiments revealed that GECIs are functional in LPTCs and might thus be useful for monitoring neuronal activity in the visual system. Second, I described the response properties of HS (horizontal system) cells, a prominent subgroup of LPTCs in Drosophila. There are three HS cells per hemisphere, HSN, HSE and HSS. All of them are tuned to horizontal motion in a directionally selective way. I could show that their responses are indicative of correlation-type motion detectors providing input to them. In addition, they receive information from the contralateral side most likely via other LPTCs. HS cells not only have strongly overlapping dendritic trees in the lobula plate accounting for their large and overlapping receptive fields, but are also coupled electrically with each other. Extensive electrical connections can also be found to descending neurons in their output region in the central brain. This characterization of HS cells is important for two reasons: i) Their responses can be used as a read-out for the effects of manipulating the presynaptic motion detection circuitry in the fly by genetic techniques; ii) they can be correlated with behavioral reactions induced by horizontal motion to study how optomotor responses are controlled in the fly. Third, I studied the input pathways to the LPTCs in the lamina, the first optic neuropile after the compound eye. From all lamina cells, L1 and L2 are the most prominent neurons and were previously shown to provide the major input to the motion detection circuits. By genetically restoring synaptic input to either one of the two pathways I revealed that these two types of cells indeed provide the major input to LPTCs. However, their functional specialization for light increments and light decrements, disclosed by blocking their synaptic output, could not be revealed in these experiments. As L1 and L2 turned out to be electrically coupled with each other restoring the input to only one cell type also restores the input to the other one. Finally, I analyzed response properties of HS cells whose dendritic structure has been altered by overexpression of Dscam (Down syndrome cell adhesion molecule) during development. Dscam is a protein that comes in a large number of different isoforms and is thought to play a major role in self-recognition and thus proper dendritic and axonal branching. HS cells that misexpress a single isoform develop smaller and less overlapping dendritic trees in the lobula plate. These anatomical defects are accompanied by smaller receptive fields but otherwise normal motion responses. All these experiments show that the combination of physiological and genetic tools is a promising approach for dissecting neural circuits and gaining new insights into information processing in the brain. Continuation of this approach will hopefully bridge the gap between neurons of the lamina and the lobula plate by revealing the local motion detectors in the intermediate neuropile, the medulla.

Lateral inhibition in proneuronal clusters in the fruit fly relies on competition between cis and trans Notch signaling.

Thu, 26 Nov 2009 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/12049/ https://edoc.ub.uni-muenchen.de/12049/1/Joesch-Krotki_Maximilian.pdf Jösch Krotki, Maximilian Albert ddc:570, ddc:500, Fakultät für Biologie

Thu, 22 Oct 2009 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/10890/ https://edoc.ub.uni-muenchen.de/10890/1/Loiberstetter_Angelika.pdf Loiberstetter, Angelika ddc:610, ddc:600, Medizinische

The aim of the present work was to identify the genes that played a role in ecological adaptation in D. melanogaster. This species, which originated in Africa, successfully adapted to a broad range of climates during the last 100.000 years. To find the genes involved, I used two different approaches: (1) a genomic region containing several ecologically relevant candidate genes and putatively carrying footprints of selection was investigated using selective sweep mapping, and (2) cold tolerance that might have been an important phenotype for the adaptation to the temperate climates was investigated using a QTL analysis. Using the technique of selective sweep mapping pioneered in the Stephan’s group, I detected evidence that recent strong positive selection has been acting on a small DNA region of 2.7 kb overlapping with the 3’ end of the HDAC6 gene in the ancestral African population. This gene codes for a newly characterized cell stress surveillance factor. HDAC6 is an unusual histone-deacetylase. It is localized in the cytoplasm and has a ubiquitin-binding and a tubulin-deacetylase activity. These properties make HDAC6 a key regulator of cytotoxic stress resistance. The phenotypic analyses show that the African and the European populations have very strong cold tolerance differences. By removing the effects of the autosomes, I showed that a significant amount of the phenotypic variance is due to genetic factors carried by the X chromosome. These factors were then more precisely mapped to two genomic regions of the X chromosome. By comparing the present results with other association studies and the Gene Ontology database, it was possible to determine a list of candidate genes influencing cold tolerance in D. melanogaster. As this list is limited to a very small number of genes, additional investigations for footprints of selection in these regions may be used to confirm their role in ecological adaptation.

Die in dieser Dissertation präsentierten Ergebnisse tragen aus dem Blickwinkel der Evolutionsbiologie zu unserem Verständnis der Regulation von Genexpression bei. Ich verwende einen bestens bekannten Modellorganismus, die Fruchtfliege Drosophila melanogaster, nicht nur als Objekt der Beobachtung, sondern auch als ein genetisches Manipulationswerkzeug, und untersuche drei verschiedene Aspekte des Prozesses, durch den die in der DNA gespeicherte Information förmlich „entfesselt“ oder umgesetzt wird zu biologischem Sinn, letztlich also zu Form und Funktion. In Kapitel 1 zeige ich zunächst, dass eine Inaktivierung des X-Chromosomes (und somit Genregulation auf chromosomaler Ebene) in der männlichen Keimbahn von D. melanogaster stattfindet. Im Gegensatz zur X-Inaktivierung in weiblichen Säugetieren, wo dies in den somatischen Zellen als Mechanismus zur Dosiskompensation auftritt, ist diese Art der Inaktivierung auf die Spermatogenese beschränkt und wurde wahrscheinlich während der Genomevolution als eine Möglichkeit etabliert, schädliche Auswirkungen in Zusammenhang mit Sexualantagonismus zu umgehen. Durch P-Element-vermittelte Keimbahntransformation erhielt ich fast 50 unabhängige Insertionen eines testisspezifischen Reportergenkonstrukts und untersuchte die dazugehörigen Reportergenaktivitäten durch Messung der Enzymaktivität und durch quantitative RT-PCR. Autosomale Insertionen dieses Konstrukts zeigten das erwartete Muster hoher männchen- und testisspezifischer Expression. Insertionen auf dem X-Chromosom zeigten dagegen wenig bzw. gar keine Expression des Transgens. Da die X-chromosomalen Insertionen die euchromatischen Abschnitte des Chromosoms abdeckten (bestimmt durch inverse PCR), konnte eine systematische Bevorzugung bestimmter Regionen bei Insertionen, die ein Fehlen von Expression auf dem X-Chromosom hätte erklären können, ausgeschlossen werden. Der Effekt scheint eine globale Eigenschaft des X-Chromosomes zu sein. Lediglich die Testisspezifität des transgenen Konstrukts ist für das Erscheinen des Effekts erforderlich, was somit eine Selektionshypothese für die X-Inaktivierung erhärtet sowie einige Beobachtungen erklären könnte, die im Zusammenhang mit der Verteilung von im Männchen und Testis exprimierten Genen im Drosophila-Genom gemacht wurden. In Kapitel 2 untersuche ich dann mutmaßliche cis-regulatorische Sequenzen und ihr Vermögen, allelspezifische Genexpression zu steuern. Nachdem Microarray-Studien umfangreiche Variabilität im Primärmerkmal Genexpression in unterschiedlichsten Taxa aufgedeckt haben, ist eine naheliegende Frage, mit der sich Evolutionsbiologen konfrontiert sehen, die nach der dieser Variabilität zugrunde liegenden genetischen Quelle. Neben epigenetischen Mechanismen gibt es einen Disput darüber, ob regulatorische Sequenzen nahe des exprimierten Gens (cis-Faktoren) und anderswo im Genom kodierte Faktoren (trans-Faktoren) einen qualitativ und quantitativ unterschiedlichen Beitrag zur Variabilität der Genexpression liefern. Hierzu wählte ich ein Gen von D. melanogaster, das nachweislich konsistente Expressionsunterschiede zwischen afrikanischen und nicht-afrikanischen („kosmopolitischen“) Stämmen zeigt, und klonierte die entsprechenden stromaufwärts flankierend gelegenen Teile jeweils in ein bakterielles Reportergenkonstrukt, um – nach erfolgreicher Integration ins Fruchtfliegengenom – direkt die von ihnen gesteuerte Auswirkung auf die Genexpression zu vergleichen. Der beobachtete Effekt war klein, jedoch signifikant, und zeigte sich nur in transgenen Fliegen, die ein X-Chromosom des afrikanischen Ausgangsstammes besaßen. Dies legt den Schluss nahe, dass zusätzlich zu den cis-regulatorischen Faktoren auch noch trans-Faktoren (vor allem auf dem X-Chromosom) zu dem zwischen den Stämmen beobachteten Expressionsunterschied beitragen. Letztendlich untersuche ich in Kapitel 3 das Phänomen des Codon bias durch seinen Zusammenhang mit Genexpression. Aufgrund der Redundanz des genetischen Codes werden viele der proteinogenen Aminosäuren durch mehr als ein Codon kodiert. Dies ermöglicht es, synonyme Codons in einer kodierenden Gensequenz auszutauschen, ohne dabei die Aminosäurensequenz des kodierten Polypeptids zu verändern. Ob dies Konsequenzen für die produzierte Proteinmenge hat (Translationseffizienz) ist Gegenstand dieses Kapitels. Ich verglich dabei die von zwei Allelen des Gens Alkoholdehydogenase (Adh) (von D. melanogaster) vermittelte Enzymaktivität direkt miteinander, welche sich in sieben Leucin-Codons unterschieden. Es ergab sich nahezu kein Unterschied in der ADH-Enzymaktivität, obwohl eines der Allele aus gänzlich optimalen Leucin-Codons bestand und das andere sieben suboptimale Leucin-Codons enthielt. Da Letzteres die Wildtypform von Adh war, legen die Ergebnisse den Schluss nahe, dass das Adh-Gen in seiner Leucin-Codonzusammensetzung (und vielleicht auch in seiner Codonzusammensetzung allgemein) bereits ausreichend optimiert ist. Weitere Versuche, die Zahl der optimalen Leucin-Codons zu erhöhen, können sogar einen Negativeffekt hinsichtlich der Enzymproduktion haben; dies möglicherweise aufgrund einer Sättigung des tRNA-Pools und/oder der Konsequenzen veränderter mRNA-Sekundärstrukturen.

Thu, 18 Dec 2008 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/10675/ https://edoc.ub.uni-muenchen.de/10675/1/Langer_Diana.pdf Langer, Diana ddc:540, ddc:500, Fakultät für

In this work, I investigate the role of gene regulatory changes in the evolution of Drosophila melanogaster. As a first step, I performed a survey of gene expression variation in the species using whole-genome microarrays. I surveyed eight strains from an ancestral African population and eight strains from a derived European population using an experimental design that allowed for the detection of expression differences within and between populations. Levels of gene expression variation were nearly equal within the two populations, but a higher amount of variation was detected in comparisons between the two populations. Most gene expression variation within populations appears to be limited by stabilizing selection. However, some genes that are differentially expressed between the two populations might be targets of positive selection. Some of these encode proteins associated with insecticide resistance, food choice, lipid metabolism, and flight. These genes are good candidates for studying adaptive regulatory evolution that accompanied the out-of-Africa migration of D. melanogaster. To verify the accuracy of the microarray experiments, I performed quantitative Real-Time PCR (qPCR), which is another method to measure gene expression, on a subset of genes. I compared the fold-changes in gene expression between pairs of strains determined by the two methods. I also compared the pattern of expression variation in male and female flies. The qPCR approach supported the general accuracy of the microarray experiments, as the fold-changes measured by the two techniques were highly correlated. Expression differences among the strains tended to be similar for male and females. However, exceptions to this general pattern could be found by looking at the pairwise fold-changes for individual genes, some of which differed in expression pattern between males and females. I also investigated the molecular evolution and population genetics of the protein-encoding and upstream regulatory regions of genes that have potentially undergone adaptive evolution at the gene-regulatory level. These genes represent a subset of the genes that showed a significant difference in gene expression between the African and European populations. A set of control genes, which showed no significant difference in expression between the two populations, was also included in the analysis. Overall, I found evidence for both positive and purifying selection in the coding and non-coding regions. However, patterns of polymorphism and divergence did not differ significantly between the candidate genes and the control genes. One of the genes that showed an interesting pattern of expression in the microarray and qPCR experiments was subjected to further, more detailed population genetic analysis. This gene, CG9509, has twofold higher expression in the European strains than in the African strains. The coding and the upstream regions of this gene show evidence of recurrent positive selection since the split of D. melanogaster and its close relative, D. sechellia. A polymorphism survey of the CG9509 region uncovered a 1.2-kb segment, which included the putative CG9509 promoter that showed no polymorphism in the European population. The European population also has several fixed or nearly-fixed derived mutations in this region. These observations, coupled with statistical analysis, provide evidence for a selective sweep in the European population. The selective sweep was likely driven by local adaptation at the level of gene expression.

Thu, 31 Jul 2008 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/8886/ https://edoc.ub.uni-muenchen.de/8886/1/Greiner_Dorothea_E.pdf Greiner, Dorothea ddc:600, ddc:61

Wed, 12 Sep 2007 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/7427/ https://edoc.ub.uni-muenchen.de/7427/1/Shivalkar_Madhuri.pdf Shivalkar, Madhuri

This work is dedicated to studying natural variation in D. melanogaster at the DNA sequence and gene expression level. In addition I present a new version of the DNA polymorphism analysis program VariScan, which includes significant improvements. In CHAPTER 1 I describe a genome scan of single nucleotide polymorphism in two natural D. melanogaster populations (from Africa and Europe) on the third chromosome. Together with polymorphism data previously published for the X chromosome of the same populations, this allows a comparative study of the polymorphism patterns of the X chromosome and an autosome. The frequency spectrum of mutations and the patterns of linkage disequilibrium are investigated. The observed patterns indicate that there is a significant difference in the behavior of the two chromosomes, as has already been suggested by previous studies. To uncover the reasons for this a coalescent based maximum likelihood method is applied that incorporates the effects of demographic history and unequal sex ratios. For the African population the differential behavior of the chromosomes can be explained by its demographic history and an excess of females. In Europe, a population bottleneck and an excess of males alone cannot explain the patterns we observe. The additional action of positive selection in this population is proposed as a possible explanation. In CHAPTER 2 I investigate the variation in gene expression of the two aforementioned populations. Whole-genome microarrays are used to study levels of expression for 88% of all known genes in eight adult males from both populations. The observed levels of expression variation are equal in Africa and Europe, despite the fact that DNA sequence variation is much higher in Africa. This is evidence for the action of stabilizing selection governing levels of expression polymorphism. Supporting this view, genes involved in many different functions, and are therefore on strong selective constraint, show less variation than do genes with only few functions. The experimental design allows the search for genes which differ in their expression patterns between Europe and Africa and might therefore have undergone adaptive evolution. Detected candidates include genes putatively involved in insecticide resistance and food choice. Surprisingly, many genes over-expressed in Africa are involved in the formation and function of the flying apparatus. In CHAPTER 3 I present version 2 of the program VariScan. This program was designed to analyse patterns of DNA sequence polymorphism on a chromosomal scale. The functionality of the core analysis tool, the wavelet decomposition, is described. In addition, multiple improvements to the previous version are presented. The program now supports the “pairwise deletion” option. This is essential for analysing data at the chromosome scale, since such data often contains incomplete information. It is now possible to add outgroup information, which allows the calculation of additional statistics. Furthermore, the separate analysis of different predefined chromosomal regions is added as an option. To increase the user friendliness, a graphical user interface is now included as part of the software package. Finally, VariScan is applied to published and computer-generated data and the ability of the wavelet-based analysis to uncover chromosomal regions with interesting DNA polymorphism patterns is demonstrated.

The development of dendrites leads to the establishment of cell-type specific morphology of dendritic trees that eventually determines the way in which synaptic information is processed within the nervous system. The aim of this study was to investigate dendritogenesis of Drosophila motion-sensitive Lobula Plate Tangential Cells (LPTCs) and to understand the role of cytoskeletal molecules in these developmental processes. I employed genetic techniques to obtain fluorescent labeling exclusively in the neurons of interest. In order to visualize the LPTCs confocal imaging was applied. Time point analysis allowed me to follow and describe the phases of LPTC differentiation in the intact Drosophila brain starting from the third instar larva throughout the pupal stages until adulthood. I determined the time when the initial growth of LPTC dendrites starts and showed it to be directional from the beginning. Additionally, I demonstrated that the phase of extensive dendritic growth and branching precedes reorganization processes that lead to establishment of the final architecture of LPTC dendritic trees. In parallel, I attempted to analyze the contribution of actin and tubulin in the shaping of the neurons. In these experiments actin-GFP localized to dendritic termini whereas tubulin-GFP was mainly observed in the primary dendritic branches. These data showed clear similarities between the cytoskeletal organization of LPTCs dendrites and vertebrate neurons. The discovery of the actin enrichment in dendritic termini made me conduct a set of experiments to test if these protrusions are the counterparts of vertebrate spines. I performed a thorough quantitative analysis of spine- like protrusions present on LPTC dendrites. Morphological features like the density and shape of the LPTC spine- like protrusions appeared to be comparable to hippocampal spines. Using immunohistochemical methods I demonstrated that LPTC spine-like protrusions are sites of synaptic contacts. The ultrastructural analysis supported the immunohistochemical data and showed that synaptic transmission takes place at the LPTC spine-like protrusions. Next, I tried to genetically modify these structures by generating LPTC mutant for genes which have vertebrate homologues known to alter spine morphology. I showed that dRac1 can modulate significantly the LPTC spine-like structure density. Finally, I tried to check if Drosophila LPTC spine-like structures are motile. To conclude, I showed an initial description of LPTC dendritogenesis and the subcellular localization of actin and tubulin in these neurons. The actin enriched spine-like structures detected on the LPTC dendrites are sites of synaptic contacts, thus resemble vertebrate spines.

The Retinoblastoma protein (pRb) was the first tumor suppressor protein to be identified. It is the founding member of the so called pRb or pocket protein family, comprising two additional members (p107 and p130) in mammalian cells, and its best characterized function is the regulation of the E2F family of transcription factors. Today, the pRb-E2F network represents one of the best understood pathways implicated in cell cycle regulation and differentiation. Pocket proteins negatively regulate the transactivation properties of E2F proteins by two mechanisms: First, binding of pocket proteins to E2F masks the E2F transactivation domain and thereby impairs transcriptional activation. Second, pocket proteins interact with several chromatin modifying and chromatin binding proteins and recruit these proteins to E2F target genes, where they help to establish a repressive chromatin conformation. In this work, advantage was taken of the relative simplicity of the Drosophila melanogaster pRb-E2F network to purify and functionally characterize native pRb repressor complexes. Two related multisubunit complexes that only differ in their pocket protein subunit (RBF1 or RBF2) have been purified from Drosophila embryo nuclear extract. These complexes contain several novel pocket protein-associated polypeptides and localize to transcriptionally silent regions on Drosophila polytene chromosomes. Moreover, they specifically associate with deacetylated histone tails, which are a hallmark of transcriptionally silent chromatin. In cycling Drosophila S2 cells, the purified complexes redundantly repress the expression of a certain class of E2F target genes implicated in differentiation and development, whereas they do not control the expression of cell cycle-regulated E2F targets. Interestingly, the isolated complexes seem to be highly conserved between different organisms. Genes encoding the Caenorhabditis elegans homologs of the complex subunits act within the same genetic pathway involved in vulval cell fate determination and they functionally cooperate in different developmental processes. Furthermore, a complex with striking homology to the Drosophila complexes also exists in human cells. In the light of the specific repression of developmentally regulated E2F target genes in cycling Drosophila cells, it is conceivable that the complexes prevent the uncontrolled expression of genes important during differentiation. Since the C. elegans homologs of the complex subunits are also involved in cell fate determination, this might be a highly conserved feature of the isolated complexes.

The goal of the work was to address the role of higher order nuclear architecture in the functional regulation of endogenous genes and transgenes in different species. In the first part of the work, 3D distance measurements were performed to analyze in WT flies and in different transgenic lines of Drosophila melanogaster (Cavalli and Paro, 1998; Zink and Paro, 1995) the nuclear localization of endogenous genes and transgenic constructs containing the Polycomb Response Element (PRE) Fab-7 relative to the nuclear periphery and heterochromatin. Transgenic constructs containing the Fab-7 element and three endogenous genes, Abd-B, sd, and Ubx, were first analyzed at their inactive state. The results showed that they were preferentially associated with the nuclear periphery and did not display specific associations with heterochromatin. The localization of the transgenic Fab-7 element was further analyzed at different states of activity. Activation of the transgenic Fab-7 element resulted in frequent (up to ~50%) association with the boundary of the heterochromatic domain. The percentages of such associations were tissue- and fl y line-dependent. Further investigations of the boundary of heterochromatin showed that this region has a complex organization, where euchromatic sites enriched in the active form of RNA pol II and trimH3K4, sites enriched in dimH3K4 and Pc-binding sites, as well as pericentromeric satellite DNA are exposed and juxtaposed towards each other. The concentration and specific architecture of such sites at the boundary of heterochromatin might help to maintain the equilibrium between activation and repression at this boundary. This specific environment might be favourable for maintaining PREs in the active state. I also investigated in three transgenic lines whether endogenous and transgenic copies of the Fab-7 element interact physically, using 3D distance measurements. In five tissues analyzed, no pairing between endogenous and transgenic copies of the Fab-7 element was observed. Also enhancement of Pc-mediated silencing did not induce pairing. Additionally, the general organization of Pc-binding sites was addressed in six larval tissues. We used different methods of microscopy and image analyses to count the numbers of Pc foci in nuclei from these tissues. Our data did not indicate clustering of Pc-binding sites and formation of so called „Pc bodies“. However, corresponding analyses are not without problems at the current stage of methodology and results must be interpreted carefully. Together with the results of previously published studies, which investigated pairing between PREs (Bantignies et al., 2003; Vazquez et al., 2006), my data demonstrated that such pairing is a highly tissue-specific phenomenon and is likely not involved in the regulation of PREs in various tissues. Activity-related positioning of transgenes was also addressed in transgenic porcine cell lines (Hofmann et al., 2003). Results of 2D erosion analyses showed that the LV-PGK transgene was associated with the nuclear periphery in its inactive state, while it occupied more interior positions in its active state. This corresponds to my results obtained with transgenic Drosophila lines. My data also suggested that the active LV-PGK construct might be associated with heterochromatin in one case. However, further experiments would be necessary to confirm such associations. The results obtained with Drosophila and porcine cells suggested conserved mechanisms for tethering inactive loci to the nuclear periphery. These were further addressed using the human CFTR locus as a model, which is closely associated with the nuclear periphery in its inactive state (Zink et al., 2004). The question was addressed whether Tpr, a protein associated with the nuclear basket, plays a role in the perinuclear localization of the inactive CFTR locus. CFTR showed a high degree of association with the nuclear periphery in control cells in accordance with previous data (Zink et al., 2004). After knock-down of Tpr via RNAi CFTR displayed a more interior positioning. This suggests that Tpr is involved in the organization of inactive gene loci at the nuclear periphery. Moreover, since Drosophila Tpr has a high level of homology to the mammalian Tpr (Zimowska et al., 1997) and as it has been shown that the yeast Tpr homologs Mlp1 and Mlp2 are involved in tethering of inactive loci to the nuclear periphery in yeast cells (Galy et al., 2000), it is possible that Tpr is a part of a conserved mechanism anchoring inactive loci to the nuclear periphery in eukaryotic cells.

In this thesis I scrutinized a specific region of the X chromosome of Drosophila melanogaster for evidence of positive directional selection. In addition, I analyzed the structure of six Southeast (SE) Asian populations of this species. In the first chapter, I analyzed a region that showed no polymorphism in a previous scan of the X chromosome in a European D. melanogaster population. This region, which I named the wapl region, is located on the distal part of the X chromosome, in cytological division 2C10 - 2E1. I observed a 60.5 - kb stretch of DNA encompassing the genes ph-d, ph-p, CG3835, bcn92, Pgd, wapl and Cyp4d1 that almost completely lacks variation in the European sample. Loci flanking this region show a skewed frequency spectrum at segregating sites, strong haplotype structure, and high levels of linkage disequilibrium. Neutrality tests revealed that these patterns of variation are unlikely under the neutral equilibrium model or simple bottleneck scenarios. In contrast, newly developed likelihood ratio tests suggest that strong positive selection has acted recently on the region under investigation, resulting in a selective sweep. Evidence is presented that this sweep may have originated in an ancestral population in Africa. In the second chapter, I revisited the center of the wapl region analyzed in chapter 1. I concentrated on the African D. melanogaster sample, as the valley of reduced variation found in the previous study was much narrower in the African sample than in the European one, which should help to pinpoint the target of selection. About 80% of the valley of reduced nucleotide variation was sequenced. This valley is located between the genes ph-d and Pgd. I therefore termed this part the ph-d - Pgd region. The new results confirm previous conclusions about selection having shaped nucleotide variability in this part of the D. melanogaster genome. Moreover, by sequencing the center of the selective sweep I was able to establish the haplotype structure in that region and to infer the historical context of the sweep. Most likely a positively selected substitution occurred at ph-p and was fixed before the out-of-Africa expansion of D. melanogaster, possibly >30,000 years ago. This substitution might be associated with the specialization of ph-p in gene regulation. In addition, the results obtained from the European sample indicate that sequence variation was not affected by demography alone. In fact, it was found that selection affected nucleotide diversity in the ph-d - Pgd region of the European sample as well. Since heterozygosity across the whole wapl region is substantially reduced, I propose that an additional selective sweep occurred at a different site in the European population. This is supported by an analysis regarding the time since the fixation of the (first) beneficial mutation at ph-p, which points toward a substitution in D. melanogaster before the colonization of Europe. In chapter 3, I obtained sequence data from six SE Asian samples for ten putatively neutrally evolving X-linked loci. Population genetic parameters were estimated and compared to those previously obtained from the European and the African sample. I observe substantially lower levels of nucleotide diversity in SE Asia than in either Africa or Europe. In particular, samples taken from more peripheral populations (e.g. Manila and Cebu, located on the Philippines) show a paucity of haplotypes. Common summary statistics indicate that genetic drift had a significant impact on these populations, which also led to considerable population substructure. One sample, i.e. Kuala Lumpur, however, shows rather high levels of heterozygosity among all SE Asian samples and is on average least differentiated from these. This indicates that the Kuala Lumpur population is ancestral to the other SE Asian populations, which is supported by a high amount of shared polymorphic sites. Finally, I revisited the wapl region, as analyzed in the first chapter, and find evidence that the selective sweep is older in Kuala Lumpur than in Europe.

Genetically encoded fluorescent indicators of neural activity represent promising tools for systems neuroscience. In the first part of my thesis, a comparative in vivo analysis of ten different genetically encoded calcium indicators as well as the pH-sensitive SynaptopHluorin is presented. The calcium indicators are either based on a single chromophore (GCaMP variants, Camgaroo variants, Pericam variants) or on two chromophores (Yellow Cameleon variants, Troponeon variants). I expressed these indicators in the cytosol of presynaptic boutons of the Drosophila larval neuromuscular junction and analyzed their fluorescence changes upon stimulation. GCaMP 1.3, GCaMP 1.6, Yellow Cameleon 2.0, 2.3, and 3.3, Inverse-Pericam, the troponin C-based calcium sensor TNL 15 and SynaptopHluorin allowed reliable detection of presynaptic fluorescence changes at the level of individual boutons. However, the response characteristics of all of these indicators differed considerably from each other. TNL 15 exhibited the most stable and fastest rising signals at lower activity rates, whereas GCaMP 1.6 produced the fastest signals at high rates of nerve activity with largest fluorescence changes. GCaMP 1.6 and GCaMP 1.3 signals, however, were complicated by bleaching, as was the case for Inverse Pericam. The fluorescence signals of the double-chromophore indicators were in general smaller but more photostable and reproducible. Camgaroo-1 and Camgaroo-2 showed little or no response, and Flash Pericam did not result in any detectable fluorescence. GCaMP 1.3 and YC 3.3 revealed fairly linear fluorescence changes and a corresponding linear increase in the signal-to-noise ratio (SNR) over an expanded range of neural activity. As expected, the expression level of the indicator had an influence on the signal kinetics and the SNR, whereas the signal amplitude was independent. In the second part of my thesis work I fused several genetically encoded calcium indicators to different signal sequences. The targeting of the indicators to distinct parts of the cell such as the membrane, vesicles or ion channels allows detection of calcium ions before they disperse in the cytosol. Specific signals can be extracted more efficiently and in a more relevant physiological context. Tagging of YC 2.3, GCaMP 1.6 and TNL 15 to transmembrane domains or proteins involved in the synaptic vesicle cycle did not result in functional targeting. TN XL fused to the transmembrane domain mCD8 at the N-terminus and eight amino acids from a calcium channel subunit at the C-terminus resulted in membrane association at the NMJ. Fractional fluorescence changes up to 6.5 % were recorded upon stimulation. In cells of the fly visual system scattered fluorescent puncta were observed. This fusion protein has the potential for monitoring calcium dynamics in close proximity of ion influx. The presented data will be useful for in vivo experiments with respect to the selection of an appropriate indicator, as well as for the correct interpretation of optical signals.

2. Summary In Drosophila melanogaster the transcriptional activity of the male X chromosome is upregulated to compensate for the reduced dosage of X-linked genes as compared to the two X chromosomes in females. This process is mediated by the Dosage Compensation Complex (DCC), a ribonucleoprotein complex consisting of five proteins (MSL1, MSL2, MSL3, MOF and MLE) and two non-coding RNAs (roX1 and roX2). The DCC preferentially localizes on the X chromosomes in males where it doubles its transcription rate. Two enzymes are associated with the DCC: the acetyltransferase MOF, specific for the lysine 16 of H4 (H4-K16), and the DNA/RNA helicase MLE. Genetic experiments demonstrated that both activities are required for dosage compensation in male flies. However, the weak association of MLE to the DCC has complicated its biochemical analysis and, so far, the involvement of MLE RNA helicase in dosage compensation has only been demonstrated genetically. Using different in vivo and in vitro approaches the physical and functional interactions of MLE with the other MSL proteins and with the roX RNAs was addressed. Monoclonal antibodies, specifically recognizing MLE, were raised in rats, offering a new tool for MLE characterization. By coexpression of the DCC subunits in SF9 cells, a recombinant complex containing MSL1-2-3, MOF, MLE and the roX2 RNA was reconstituted and purified. A specific integration of roX2 into the DCC could be observed only in the absence of MLE. Non specific RNA binding properties seemed instead associated to MLE RNA helicase. Moreover, the purified MSL complex did not affect the ATPase activity of MLE in the presence or absence of roX2 RNA. In vitro, MLE showed a preferential association with MSL1 and MSL2 and MLE interaction with both MSL proteins were not RNA mediated. In view of these results we suggest that binding to roX2 is not the main determinant for MLE integration into the DCC complex and protein-protein interactions might instead contribute to its proper recruitment to the X chromosome. MLE is a member of the DEAD-box RNA helicase family and it shares with the other members the same domain organization. In addition to a central ATPase/helicase domain, two predicted N-terminal double strand (ds) RNA-binding motifs (dsRBM1 and dsRBM2) and a predicted C-terminal single strand (ss) RNA/DNA-binding domain (RGG-box) are also present in MLE protein. These domains have been extensively characterized in RHA, human ortholog of MLE, and their RNA binding properties confirmed. However, it is not known how MLE binds RNA and how the different RNA binding modules contribute to stimulate its enzymatic activities. A preferential binding of MLE to dsRNA compared to ssRNA was shown by binding assays. In addition, changes in the affinity of MLE for both ssRNA and dsRNA were observed in the presence of different nucleotides. Deletion mutants of MLE were produced and purified from insect cells in order to address the contribution of the different RNA binding domains to MLE enzymatic activities. By transient expression in Drosophila cells of the same deletion mutants fused to GFP, the effects of individual domains on MLE recruitment to the X chromosome were also determined. The results show that unlike RHA, the dsRB1 and the RGG domains are dispensable for MLE RNA binding and unwinding, whereas dsRB2 seems to play the major role in coordinating both activities. However, the enzymatic activities alone are not sufficient to properly target MLE to the X chromosome. These results provide new data on the functional properties of MLE RNA helicase that may help to elucidate its molecular mechanisms of action.

A species’ evolutionary history is influenced by both neutral and selective processes. The effects that these forces have on genetic variation depend on their relative contributions. It is therefore important to be able to disentangle them. I conducted a comprehensive population genetics analysis of DNA polymorphism in Drosophila melanogaster, based on data collected from more than 250 loci spanning the entire X chromosome. Part of my work was dedicated to unraveling the relative roles of natural selection and demography in the recent history of a European population. First, I found evidence of a large impact of the population-size bottleneck associated with the colonization of Europe by the ancestral sub-Saharan populations. The multi-locus approach was crucial to disentangle neutral and selective forces, since theory predicts that demography has genome-wide effects, whereas selection acts only locally. Hence, I developed a coalescent-based maximum-likelihood method that estimated the population-size bottleneck to be ~4,000–16,000 years old. While this can account for most of the reduction of variation observed in the European sample, I could identify several loci and regions whose polymorphism pattern departs from the expectations under such a demographic scenario. One of these candidate regions was studied further in detail, revealing a pronounced valley of reduced nucleotide variation that is incompatible with a simple bottleneck model. Rather, this finding and the associated skew in the allelic frequency spectrum support the recent action of positive selection. Taken together, these results suggest that the European population experienced numerous episodes of natural selection to adapt to the new environment. A second goal of my research was to investigate the evolutionary patterns of non-coding DNA and detect signatures of selective constraint. I found that in this species functional constraints limit the accumulation of nucleotide mutations and of insertion/deletions in both intergenic and intronic regions. In particular, I showed that insertions have smaller sizes and higher frequencies than deletions, supporting the hypothesis that they are selected to compensate for the loss of DNA caused by deletion bias. Analysis of a simple model of selective constraints suggests that the blocks of functional elements located in intergenic sequences are on average larger than those in introns, while the length distribution of relatively unconstrained sequences interspaced between these blocks is similar in the two non-coding regions. Consistently, sequences conserved across species (i.e., free of deletions and/or insertions) have lower variation and divergence compared to the remaining fraction of DNA, supporting the presence of evolutionary constraints in these blocks. Moreover, I show that the base composition of intergenic and intronic regions is shaped by a complex interaction of neutral and non-neutral processes. Remarkably, GC content seems to be an important determinant of genetic diversity.

Thu, 30 Jun 2005 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/4233/ https://edoc.ub.uni-muenchen.de/4233/1/neumann_martin.pdf Neumann, Martin ddc:610, ddc:600, Medizin

This thesis intends to detect evidence of Darwinian selection and ultimately identify genes and substitutions that were involved in adaptation. The model organism Drosophila melanogaster was chosen as the study object, since the availability of the genome sequence and its evolutionary history allows us to investigate ancestral and derived populations. To identify the footprints of natural selection, the first objective was to locate genomic regions subject to selection. Such footprints involve a reduction in genetic variation along a recombining chromosome caused by a fixation of a beneficial mutation (i.e., so-called “selective sweep”) in a population under study. Single nucleotide polymorphisms of non-coding regions (i.e., 105 fragments) of the X chromosome in a putatively ancestral population of D. melanogaster from Zimbabwe were surveyed and compared to a derived European population in the first chapter. In contrast to the European population, evidence of selection was weak in the African population, but a strong signature of a population size expansion was observed. To examine the impact of demography and selection more deeply, an analysis of an enlarged DNA sequencing data set (i.e., 253 fragments) of the African population is presented in chapter two. A clear signature of a recent size expansion was observed and the time estimated of the expansion is 15,000 years before present, which was probably caused by drastic climatic changes. The enlarged data set revealed, in addition, that recombination is mutagenic in D. melanogaster. In the second part of this thesis, candidate regions of selective sweeps detected in the genome scan in both populations of D. melanogaster were investigated. In chapter three, a more detailed analysis of the region comprising an observed local reduction in variation in one X-linked fragment in the derived European population revealed significant evidence of recent Darwinian selection. The target of selection was attributed to three replacement sites leading to amino acid changes in two predicted genes, CG1677 and CG2059. In contrast, a lower number of haplotypes and a trend for low haplotype diversity suggesting the recent action of a selective sweep was examined in chapter four in the ancestral D. melanogaster population. An enlarged DNA sequencing data set revealed another feature unique to a selective sweep, namely the decay in haplotype structure. The target of selection was localized at the 5’ region of gene CG4661. In the third part of this thesis, the genetic variation of D. melanogaster populations from Southeast Asia were examined to provide first insights into these derived populations and the groundwork for future studies. Since no population genetic approach was done in natural D. melanogaster populations from this region, inversions were used as genetic markers. Other than a high frequency of the four common cosmopolitan inversions, there were neither signs for genetic differentiation between populations nor for natural selection. These findings can best be explained by a homogeneous habitat and a joint history of these populations revealing the existence of a panmictic population on Sundaland ~18,000 years ago.

In der vorliegenden Arbeit wurden die beiden Histon-Methyltransferasen Su(var)3-9 und E(Z) aus Drosophila melanogaster charakterisiert. Die Histonmethylierung als Modifikation war schon länger bekannt gewesen, bis zum Jahr 2000 war jedoch vor allem die Acetylierung etwas genauer untersucht worden. Su(var)3-9 war die einzige bekannte Histon-Lysin-Methyltransferase, als diese Arbeit begonnen wurde. Zur Charakterisierung wurde das myc-getagte Enzym aus Drosophila-Kernextrakt durch Affinitätschromatographie aufgereinigt und zunächst die Substratspezifität festgestellt. Wie das humane Enzym Suv39H1 methyliert es ebenfalls spezifisch H3-K9 (Lysin 9 im Histon H3). Das aus den Kernextrakten aufgereinigte Enzym besitzt aber auch die Fähigkeit, ein an H3-K9 präacetyliertes Substrat zu methylieren. Die Vermutung, dass Su(var)3-9 mit einer Histondeacetylase assoziiert ist, konnte durch Verwendung von TSA als HDAC-Inhibitor bestätigt werden. Es stellte sich heraus, dass HDAC1 (Rpd3) mit Su(var)3-9 assoziiert ist. Um das Enzym besser untersuchen zu können, wurde es als Volllängenprotein und als Deletionsmutante in E. coli exprimiert. Die Aufreinigung des rekombinanten Enzyms sowie seine Lagerbedingungen wurden optimiert. Das Volllängenprotein Su(var)3-9 liegt – wie durch Gelfiltration festgestellt - als Dimer vor, die Interaktion mit sich selbst ist über den N-Terminus vermittelt. Su(var)3-9 bindet an sein eigenes, bereits methyliertes Substrat. Dies wurde an Peptiden untersucht, die den ersten 20 Aminosäuren des Histons H3 entsprechen, und entweder an Lysin 9 dimethyliert oder unmodifiziert waren. Die Interaktion mit dem methylierten Substrat ist auf die Chromodomäne von Su(var)3-9 zurückzuführen, ist jedoch schwächer als die Wechselwirkung von HP1 mit methyliertem H3-K9. Des weiteren wurde eine Drosophila-Zelllinie stabil mit Su(var)3-9 transfiziert. Das überexprimierte Protein ist jedoch nur schwach aktiv. Die Tatsachen, dass Su(var)3-9 mit HDAC1 interagiert sowie mit seinem eigenen Substrat assoziiert, ermöglichen die Aufstellung von Hypothesen über die bis jetzt kaum erhellte Ausbreitung von Heterochromatin in euchromatische Bereiche. Durch die Wechselwirkung mit der Deacetylase könnte Su(var)3-9 auch in aktiv transkribierte Bereiche vordringen und diese methylieren. Die Acetylierung, Zeichen für aktive Transkription, würde durch die Methylierung ersetzt werden. Die Interaktion mit seinem umgesetzten Substrat könnte verhindern, dass das Enzym sich nach der Reaktion entfernt, vielmehr könnte Su(var)3-9 entlang eines DNA-Stranges sukzessive alle Nukleosomen methylieren. Die darauffolgende Bindung von HP1 an methyliertes H3-K9 könnte den heterochromatischen Charakter des Chromatins verstärken und für längere Zeit festlegen. Aus Drosophila-Kernextrakten gelang es weiterhin, den E(Z)/ESC-Komplex über Säulenchromatographie aufzureinigen. Dieser enthält neben E(Z), ESC, p55 und Rpd3 auch Su(z)12. E(Z), ESC und Su(z)12 gehören der Polycomb-Gruppe an. Deren Funktion ist die dauerhafte Repression der homöotischen Gene. Sie spielen daher eine wichtige Rolle im „Zellgedächtnis“ während der frühen Entwicklung von Drosophila. Es konnte gezeigt werden, dass der E(Z)/ESC-Komplex Lysin 9 sowie Lysin 27 im Histon H3 methyliert. Außerdem wurde in vitro ein Teilkomplex aus rekombinantem E(Z), p55 und ESC rekonstituiert, der das Histon H3 methylieren kann. Ein Teilkomplex, der E(Z) mit mutierter SET-Domäne enthält, ist nicht in der Lage, H3 zu methylieren. Die Vorhersage, dass E(Z) aufgrund seiner SET-Domäne eine Methyltransferase sein müsse, konnte durch vorliegende Untersuchungen bestätigt werden. Polycomb ist ein weiteres Protein aus der Polycomb-Gruppe. In dieser Arbeit konnte gezeigt werden, dass dieses Protein spezifisch an das Histon H3 bindet, das an K27 trimethyliert ist. Polycomb besitzt wie HP1 eine Chromodomäne. Aus den vorliegenden Daten kann folgendes Modell aufgestellt werden: Nach der Methylierung von H3-K9 sowie H3-K27 durch den E(Z)/ESC-Komplex in homöotischen Genen, die schon abgeschaltet sind und weiterhin reprimiert werden müssen, bindet Polycomb an dieses Methylierungsmuster. Polycomb befindet sich in einem großen Komplex mit weiteren Polycomb-Gruppen-Proteinen. Die Bindung dieses Komplexes an Chromatin könnte ein denkbarer Mechanismus sein, wie die dauerhafte Repression der homöotischen Gene vermittelt wird. Um den E(Z)/ESC-Komplex genauer untersuchen zu können, wurden Viren für das Baculosystem hergestellt, so dass eine Einzel- oder auch Coexpression der Proteine möglich ist. Die Aktivität von E(Z), das im Baculosystem exprimiert wurde, ist nicht besonders hoch. Es bindet unter den in dieser Arbeit verwendeten Bedingungen weder an DNA, noch an Histone noch an H3-Peptide, die methyliert sind. Innerhalb des E(Z)/ESC-Komplexes bindet E(Z) an p55, Rpd3, ESC sowie Su(z)12. Su(z)12 interagiert mit p55, Rpd3 und E(Z). Die weiteren Interaktionen werden am besten durch eine bildliche Darstellung (siehe Abb. 86) vermittelt. In einem Luciferase-Assay wurde eine repressive Wirkung von E(Z) festgestellt. Dieses Experiment bedarf allerdings eines aktivierten Systems. Ferner muss durch Mutationsanalysen sichergestellt werden, dass die repressive Wirkung auf die Methyltransferase-Aktivität von E(Z) zurückzuführen ist. Kürzlich wurde entdeckt, dass E(Z) sowie Su(z)12 in verschiedenen Tumoren überexprimiert sind. Noch ist weder deren Funktion in den Tumorzellen klar, noch weiss man, ob die Überexpression der Grund oder eine Folge der Tumorbildung ist, noch kennt man alle Zielgene, die durch eine Überexpression von E(Z) und Su(z)12 beeinflusst werden. In nächster Zeit sind hier Einsichten in die Wirkungsweise von E(Z), Su(z)12 und anderen Polycomb-Gruppen-Proteinen zu erwarten.