Acute Synaptic Activity Causes Differential miRNA Expression in The Drosophila melanogaster Larval Central Nervous System

The primary goal of this thesis was to determine if spaced synaptic stimulation induced the differential expression of microRNAs (miRNAs) in the Drosophila melanogaster central nervous system (CNS). Prior to attaining this goal, we needed to identify and validate a spaced stimulation paradigm that could induce the formation of new synaptic growth at a model synapse, the larval neuromuscular junction (NMJ). Both Channelrhodopsin- and high potassium-based stimulation paradigms adapted from (Ataman, et al. 2008) were tested. Once validation of these paradigms was complete, we sought to characterize the miRNA expression profile of the larval CNS by miRNA array. Following attainment of these data, we used quantitative real-time PCR (RT-qPCR) to determine if acute synaptic stimulation caused the differential expression of neuronal miRNAs. We found that upon high potassium spaced training in a wild type (Canton S) genotype, 5 miRNAs showed significant differential expression when normalized to a validated reference gene, the U1 snRNA. Moreover, absolute quantification of our RT-qPCR study implicated one miRNA: miR-958 as being significantly regulated by activity. Investigation into potential targets for miR-958 revealed it to be a potential regular of Dlar, a protein tyrosine phosphatase implicated in synapse development. This investigation provides the foundation to directly test our underlying hypothesis that, following spaced training, differential expression of miRNAs alters the translation of proteins required to induce and maintain these structural changes at the synapse.

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.

Drosophila melanogaster larvae make nutritional choices that minimize developmental time

Organisms from slime moulds to humans carefully regulate their macronutrient intake to optimize a wide range of life history characters including survival, stress resistance, and reproductive success. However, life history characters often differ in their response to nutrition, forcing organisms to make foraging decisions while balancing the trade-offs between these effects. To date, we have a limited understanding of how the nutritional environment shapes the relationship between life history characters and foraging decisions. To gain insight into the problem, we used a geometric framework for nutrition to assess how the protein and carbohydrate content of the larval diet affected key life history traits in the fruit fly, Drosophila melanogaster. In no-choice assays, survival from egg to pupae, female and male body size, and ovariole number - a proxy for female fecundity - were maximized at the highest protein to carbohydrate (P:C) ratio (1.5:1). In contrast, development time was minimized at intermediate P:C ratios, around 1:2. Next, we subjected larvae to two-choice tests to determine how they regulated their protein and carbohydrate intake in relation to these life history traits. Our results show that larvae targeted their consumption to P:C ratios that minimized development time. Finally, we examined whether adult females also chose to lay their eggs in the P:C ratios that minimized developmental time. Using a three-choice assay, we found that adult females preferentially laid their eggs in food P:C ratios that were suboptimal for all larval life history traits. Our results demonstrate that D. melanogaster larvae make foraging decisions that trade-off developmental time with body size, ovariole number, and survival. In addition, adult females make oviposition decisions that do not appear to benefit the larvae. We propose that these decisions may reflect the living nature of the larval nutritional environment in rotting fruit. These studies illustrate the interaction between the nutritional environment, life history traits, and foraging choices in D. melanogaster, and lend insight into the ecology of their foraging decisions.

Drosophila melanogaster as a model for basal body research

The fruit fly, Drosophila melanogaster, is one of the most extensively studied organisms in biological research and has centrioles/basal bodies and cilia that can be modelled to investigate their functions in animals generally. Centrioles are nine-fold symmetrical microtubule-based cylindrical structures required to form centrosomes and also to nucleate the formation of cilia and flagella. When they function to template cilia, centrioles transition into basal bodies. The fruit fly has various types of basal bodies and cilia, which are needed for sensory neuron and sperm function. Genetics, cell biology and behaviour studies in the fruit fly have unveiled new basal body components and revealed different modes of assembly and functions of basal bodies that are conserved in many other organisms, including human, green algae and plasmodium. Here we describe the various basal bodies of Drosophila, what is known about their composition, structure and function.

Os efeitos do plasma não térmico à pressão atmosférica no desenvolvimento larval e anomalias morfológicas de Drosophila Melanogaster

O plasma não térmico nos cuidados de saúde é um campo emergente que tem as suas raízes na ciência de plasmas. Este tipo de investigação tem crescido rapidamente e é agora objeto de um amplo esforço de pesquisa interdisciplinar envolvendo a medicina, a biologia, a física, a química e a engenharia. Têm sido feitos vários trabalhos de modo a elucidar quais as interações das espécies produzidas pelo plasma com os sistemas vivos. É evidente que o mecanismo da interação do plasma com os sistemas vivos é complexo, em parte devido à complexidade do plasma mas principalmente devido à enorme complexidade da biologia. O principal objetivo desta dissertação foi observar os efeitos do plasma não térmico à pressão atmosférica (PNTPAs) no desenvolvimento larval e anomalias morfológicas de Drosophila melanogaster. Para o efeito, foram expostas e analisadas fenotipicamente 2.566 larvas após exposição, dos diferentes estádios (1.º, 2.º e 3.º) de desenvolvimento. Os testes foram realizados com aplicações de plasma com e sem ultra violeta, em duas linhas diferentes de Drosophila; uma linha selvagem preparada por nós e uma linha laboratorial. A análise fenotípica revelou que após exposição as larvas apresentavam alterações no fenótipo e no comportamento que não foram observadas no controlo, nomeadamente anomalias nas mudas, traqueias partidas, formação de massas melanóticas que podiam persistir até à fase adulta, excesso de gotículas lipídicas, atraso no desenvolvimento, comportamento de não alimentação e formação de pupa imatura que levava à formação de pupa precoce e morte pupal. Na fase pupal, as anomalias mais comuns estavam relacionadas com a forma do pupário (causadas pela pipação prematura), apresentando um desenvolvimento aberrante. Entre os vários fenótipos observados, o mais significativo foi o criptocefálico (alterações na eversão dos discos imaginais) levando à morte pupal. Nos adultos, as principais anomalias morfológicas foram registadas na formação e segmentação das patas, na forma e padrão das nervuras das asas e na formação do tórax. A similaridade destes resultados com trabalhos publicados relacionados com a hormona esteróide ecdisona indicam que provavelmente o PNTPA poderá ter influenciado a biossíntese e/ou a regulação da ecdisona, a principal hormona que regula o desenvolvimento e a metamorfose em Drosophila.

Regulation of dendrite growth, placement, and patterning in Drosophila melanogaster class 4 dendrite arborization neurons

Thesis (Ph.D.)--University of Washington, 2016-06

Functional dissection of the Suppressor of UnderReplication protein of Drosophila melanogaster: identification of domains influencing chromosome binding and DNA replication

The Suppressor of UnderReplication (SuUR) gene controls the DNA underreplication in intercalary and pericentric heterochromatin of Drosophila melanogaster salivary gland polytene chromosomes. In the present work, we investigate the functional importance of different regions of the SUUR protein by expressing truncations of the protein in an UAS-GAL4 system. We find that SUUR has at least two separate chromosome-binding regions that are able to recognize intercalary and pericentric heterochromatin specifically. The C-terminal part controls DNA underreplication in intercalary heterochromatin and partially in pericentric heterochromatin regions. The C-terminal half of SUUR suppresses endoreplication when ectopically expressed in the salivary gland. Ectopic expression of the N-terminal fragments of SUUR depletes endogenous SUUR from polytene chromosomes, causes the SuUR(-) stopphenotype and induces specific swellings in heterochromatin.

EFFECTS OF A VISUAL HORIZON ON DROSOPHILA MELANOGASTER OBJECT-TRACKING RESPONSES

To navigate effectively in three-dimensional space, flying insects must approximate distances to nearby objects. Humans are able to use an array of cues to guide depth perception in the visual world. However, some of these cues are not available to insects that are constrained by their rigid eyes and relatively small body size. Flying fruit flies can use motion parallax to gauge the distance of nearby objects, but using this cue becomes a less effective strategy as objects become more remote. Humans are able to infer depth across far distances by comparing the angular distance of an object to the horizon. This study tested if flying fruit flies, like humans, use the relative position of the horizon as a depth cue. Fruit flies in tethered flight were stimulated with a virtual environment that displayed vertical bars of varying elevation relative to a horizon, and their tracking responses were recorded. This study showed that tracking responses of the flies were strongly increased by reducing the apparent elevation of the bar against the horizon, indicating that fruit flies may be able to assess the distance of far off objects in the natural world by comparing them against a visual horizon.

Using Doubly-Labeled Water to Measure Energy Expenditure in an Important Small Ectotherm Drosophila melanogaster

Open Access funded by Wellcome Trust for University College London (UCL) authors. Acknowledegements Funding for this work was provided by the Wellcome Trust (MDWP, LP). John Speakman was supported by the Strategic Priority Research Program of the Chinese Academy of Sciences (No. XDB13030000) a 1000 talents professorship. We are grateful to Peter Thomson and Paula Redman for technical assistance with isotope analysis

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.