Characterization in Drosophila melanogaster of dPDZ-GEF, a Rap GTPase activator

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Defining the Role of the Histone Methyltransferase, PR-Set7, in Maintaining the Genome Integrity of Drosophila Melanogaster

The complete and faithful duplication of the genome is essential to ensure normal cell division and organismal development. Eukaryotic DNA replication is initiated at multiple sites termed origins of replication that are activated at different time through S phase. The replication timing program is regulated by the S-phase checkpoint, which signals and repairs replicative stress. Eukaryotic DNA is packaged with histones into chromatin, thus DNA-templated processes including replication are modulated by the local chromatin environment such as post-translational modifications (PTMs) of histones.

One such epigenetic mark, methylation of lysine 20 on histone H4 (H4K20), has been linked to chromatin compaction, transcription, DNA repair and DNA replication. H4K20 can be mono-, di- and tri-methylated. Monomethylation of H4K20 (H4K20me1) is mediated by the cell cycle-regulated histone methyltransferase PR-Set7 and subsequent di-/tri- methylation is catalyzed by Suv4-20. Prior studies have shown that PR-Set7 depletion in mammalian cells results in defective S phase progression and the accumulation of DNA damage, which may be partially attributed to defects in origin selection and activation. Meanwhile, overexpression of mammalian PR-Set7 recruits components of pre-Replication Complex (pre-RC) onto chromatin and licenses replication origins for re-replication. However, these studies were limited to only a handful of mammalian origins, and it remains unclear how PR-Set7 impacts the replication program on a genomic scale. Finally, the methylation substrates of PR-Set7 include both histone (H4K20) and non-histone targets, therefore it is necessary to directly test the role of H4K20 methylation in PR-Set7 regulated phenotypes.

I employed genetic, cytological, and genomic approaches to better understand the role of H4K20 methylation in regulating DNA replication and genome stability in Drosophila melanogaster cells. Depletion of Drosophila PR-Set7 by RNAi in cultured Kc167 cells led to an ATR-dependent cell cycle arrest with near 4N DNA content and the accumulation of DNA damage, indicating a defect in completing S phase. The cells were arrested at the second S phase following PR-Set7 downregulation, suggesting that it was an epigenetic effect that coupled to the dilution of histone modification over multiple cell cycles. To directly test the role of H4K20 methylation in regulating genome integrity, I collaborated with the Duronio Lab and observed spontaneous DNA damage on the imaginal wing discs of third instar mutant larvae that had an alanine substitution on H4K20 (H4K20A) thus unable to be methylated, confirming that H4K20 is a bona fide target of PR-Set7 in maintaining genome integrity.

One possible source of DNA damage due to loss of PR-Set7 is reduced origin activity. I used BrdU-seq to profile the genome-wide origin activation pattern. However, I found that deregulation of H4K20 methylation states by manipulating the H4K20 methyltransferases PR-Set7 and Suv4-20 had no impact on origin activation throughout the genome. I then mapped the genomic distribution of DNA damage upon PR-Set7 depletion. Surprisingly, ChIP-seq of the DNA damage marker γ-H2A.v located the DNA damage to late replicating euchromatic regions of the Drosophila genome, and the strength of γ-H2A.v signal was uniformly distributed and spanned the entire late replication domain, implying stochastic replication fork collapse within late replicating regions. Together these data suggest that PR-Set7-mediated monomethylation of H4K20 is critical for maintaining the genomic integrity of late replicating domains, presumably via stabilization of late replicating forks.

In addition to investigating the function of H4K20me, I also used immunofluorescence to characterize the cell cycle regulated chromatin loading of Mcm2-7 complex, the DNA helicase that licenses replication origins, using H4K20me1 level as a proxy for cell cycle stages. In parallel with chromatin spindown data by Powell et al. (Powell et al. 2015), we showed a continuous loading of Mcm2-7 during G1 and a progressive removal from chromatin through S phase.

Descending Control of Limb Movements in Drosophila melanogaster

Because the interactions between feedforward influences are inextricably linked during many motor outputs (including but not limited to walking), the contribution of descending inputs to the generation of movements is difficult to study. Here we take advantage of the relatively small number of descending neurons (DNs) in the Drosophila melanogaster model system. We first characterize the number and distribution of the DN populations, then present a novel load free preparation, which enables the study of descending control on limb movements in a context where sensory feedback can be is reduced while leaving the nervous system, musculature, and cuticle of the animal relatively intact. Lastly we use in-vivo whole cell patch clamp electrophysiology to characterize the role of individual DNs in response to specific sensory stimuli and in relationship to movement. We find that there are approximately 1100 DNs in Drosophila that are distributed across six clusters. Input from these DNs is not necessary for coordinated motor activity, which can be generated by the thoracic ganglion, but is necessary for the specific combinations of joint movements typically observed in walking. Lastly, we identify a particular cluster of DNs that are tuned to sensory stimuli and innervate the leg neuromeres. We propose that a multi-layered interaction between these DNs, other DNs, and motor circuits in the thoracic ganglia enable the diverse but well-coordinated range of motor outputs an animal might exhibit.

Characterization in Drosophila melanogaster of dPDZ-GEF, a Rap GTPase activator

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

The white gene and locomotor recovery from anoxia in Drosophila melanogaster

Locomotor recovery from anoxia is complicated and little is known about the molecular and cellular mechanisms regulating anoxic recovery in Drosophila. For this thesis I established a protocol for large-scale analysis of locomotor activity in adult flies with exposure to a transient anoxia. Using this protocol I observed that wild-type Canton-S flies recovered faster and more consistently from anoxia than the white-eyed mutant w1118, which carries a null allele of w1118 in an isogenic genetic background. Both Canton-S and w1118 are commonly used controls in the Drosophila community. Genetic analysis including serial backcrossing, RNAi knockdown, w+ duplication to Y chromosome as well as gene mutation revealed a strong association between the white gene and the timing of locomotor recovery. I also found that the locomotor recovery phenotype is independent of white-associated eye pigmentation, that heterozygous w+ allele was haplo-insufficient to induce fast and consistent locomotor recovery from anoxia in female flies, and that mini-white is insufficient to promote fast and consistent locomotor recovery. Moreover, locomotor recovery was delayed in flies with RNAi knockdown of white in subsets of serotonin neurons in the central nervous system. I further demonstrated that mutations of phosphodiesterase genes (PDE) displayed wild-type-like fast and consistent locomotor recovery, and that locomotor recovery was light-sensitive in the night in w1118. The delayed locomotor recovery and the light sensitivity were eliminated in PDE mutants that were dual-specific or cyclic guanosine monophosphate (cGMP)-specific. Up-regulation of cGMP using multiple approaches including PDE mutation, sildenafil feeding or specific expression of an atypical soluble guanylyl cyclase (Gyc88E) was sufficient to suppress w-RNAi induced delay of locomotor recovery. Taken together, these data strongly support the hypothesis that White transports cGMP and promotes fast and consistent locomotor recovery from anoxia.

Ultrastructural and Histological Study of Oogenesis and Oocyte Degeneration in the PenshellAtrina maura(Bivalvia: Pinnidae)

The successive stages of oogenesis and the changes involved in the oocyte degeneration process in the penshell Atrina maura were examined using light and transmission electron microscopy. The ovarian maturation process is asynchronous, as oocytes at different developmental stages can be found simultaneously. Oocytes develop from oogonia and then undergo three distinct stages of oogenesis: previtellogenesis, vitellogenesis and postvitellogenesis with mature oocytes. Atrina maura displays a solitary oogenesis type, in which follicular cells become associated with oocytes from the earliest stages of development and seem to play an integral role in vitellogenesis. The cytoplasm of vitellogenic oocytes contains numerous whorls of rough endoplasmic reticulum and Golgi bodies, suggesting that auto-synthetic vitellogenesis may occur in this species. In addition, the degeneration process of postvitellogenic oocytes triggered by a seasonal increase in water temperature (> 25°C) is described.

Ultrastructural and Histological Study of Oogenesis and Oocyte Degeneration in the PenshellAtrina maura(Bivalvia: Pinnidae)

The successive stages of oogenesis and the changes involved in the oocyte degeneration process in the penshell Atrina maura were examined using light and transmission electron microscopy. The ovarian maturation process is asynchronous, as oocytes at different developmental stages can be found simultaneously. Oocytes develop from oogonia and then undergo three distinct stages of oogenesis: previtellogenesis, vitellogenesis and postvitellogenesis with mature oocytes. Atrina maura displays a solitary oogenesis type, in which follicular cells become associated with oocytes from the earliest stages of development and seem to play an integral role in vitellogenesis. The cytoplasm of vitellogenic oocytes contains numerous whorls of rough endoplasmic reticulum and Golgi bodies, suggesting that auto-synthetic vitellogenesis may occur in this species. In addition, the degeneration process of postvitellogenic oocytes triggered by a seasonal increase in water temperature (> 25°C) is described.

Genregulation in der Drosophila Spermatogenese am Beispiel der Mst(3)CGP-Genfamilie

In dieser Dissertation wird die Rolle des zentralen Kontrollelementes TCE auf unterschiedlichen Ebenen der Genexpression untersucht. Das TCE verhindert die Translation prämeiotisch gebildeter mRNAs in der Spermatogenese von Drosophila bis zu einem späten postmeiotischen Stadium. Gleichzeitig provoziert es Transkriptionsaktivität. Das TCE wurde zunächst in einer kleinen Genfamilie identifiziert und am Beispiel des Gens Mst87F detaillierter untersucht. In EMSA-Experimenten wurde die Komplexbildung mit regulatorischen Proteinen aus Proteinextrakten des Hodengewebes am TCE der Mst87F mRNA nachgewiesen. Massenspektrometrische Analysen ergaben u.a. die Kandidatenproteine Exuperantia (Exu), dFmr1 und CG3213. Die Komplexbildung an einem zweiten Mitglied der Genfamilie - Mst98Ca -, welches sich in der Genstruktur und dem Proteinaufbau von Mst87F unterscheidet, belegt die Allgemeingültigkeit dieser Interaktion. Beim Einsatz von veränderten TCE-Sequenzen ergibt sich ein abweichendes Erscheinungsbild der Komplexe, was mit dem Verlust der Funktion korreliert. Auch die Komplexbildung mit den rekombinanten Proteinen von exuperantia und dfmr1 erfolgt an beiden RNAs in gleicher Weise. In Kombination wird ein stärkerer Shift erzeugt. In einer Exu-defizienten Mutante beobachtet man drastische Veränderungen in der Lokalisation von einem Mst87F-GFP- bzw. CG3213-GFP-Fusionsprotein. Analysen mittels der qPCR zeigen eine drastische Verringerung der Mst87F mRNA Menge. Beides lässt vermuten, dass das Fehlen von Exu bereits in frühen Stadien zu molekularen Defekten führt. Um die Translationskontrolle zu umgehen, wurden Transgene mit einer IRES (aus dem Gen reaper) an verschiedenen Positionen des 5'UTRs erzeugt. Die erwartete Translationsinitiation durch die IRES blieb aus. Northern- und qPCR-Analysen zeigen eine starke Reduktion des mRNA-Niveaus. Somit kann aufgrund der drastischen Deregulation auf Transkriptionsebene der Effekt auf die Translationskontrolle nicht mehr analysiert werden. Überraschenderweise wurden durch die Verwendung einer anderen IRES (aus der Genkassette CG31311) die Expressionscharakteristika des Ursprungsgens auf Mst87F übertragen. Das Fusionsprotein lässt sich plötzlich in den Ommatidien der Komplexaugen nachweisen. Da aus früheren Arbeiten bereits eine Rolle des TCE auf Transkriptionsebene nachgewiesen ist, wurde die Komplexbildung auf Mst87F-DNA-Fragmente mit TCE ausgedehnt. Analysen unter Verwendung der rekombinanten Proteine Exu und dFmr1 verliefen negativ. Daraufhin sollten massenspektrometrische Experimente neue Kandidaten für regulatorische Proteine auf DNA-Ebene identifizieren. Von vier weiteren Kandidaten zeigen zwei unter RNAi-Einfluß komplette Sterilität und starke Defekte in der Spermienentwicklung.

Functions of the SLC36 transporter Pathetic in growth control of Drosophila sensory neurons

Thesis (Ph.D.)--University of Washington, 2016-08

The Role of G Protein In Alcohol Related Behaviours Using Drosophila melanogaster As A Model

Ethanol, classified as a drug, affects the central nervous system, and its consumption has been linked to the development of several behaviours including tolerance and dependence. Alcohol tolerance is defined as the need for higher doses of alcohol to induce the same changes observed in the initial exposure or where repetitive exposures of the same alcohol dose induce a lower response. Ethanol has been shown to interact with numerous targets and ultimately influence both short and long term adaptation at the cellular and molecular level in brain [1]. These adaptation processes are likely to involve signalling molecules: our work has focussed on G proteins gene expression. Using both wild type and several mutant fruit fly (Drosophila melanogaster) as a model for behaviour and molecular studies, we observed significant increases in sedation time (ST50) in response to alcohol (P<0.001) Fig.A. We also observed a consistent and significant decrease of Gq protein mRNA expression in Drosophila dUNC and DopR2 mutants chronically exposed to alcohol (*P<0.05). Fig B. Method: Six male flies were observed in drosophila polystyrene 25 x 95mm transparent vial in between cotton plugs. To the top plug, 500uL of 100% ethanol was added. Time till 50% of the flies were sedated was recorded on each day following the schedule. Fig. C (n=4-6). Using RT-PCR, we also quantified G protein mRNA expression levels one hour post initial 30 minutes of ethanol expression on day 1 and day 3 relative to expression in naïve flies.(n=2) [A] Increase in sedation time indicative of tolerance in different mutant lines and wild type flies. Six male flies were used in each experiment and (n= 4-6. ***P<0.001 unpaired t tests). [B] RT-PCR results showing significant reduction in Gq mRNA in flies chronically exposed to alcohol. (n=2. *P<0.05) [C] Alcohol exposure schedule. (1) Kaun K.R., R. Azanchi, Z. Maung, J. Hirsh, U. Heberlein. (2011). A Drosophila model for alcohol reward. Nature Neuroscience. 14 (5), 612–619.

Caracterização da fosfatase String/Cdc25 como um regulador da neurotoxicidade de Tau em Drosophila

As tauopatias, grupo onde se inclui a doença de Alzheimer (AD), são caracterizadas pela deposição intracelular de emaranhados neurofibrilares (NFTs), compostos principalmente por formas hiperfosforiladas da proteína Tau, uma proteína que se associa aos microtúbulos. Os mecanismos moleculares subjacentes à neurotoxicidade induzida por Tau não são ainda claros. Drosophila melanogaster tem sido usada para modelar diversas doenças neurodegenerativas humanas, incluindo as tauopatias. Neste trabalho foi usado o sistema visual de Drosophila como modelo para identificar os passos que podem levar à acumulação de Tau em Tauopatias. Durante o desenvolvimento do olho de Drosophila, a expressão ectópica de hTau induz um olho rugoso, em consequência da neurotoxicidade, e que pode ser utilizado para identificar modificadores do fenótipo. A fosfatase codificada por string /cdc25 (stg), um regulador universal da transição G2/M, foi previamente identificada como um supressor da neurotoxicidade associada à expressão da proteina Tau. No entanto, os mecanismos moleculares que estão na base desta interação genética nunca foram estudados, desconhecendo-se também se a atividade fosfatase de Stg/Cdc25 é essencial para modular os níveis de fosforilação de Tau. O objetivo deste projeto consistiu em elucidar os mecanismos que se encontram na base da interação Stg-Tau. Para alcançar este objectivo, usou-se uma abordagem genética e bioquímica. Os resultados obtidos sugerem que Stg é um possível modulador da neurotoxicidade de Tau.

The complexity of Drosophila innate immunity

Metazoans rely on efficient mechanisms to oppose infections caused by pathogens. The immediate and first-line defense mechanism(s) in metazoans, referred to as the innate immune system, is initiated upon recognition of microbial intruders by germline encoded receptors and is executed by a set of rapid effector mechanisms. Adaptive immunity is restricted to vertebrate species and it is controlled and assisted by the innate immune system. Interestingly, most of the basic signaling cascades that regulate the primeval innate defense mechanism(s) have been well conserved during evolution, for instance between humans and the fruit fly, Drosophila melanogaster. Being devoid of adaptive signaling and effector systems, Drosophila has become an established model system for studying pristine innate immune cascades and reactions. In general, an immune response is evoked when microorganisms pass the fruit fly’s physical barriers (e.g. cuticle, epithelial lining of gut and trachea), and it is mainly executed in the hemolymph, the equivalent of the mammalian blood. Innate immunity in the fruit fly consists of a phenoloxidase (PO) response, a cellular response (hemocytes), an antiviral response, and the NF-κB dependent production of antimicrobial peptides referred to as the humoral response. The JAK/STAT and Jun kinase signaling cascades are also implicated in the defence against pathogens.

Phylum-wide transcriptome analysis of oogenesis and early embryogenesis in selected nematode species

Oogenesis is a prerequisite for embryogenesis in Metazoa. During both biological processes important decisions must be made to form the embryo and hence ensure the next generation: (1) Maternal gene products (mRNAs, proteins and nutrients) must be supplied to the embryo. (2) Polarity must be established and axes must be specified. While incorporation of maternal gene products occurs during oogenesis, the time point of polarity establishment and axis specification varies among species, as it is accomplished either prior, during, or after fertilisation. But not only the time point when these events take place varies among species but also the underlying mechanisms by which they are triggered. For the nematode model Caenorhabditis elegans the underlying pathways and gene regulatory networks (GRNs) are well understood. It is known that there the sperm entry point initiates a primary polarity in the 1-celled egg and with it the establishment of the anteroposterior axis. However, studies of other nematodes demonstrated that polarity establishment can be independent of sperm entry (Goldstein et al., 1998; Lahl et al., 2006) and that cleavage patterns, symmetry formation and cell specification also differ from C. elegans. In contrast to the studied Chromadorea (more derived nematodes including C. elegans), embryos of some marine Enoplea (more basal representatives) even show no discernible early polarity and blastomeres can adopt variable cell fates (Voronov and Panchin 1998). The underlying pathways controlling the obviously variant embryonic processes in non-Caenorhabditis nematodes are essentially unknown. In this thesis I addressed this issue by performing a detailed unbiased comparative transcriptome analysis based on microarrays and RNA sequencing of selected developmental stages in a variety of nematodes from different phylogenetic branches with C. elegans as a reference system and a nematomorph as an outgroup representative. In addition, I made use of available genomic data to determine the presence or absence of genes for which no expression had been detected. In particular, I focussed on components of selected pathways or GRNs which are known to play essential roles during C. elegans development and/or other invertebrate or vertebrate model systems. Oogenesis must be regulated differently in non-Caenorhabditis nematodes, as crucial controlling components of Wnt and sex determination signaling are absent in these species. In this respect, I identified female-specific expression of potential polarity associated genes during gonad development and oogenesis in the Enoplean nematode Romanomermis culicivorax. I could show that known downstream components of the polarity complexes PAR-3/-6/PKC-3 and PAR-1/-2 are absent in non-Caenorhabditis species. Even PAR-2 as part of the polarity complex does not exist in these nematodes. Instead, transcriptomes of nematodes (including C. elegans), show expression of other polarity-associated complexes such as the Lgl (Lethal giant larvae) complex. This result could pose an alternative route for nematodes and nematomorphs to initiate polarity during early embryogenesis. I could show that crucial pathways of axis specification, such as Wnt and BMP are very different in C. elegans compared to other nematodes. In the former, Wnt signaling, for instance, is mediated by four paralogous beta-catenins, while other Chromadorea have fewer and Enoplea only one beta-catenin. The transcriptomes of R. culicivorax and the nematomorph show that regulators of BMP (e.g. Chordin), are specifically expressed during early embryogenesis only in Enoplea and the close outgroup of nematomorphs. In conclusion, my results demonstrate that the molecular machinery controlling oogenesis and embryogenesis in nematodes is unexpectedly variable and C. elegans cannot be taken as a general model for nematode development. Under this perspective, Enoplean nematodes show more similarities with outgroups than with C. elegans. It appears that certain pathway components were lost or gained during evolution and others adopted new functions. Based on my findings I can conjecture, which pathway components may be ancestral and which were newly acquired in the course of nematode evolution.